Ants and agricultural pests in the Mediterranean: exploring antagonism and mutualism paradigms for biological control

UNIVERSITA’ DEGLI STUDI DI PARMA DOTTORATO DI RICERCA IN Biologia Evoluzionistica ed Ecologia CICLO XXXVI Ants and agricultural pests in the Mediterranean: exploring antagonism and mutualism paradigms for biological control Coordinatore: Chiar.mo Prof. Pierluigi Viaroli Tutore: Chiar.mo Prof. Donato A. Grasso Dottorando: Dott. Enrico Schifani Anni Accademici 2020/2021 – 2022/2023 Abstract Ants are insects with an almost unparalleled ecological impact on terrestrial ecosystems, providing many important ecosystem services through their complex interactions with countless other organisms. Ant role in agroecosystems has drawn significant attention by humans for centuries but is still better investigated for the tropics as compared to temperate regions. Most ants are generalist predators, and their predatory abilities have also been appreciated for their antagonistic effects on many agricultural pest insects. At the same time, ants establish mutualistic relationships with several honeydew-producing hemipterans, forming keystone ecological associations that can also develop into a problem for agricultural settings in which some ant-mutualist hemipterans are important pests. We explore ecological and behavioral interactions between ants and agricultural pest insects through antagonism and mutualism paradigms using laboratory and field experiments, while also gathering baseline data on the diversity and distribution of ant species in agroecosystems across Italy. We present our results through eleven chapters grouped in three sections: i) Ants as biological control agents of insect pests; ii) Ants as mutualist partners of insect pests; iii) First data on ant diversity and distribution in Italian agroecosystems. In the first section, we describe for the first time the role of ants as natural enemies of key agricultural pests accounting both direct and direct interactions and analyzing potential conflicts with other natural enemies, while also presenting encouraging results on behavioral manipulation of ants through artificial nectaries. In the second section, we describe ecological and behavioral aspects of the interactions between ants, honeydew-producing mutualist hemipterans, and their natural enemies, emphasizing the variety of outcomes that the species-specific characteristics of the involved actors may produce. Furthermore, we present a comparative review of the methodologies developed to manage ant-hemipteran mutualism when this is responsible for significant pest outbreaks. In the third section, we present first data on ant diversity in Italian agroecosystems as well as the inclusion of the ant species studied in this thesis in the DNA barcoding library of European ants. 1 Riassunto Le formiche sono responsabili di importanti servizi ecosistemici attraverso le complesse interazioni con innumerevoli altri organismi, rendendosi insetti dall’impatto ecologico quasi senza pari per quanto riguarda gli ecosistemi terrestri. Il ruolo delle formiche negli agroecosistemi ha attratto significativa attenzione da parte dell’uomo per secoli, ma è ancora meglio noto nelle regioni tropicali rispetto a quelle temperate. La maggior parte delle formiche sono predatori generalisti, e le loro abilità predatrici sono state apprezzate anche per il loro effetto antagonistico nei confronti di molte specie dannose di insetti. Allo stesso tempo, le formiche instaurano relazioni mutualistiche con molti emitteri produttori di melata, formando associazioni chiave di volta dal punto di vista ecologico che possono divenire problematiche nei contesti agricoli in cui alcuni emitteri mutualisti delle formiche hanno un ruolo importante come insetti dannosi. Abbiamo esplorato le interazioni ecologiche e comportamentali fra formiche e insetti dannosi in agricoltura attraverso i paradigmi di antagonismo e mutualismo, utilizzando esperimenti sia di campo che di laboratorio, e raccogliendo al contempo dati di base sulla diversità e distribuzione delle specie di formiche degli agroecosistemi italiani. Presentiamo i nostri risultati in undici capitoli, raggruppati in tre sezioni: i) Le formiche come agenti di controllo biologico di insetti dannosi; ii) Le formiche come partner mutualisti di insetti dannosi; iii) Primi dati sulla diversità e distribuzione delle formiche negli agroecosistemi italiani. Nella prima sezione descriviamo per la prima volta il ruolo delle formiche come nemici naturali di importanti insetti nocivi in agricoltura, tenendo conto sia delle interazioni dirette che di quelle indirette, analizzando possibili conflitti con altri antagonisti naturali e presentando risultati incoraggianti sulla manipolazione comportamentale delle formiche tramite nettàri artificiali. Nella seconda sezione descriviamo aspetti ecologici e comportamentali delle interazioni fra formiche, emitteri mutualisti produttori di melata, ed i loro antagonisti naturali, enfatizzando la varietà di risultati che possono scaturire dalle caratteristiche specie-specifiche degli attori coinvolti. Infine, presentiamo una revisione comparativa delle metodologie sviluppate per la gestione del mutualismo formiche-emitteri 2 quando questo è responsabile di significativi incrementi di insetti nocivi. Nella terza sezione, presentiamo primi dati sulla diversità delle formiche negli agroecosistemi italiani, e l’inclusione delle specie di formiche studiate in questa tesi all’interno della libreria di DNA barcoding delle formiche europee. 3 1. Introduction 1.1 Ants and their ecological role in terrestrial ecosystems………………………..……………..7 1.2 The intricate relationship between ants and agriculture: consequences for biological control……………………………………………………………………………….………..9 1.3 Aims……………………………………………………………………………..…………..11 2. Section I: Ants as biocontrol agents of insect pests…………………………………………12 2.1 Chapter 1 – New tools for conservation biological control: testing ant-attracting artificial nectaries to employ ants as plant defenders…………………………………………………15 2.2 Chapter 2 – Predatory abilities of Two Mediterranean ants on the eggs and larvae of the codling moth Cydia pomonella……………………………………………………………...37 2.3 Chapter 3 – Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus…….…………………..….45 2.4 Chapter 4 – Interactions between egg parasitoids and predatory ants for the biocontrol of the invasive brown marmorated stink bug Halyomorpha halys……………………………..…..54 2.5 Chapter 5 – Mediterranean ants can increase nymph mortality in the stink bug Nezara viridula without interfering with its egg parasitoid Trissolcus basalis……………………………….62 3. Section II: Ants as mutualist partners of insect pests………………………………...……69 3.1 Chapter 6 – Ant attendance does not necessarily imply protection of aphids from their arthropod natural enemies……………………………………………………………...……72 3.2 Chapter 7 – Fight and rescue or give up and flee? Behavioral responses of different ant species tending the mutualist walnut aphid Panaphis juglandis to native and exotic ladybugs…...….78 3.3 Chapter 8 – Toward sustainable management of ant-hemipteran mutualism in agricultural settings: a comparison of different approaches………………………………………………85 4. Section III: First data on ant diversity and distribution in Italian agroecosystems…..……94 4.1 Chapter 9 – Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley……………………………………………………………………………...….96 4.2 Chapter 10 – Trunk size influences species richness and functional composition of biogeographically different tree-visiting ant communities in pear orchards……………….110 4.3 Chapter 11 – Developing the DNA barcoding library of European ants: the species of the Italian agroecosystems……………………………………………………………..………117 5. Discussion………………….……………………………………………………...………124 6. Acknowledgements………………………………………………………………..………129 7. References………………………………………………………………………...….……130 8. Index of other articles produced during the years 2020-2023…………………….………136 4 1. INTRODUCTION 5 1.1 Ants and their ecological role in terrestrial ecosystems Ants (Hymenoptera: Formicoidea: Formicidae) are an eusocial insect family that evolved during the Cretaceous period, closely related to the Apoidea and Scolioidea superfamilies (Branstetter et al. 2022; Romiguier et al. 2022). Today, they count around 14,150 described species (Bolton 2023), being the largest of all eusocial animal groups (Wilson & Hölldobler 1990), while a large portion of their diversity is thought to be still undescribed (Kass et al. 2022). Latest estimates suggest there are around 20 × 1015 (20 quadrillion) ants on Earth, accounting for a remarkable biomass of 12 megatons of dry carbon, which is more than wild mammals and birds combined and around 20% of human biomass (Schultheiss et al. 2022). While their origins were most likely tropical, ants have become almost ubiquitous in all terrestrial habitats excluding the poles (Guénard et al. 2017), and in this wide geographic range, they have an important and multifaced ecological role (Wilson & Hölldobler 1990; Lach et al. 2010; Parker & Kronauer 2021). Ancestral ants were probably ground-nesting predators and scavengers of tropical rainforests, and most of the extant species retain both characteristics (Wilson & Hölldobler 1990; 2008). While a large part of them diversified their diet by including food sources other than other small animals, almost all ants are at least occasionally predators and scavengers of other arthropods, and entire lineages are exclusively predatory, including several specialized predators (Wilson & Hölldobler 1990; 2008). The predatory activity of generalist and specialized ants has acted as a evolutionary pressure selecting ecosystem-wide defensive or evasive adaptations to coexist with ants across terrestrial arthropods (Parker & Kronauer 2021). Diet diversification, however, was especially important in the formicoid clade, which comprises most of the actual species diversity of ants, with honeydew and seed consumption becoming particularly important outside the tropics (Wilson & Hölldobler 2008). Both represent are the foundation of some of the most frequent interactions between ants and plants, which also encompass several other mechanisms, from indirect herbivory by fungus-growing Attini species to strong mutualistic relationships involving several different lineages of myrmecophytes or 6 myrmecophilous plants (Wilson & Hölldobler 1990; Rico-Gray & Oliveira 2008). Ants eat the honeydew produced by several phytophagous insects, mostly aphids and scale bugs, often in exchange for protecting them against predators or parasitoids, pathogen fungi, and microbes, or adverse climatic conditions (Wilson & Hölldobler 1990). This type of mutualistic relationship between ants and honeydew-producing herbivores, known as trophobiosis, is considered an ecological keystone, being extremely successful across the world and part of larger multitrophic networks (Parker & Kronauer 2021). Other sugary liquid foods can be directly provided by myrmecophilous plants to their ant partners in the form of nectar produced by extrafloral nectaries (Rico-Gray & Oliveira 2008). On the other hand, seed consumption can be both beneficial or negative for different plant species, based on whether it contributes positively to seed dispersal (myrmecochory) as in the case of ant-adapted seeds with elaiosomes, and can have a deep influence over plant communities in some habitats (Rico-Gray & Oliveira 2008; Lach et al. 2010). Most extant ant species build their nest on the ground, either digging in the soil, rotten wood, or living in rock crevices, but a considerable number of species build their nests on the trunk or canopy of trees and other plants (Wilson & Hölldobler 1990). In all cases, ants may act as important ecosystem engineers, chemically enriching the soil and participating in bioturbation processes, while also speeding the degradation of deadwood (De Bruyn & Conacher 1990; Lach et al. 2010). Ant nests are typically essential to the life of a plethora of myrmecophilous animals, mostly insects, and spiders, that have evolved special adaptations to live as inquilines or parasites of ant colonies, exploiting their resources and the protection the nests and colonies can offer (Parker & Kronauer 2021). Ant communities, which may strongly vary in species richness and composition based on biogeographical and habitat characteristics, are structured along hierarchical structures based on behavioral dominance, colony size, foraging strategies, and other key biological characteristics, and can be used as a monitoring tool to track environmental changes (Lach et al. 2010). 7 1.2 The intricate relationship between ants and agriculture Ants play a significant but ambivalent role in agricultural settings, where they offer both services and disservices, in a complex balance that is overall favorable in most cases but may significantly vary based on local ecological conditions and on agricultural management (e.g., Styrsky & Eubanks 2007; Anjos et al. 2022). The service ants provide that has attained the most attention is the control of numerous insect pests (Way & Khoo 1992; Choate & Drummond 2011; Offenberg 2015; Anjos et al. 2022). At the very least since 304 A.D., farmers in Asia appreciated the favorable role of ants as enemies of phytophagous insects to the point of making active use of them by placing weaver ant (Oecophylla smaragdina Fabricius, 1775) nests on the canopy fruit trees (Huang & Yang 1987; Van Mele 2008; Offenberg 2015). The earliest reference is contained in the flora text Nanfang caomu zhuang (南方 草木狀, Plants of the Southern Regions), attributed to the Chinese botanist Ji Han (嵇含, 263-307), who describes how farmers in southern China would by weaver ant nests in local markets and place them on their citrus tree, and how this was believed to be the only way to protect the fruits from the attacks of insects (Huang & Yang 1987; Van Mele 2008; Offenberg 2015). The use of weaver ants is a traditional farming practice, and the Chinese testimony represents the earliest known case of biological control. It has been the subject of extensive research efforts and is still exercised in several regions of Asia and some parts of Africa (Van Mele 2008; Lach et al. 2010; Offenberg 2015). However, while weaver ants possess some unique characteristics, they share with most other ant species across the world several key traits that are favorable for biological control (Risch & Carrol 1982; Way & Khoo 1992; Philpott & Foster 2005; Benckiser 2010; Choate & Drummond 2011; Offenberg 2015): i) being polyphagous generalist predators in most cases, and often territorially aggressive, they can prey upon a wide range of pests while still displacing others; ii) their permanent colonies can withstand periods of food shortage by building food stocks, and at the same time respond to pest outbreaks with mass recruitment capabilities; iii) their activity can be manipulated by 8 insects Article New Tools for Conservation Biological Control: Testing Ant-Attracting Artificial Nectaries to Employ Ants as Plant Defenders Enrico Schifani 1,*,†, Cristina Castracani 1,*,†, Daniele Giannetti 1, Fiorenza Augusta Spotti 1, Roberto Reggiani 2, Stefano Leonardi 1, Alessandra Mori 1and Donato Antonio Grasso 1 1Department of Chemistry, Life Sciences & Environmental Sustainability, University of Parma, Parco Area delle Scienze, 11/a, 43124 Parma, Italy; [email protected] (D.G.); [email protected] (F.A.S.); [email protected] (S.L.); [email protected] (A.M.); [email protected] (D.A.G.) 2Azienda Agraria Sperimentale Stuard, Strada Madonna dell’Aiuto, 7/a, 43126 San Pancrazio, Parma, Italy; [email protected] *Correspondence: [email protected] (E.S.); [email protected] (C.C.) †These authors contributed equally. Received: 8 November 2019; Accepted: 13 February 2020; Published: 17 February 2020   Abstract: Knowledge of the role of ants in many agroecosystems is relatively scarce, and in temperate regions the possibility to exploit ants as biocontrol agents for crop protection is still largely unexplored. Drawing inspiration from mutualistic ant–plant relationships mediated by extrafloral nectaries (EFNs), we tested the use of artificial nectaries (ANs) in order to increase ant activity on pear trees and to evaluate the effects on the arthropods, plant health and fruit production. While EFNs secrete a complex solution mainly composed of sugars and amino acids, ANs were filled with water and sucrose only. The results suggest that ANs can be used as manipulative instruments to increase ant activity over long periods of time. High ant activity was significantly linked to lower incidence of the pathogen fungus Venturia pyrina (pear scab) on pear leaves, and of the presence of Cydia pomonella (codling moth) caterpillars on pear fruit production. These results further encourage exploring underrated possibilities in the development of new tools for conservation biological control (CBC). Keywords: Integrated Pest Management (IPM); Conservation Biological Control (CBC); ant-plant relationships; multitrophic interactions; applied myrmecology; agroecology; mutualism; indirect defense; Pyrus orchard; plant health; pear trees 1. Introduction Plants have evolved very complex relationships with ants, and have been fundamental to the rise of many modern lineages of these insects [ 1 – 4 ]. Some of these relationships are strictly antagonistic, revolving around herbivory in a few New World species and more commonly around seed predation [ 5 ]. Many of the other relationships are beneficial to plants, and encompass several different aspects from seed dispersal and soil processing to rare cases of pollination [ 6 , 7 ], while the vast majority are based on an ant’s appetite for sugary liquids [ 1 ], which is produced in the form of honeydew by sap-feeding insects (mainly heteropterans) or directly by plants in the form of nectars. Ants may protect sap-feeding insects in exchange for honeydew [ 8 – 11 ]. Although ants may indirectly damage plants through this relationship, its cost–benefit ratio may still be beneficial overall for the plant if ant presence displaces more damaging herbivores [ 12 ]. Some plants produce nectars, other food rewards or offer shelter to ants in exchange for protection against herbivory, competing plants or even pathogens [ 13 – 17 ]. These Insects 2020,11, 129; doi:10.3390/insects11020129 www.mdpi.com/journal/insects 15 Insects 2020,11, 129 2 of 21 rewards may also be aimed at distracting ants from tending sap feeders [ 18 – 21 ]. Several cases of these ant-plant mutualisms are mediated by extrafloral nectaries (EFNs), which have been described for 4017 plant species of over 450 evolutionary lineages (21% of vascular plant families), and are expected to be found in about 8000 species [ 22 , 23 ]. EFNs are both a food reward for ants and manipulative tools to maximize plant advantages in the ant–plant relationship [ 24 – 26 ]. They produce complex solutions in which sugars and amino acids are the main components, while less abundant constituents such as secondary metabolites may also play an important role [ 25 , 26 ]. Moreover, in some plants EFNs can be an induced defense in response to herbivory [ 27 ]. However, plant defense by ants against herbivores can also be mediated by other adaptations, such as certain volatiles [ 28 – 32 ]. Although the latter mechanism has only been documented in a few cases, it is easy to speculate that ants may act as plant defenders by attacking herbivores much more often than currently reported. An urgent need to develop more-sustainable agriculture is widely recognized, and Integrated Pest Management (IPM) should reduce the use of pesticides by favoring biological control techniques [ 33 – 35 ]. The use of ants as biocontrol agents is overlooked in comparison to other insect groups, and their use has been limited mainly to equatorial and tropical regions. Nonetheless, the oldest case of biological control mediated by insects (304 A.D.) attests to the use of the ant genus Oecophylla Smith, F., 1860 as a biocontrol agent [ 36 ]. Today, Oecophylla spp. are still used in more than 20 countries of Africa, Asia and Oceania to control about 50 different pest species [ 37 , 38 ]. Although they are considered bioindicators of soil function and habitat quality in rural environments [ 39 , 40 ], both their distribution and their role in most agroecosystems are still insufficiently documented. However, ants have several characteristics that make them good candidates for biocontrol agents [ 5 , 38 , 41 – 46 ]. First, they may be generalist predators, whose polymorphism often enhances their polyphagy. Second, many ants are often territorial and aggressive, thus chasing away intruders unsuitable as prey. In addition, recruitment allows a quick reaction to increases in prey. Moreover, colonies can withstand periods of food shortage and without becoming particularly susceptible to satiety because they build food stocks and rear immature stages. Finally, ant activity in agroecosystems can be easily manipulated, for example, by transferring colonies or offering additional food sources and nest sites (e.g., [ 47 , 48 ]). In addition, the manipulation of ant foraging pathways may redirect foragers towards target pests, and can be conducted using plants with EFNs [49]. Ant–sap feeder interactions are usually the main cause of concern about the role of ants in some agroecosystems. For example, in vineyards and citrus orchards, a number of studies have shown that ant presence increases populations of mutualistic aphids and coccids and decreases those of some of their natural enemies (e.g., [ 50 – 56 ]). As a result, additional research has focused on the control of ant populations in these agroecosystems, either by employing chemical substances (e.g., [ 57 – 64 ]) or distracting tending ants by providing sugary substances (e.g., [ 21 ]). The latter method may allow the positive effects of ant presence (see [ 12 ]). Nevertheless, other studies have found that the presence of ants in citrus and vine groves was weakly related or not related at all to the presence of sap feeders [ 21 , 65 ]. In addition, some natural predators of aphids and coccids may even increase under ant presence in vineyards [ 51 ]. Moreover, not all economically important sap-feeding pests are mutualists, and many ant species are not mutualists either. Even in citrus and vine groves where the phenomenon has attained particular attention, only a small proportion of ant species has been linked to pest outbreaks. In fact, of the 123 ant species inhabiting South Africa citrus orchards, only about 25 tend aphids and only three or four are considered responsible for pest outbreaks [66,67]. In Europe, the use of ants in biological control has mostly developed around wood ants (Formica rufa group). These have usually been employed in forest ecosystems [ 68 , 69 ], and much more rarely in agroecosystems, to which they had to be transported [ 48 , 70 ]. Only a few studies have dealt with ants in Italian agroecosystems (e.g., [ 71 – 77 ]). However, native ant species common in Italy and in the Mediterranean basin could be effective control agents against highly problematic pests such as the exotic brown marmorated stink bug Halyomorpha halys (Stål, 1855) [ 78 ], fungi and other herbivores [ 79 ]. In some cases, common harvester ants may have a positive role in the control of weeds [ 80 ]. Many 16 Insects 2020,11, 129 3 of 21 of the above-mentioned ant characteristics considered promising for biological control were also documented for Italian ants (e.g., [7,68,74,75,81–84]). Our paper reports on the role of ants and their use in conservation biological control (CBC) [ 85 , 86 ], and our experiment was carried out in a pear agroecosystem. Pear trees have been cultivated for at least 3000 years, and currently continue to be important for fruit production in wide regions of the planet, from Eurasia to North America [ 87 ]. Moreover, in Italy, a remarkable diversity of wild and cultivated forms has been documented [ 88 ], none of which possess EFNs or other structures aimed at attracting ants. Finally, pear orchards host a rich community of pest insects, whose control costs about USD 14 million each year worldwide, and does not usually employ ants [ 89 ]. However, ant presence in pear agroecosystems is not commonly associated with pest outbreaks. Conversely, Crematogaster subdentata Mayr, 1877 was reported to suppress a population of the San Jose scale Quadraspidiotus perniciosus Comstock, 1881 [ 90 ]. Moreover, Formica neoclara Emery, 1893 was considered to be a promising biocontrol agent of the pear psylla, Cacopsylla pyricola (Foerster, 1848) [ 91 – 93 ]. Although other authors suggest some ants may instead favor the latter [ 94 , 95 ], no clear evidence was provided. Finally, there is other evidence suggesting that Cacopsylla spp. primary parasitoids are even favored when ants are present, because they are more effective at keeping away the hyperparasitoids [96]. We introduced artificial nectaries (ANs) to study the impact of ants in a pear agroecosystem. Inspired by EFNs, ANs are manipulative tools designed to increase ant activity on trees by dispensing a liquid made of water and sugar to attract ants. Although a few studies have already tested the use of ANs or other food sources to attain agroecological benefits (e.g., [ 20 , 21 , 97 – 100 ]), none of them have studied ant effectiveness in defending plants, focusing instead on distracting ants from mutualistic sap feeders. Thus, the aim of our study was to evaluate both the functionality of ANs and the impact of different levels of ant activity on arboreal arthropodofauna, plant health and fruit production. 2. Materials and Methods 2.1. Study Area Sampling was conducted in a 1-hectare organic orchard located near Pontescodogna, Parma, Italy (44.7378, 10.1954) and part of the Regional Natural Park “Boschi di Carrega”. The orchard consisted of 430 fruit trees (mainly pear, but also apple, apricot, cherry, fig, peach and plum trees), arranged in 15 rows and partially surrounded by a deciduous broadleaved forest. The orchard management was limited to periodical lawn mowing. 2.2. Artificial Nectaries (ANs) The artificial nectary used in the experiment was made of a 1l plastic bottles (nectary tank) connected to an infusion set (Figure 1). Two holes were drilled in the bottle: one on its basal surface, later used to add or refill the liquid, and one on its cap, to connect the infusion set consisting of a flow controller and a dispenser releasing the liquid at the AN distal end. The AN created a direct slow, steady and adjustable flux of artificial nectar (set at 15–20 drops per minute) to a focus area on the plant (Nectar-releasing points). The content of the ANs consisted of a liquid solution made of 10g of sucrose in 1l of water and was refilled whenever needed (usually once a week) in order to provide an uninterrupted operation. 17 Insects 2020,11, 129 4 of 21 Insects 2020, 11, 129 4 of 21 Figure 1. Artificial nectary (AN) installed on a pear tree. In pictures (a) and (b), the main components of the artificial nectary are shown: the tank (1) and the infusion set (2). The infusion set is made of the flow controller (2a) and the dispenser (2b). In (b), two Lasius niger workers are seen drinking at the nectar released by the dispenser. 2.3. Treatments A total of 20 adult pear trees, homogenous in size (tree height: 2.5–3 m; trunk Ø: 30–40 cm), were selected for the experiment from two pear rows. They were chosen from among those plants standing far enough away from neighboring trees in order to avoid any contact and eventual passage of ants between them. Four treatment groups, each made up of five randomly selected trees, were then created. On each tree, two of the main branches departing from the trunk were selected as focal branches and used for data collection. Treatment groups differed according to the presence/absence of two different manipulations: ANs and ant-exclusion. Six ANs were placed in each tree in order to create six different release points: three points each per focal branch, one proximally to the trunk, one in the middle of the branch and the last distally to the trunk (Figure 2). Ant-exclusion consisted of the placement of sticky barriers at the base of the trunk in order to prevent soil-nesting ant access to the tree. Sticky barriers are a common system used for ant exclusion in field experiments [20,101,102]. Given the almost complete absence of arboreal-nesting species in the orchard, sticky barriers were expected to be effective in eliminating ants from targeted trees. As a result, the following treatments were created: • Treatment 1 (ANs+/Ants+): ANs were installed and ants were free to climb the tree • Treatment 2 (ANs−/Ants+): no ANs were installed and ants were free to climb the tree (trees with no manipulations) • Treatment 3 (ANs+/Ants−): ANs were installed and ants were not free to climb the tree since sticky barriers were installed • Treatment 4: (ANs−/Ants−): no ANs were installed and ants were not free to climb the tree since sticky barriers were installed Figure 1. Artificial nectary (AN) installed on a pear tree. In pictures ( a ) and ( b ), the main components of the artificial nectary are shown: the tank (1) and the infusion set (2). The infusion set is made of the flow controller (2a) and the dispenser (2b). In ( b ), two Lasius niger workers are seen drinking at the nectar released by the dispenser. 2.3. Treatments A total of 20 adult pear trees, homogenous in size (tree height: 2.5–3 m; trunk Ø: 30–40 cm), were selected for the experiment from two pear rows. They were chosen from among those plants standing far enough away from neighboring trees in order to avoid any contact and eventual passage of ants between them. Four treatment groups, each made up of five randomly selected trees, were then created. On each tree, two of the main branches departing from the trunk were selected as focal branches and used for data collection. Treatment groups differed according to the presence/absence of two different manipulations: ANs and ant-exclusion. Six ANs were placed in each tree in order to create six different release points: three points each per focal branch, one proximally to the trunk, one in the middle of the branch and the last distally to the trunk (Figure 2). Ant-exclusion consisted of the placement of sticky barriers at the base of the trunk in order to prevent soil-nesting ant access to the tree. Sticky barriers are a common system used for ant exclusion in field experiments [ 20 , 101 , 102 ]. Given the almost complete absence of arboreal-nesting species in the orchard, sticky barriers were expected to be effective in eliminating ants from targeted trees. As a result, the following treatments were created: •Treatment 1 (ANs+/Ants+): ANs were installed and ants were free to climb the tree • Treatment 2 (ANs − /Ants+): no ANs were installed and ants were free to climb the tree (trees with no manipulations) • Treatment 3 (ANs+/Ants − ): ANs were installed and ants were not free to climb the tree since sticky barriers were installed • Treatment 4: (ANs − /Ants − ): no ANs were installed and ants were not free to climb the tree since sticky barriers were installed 18 Insects 2020,11, 129 5 of 21 Insects 2020, 11, 129 5 of 21 Figure 2. Positioning of the experimental apparatus on the trunk and the two focal branches of the tree. Ant-flux counting spots were present in all the trees (n = 20), whereas nectar-releasing points and sticky barriers were present only according to treatments: ANs+ for nectar-releasing points and Ants− for barriers. 2.4. Data Collection Data were typically collected from 09:00 to 12:00 once a week during a two-month period from the beginning of July to the end of August 2018 (nine weeks). Data recording started one week after the beginning of manipulations. 2.4.1. Ant Activity Data were collected with the aim of monitoring both abundance and diversity of ants active on the trees according to the four treatments. In order to avoid specimen collection during data sampling, a checklist of ant species in the orchard was compiled before the beginning of the experiment (see Table S1). In June 2018, ants were collected by direct sampling and identified under a ZEISS Stemi 508 stereoscopic microscope. Measurements were taken with the aid of an Axiocam Erc 5 s mounted on the microscope and ZEISS Zen core Software. Taxonomic identifications were mainly made following Radchenko and Elmes [103] and Seifert [104]. For each tree, five ant-flux counting spots were selected to record ant activity: on the trunk (50 cm above the ground) (1), on each focal branch between the first and second (2, 3) and between the second and the third nectar-releasing points (4, 5). At each spot, the species and number of ants were recorded during one-minute samplings (ant flux: N ants/min). Ants were counted if crossing (both directions) an imaginary circumference of the trunk/branch at each spot (Figure 2). 2.4.2. Arthropod Abundance In order to evaluate the effect of the presence of ants and ANs on the arthropod fauna, the number of all non-ant arthropods (herein simply “arthropods”) was recorded. When possible, individuals were identified at species rank, and, if necessary, a few individuals were collected from adjacent trees (not involved in the samplings) in order to minimize any possible influence on the experiment. Taxonomic aids to identification were Chinery, Leraut and the website Araneae— Spiders of Europe [105–107]. Arthropods were counted and identified on both focal branches of each tree through direct observation, by inspecting each branch from side to side. We decided to concentrate on focal branches since the maximum effect of ANs was expected at this location. In addition, in order to produce a checklist of the arthropods of the area, data from focal branches were integrated with other direct observations on arthropod presence recorded by chance while working on the experiment. Figure 2. Positioning of the experimental apparatus on the trunk and the two focal branches of the tree. Ant-flux counting spots were present in all the trees (n=20), whereas nectar-releasing points and sticky barriers were present only according to treatments: ANs+for nectar-releasing points and Ants − for barriers. 2.4. Data Collection Data were typically collected from 09:00 to 12:00 once a week during a two-month period from the beginning of July to the end of August 2018 (nine weeks). Data recording started one week after the beginning of manipulations. 2.4.1. Ant Activity Data were collected with the aim of monitoring both abundance and diversity of ants active on the trees according to the four treatments. In order to avoid specimen collection during data sampling, a checklist of ant species in the orchard was compiled before the beginning of the experiment (see Table S1). In June 2018, ants were collected by direct sampling and identified under a ZEISS Stemi 508 stereoscopic microscope. Measurements were taken with the aid of an Axiocam Erc 5 s mounted on the microscope and ZEISS Zen core Software. Taxonomic identifications were mainly made following Radchenko and Elmes [103] and Seifert [104]. For each tree, five ant-flux counting spots were selected to record ant activity: on the trunk (50 cm above the ground) (1), on each focal branch between the first and second (2, 3) and between the second and the third nectar-releasing points (4, 5). At each spot, the species and number of ants were recorded during one-minute samplings (ant flux: N ants/min). Ants were counted if crossing (both directions) an imaginary circumference of the trunk/branch at each spot (Figure 2). 2.4.2. Arthropod Abundance In order to evaluate the effect of the presence of ants and ANs on the arthropod fauna, the number of all non-ant arthropods (herein simply “arthropods”) was recorded. When possible, individuals were identified at species rank, and, if necessary, a few individuals were collected from adjacent trees (not involved in the samplings) in order to minimize any possible influence on the experiment. Taxonomic aids to identification were Chinery, Leraut and the website Araneae—Spiders of Europe [ 105 – 107 ]. Arthropods were counted and identified on both focal branches of each tree through direct observation, by inspecting each branch from side to side. We decided to concentrate on focal branches since the maximum effect of ANs was expected at this location. 19 Insects 2020,11, 129 6 of 21 In addition, in order to produce a checklist of the arthropods of the area, data from focal branches were integrated with other direct observations on arthropod presence recorded by chance while working on the experiment. 2.4.3. Leaf Damages A preliminary assessment of the most common types of damage affecting the leaves in the study area was conducted during June. These types of damage may be linked to herbivory or pathogens, which are damage sources potentially affected by ant activity (e.g., [ 48 ]). As a result, four damage categories were established and a damage scale was set for each category (also see Figure 3): • Scab (S): presence of distinct black spots on the leaf surface, attributed to the fungus Venturia pyrina Aderh. (1896). Scores: 1 =absent; 2 =low (less than half leaf surface interested); 3 = medium (half of the leaf surface interested); 4 =high (more than half leaf surface interested) • Necrosis (N): presence of extended necrotic areas on the leaf (surfaces larger than spots and with different shapes). Scores: 0 =absent; 1 =present • Holes (H): presence of holes due to missing parts of tissue far from the edges. Scores: 0 =absent; 1=present • Damaged Edge (DE): presence of altered leaf profile due to missing parts of tissue at the edges; Scores: 1 =absent; 2 =low (less than half leaf edge interested); 3 =medium (half of the leaf edge interested); 4 =high (more than half leaf edge interested) Insects 2020, 11, 129 6 of 21 2.4.3. Leaf Damages A preliminary assessment of the most common types of damage affecting the leaves in the study area was conducted during June. These types of damage may be linked to herbivory or pathogens, which are damage sources potentially affected by ant activity (e.g., [48]). As a result, four damage categories were established and a damage scale was set for each category (also see Figure 3): • Scab (S): presence of distinct black spots on the leaf surface, attributed to the fungus Venturia pyrina Aderh. (1896). Scores: 1 = absent; 2 = low (less than half leaf surface interested); 3 = medium (half of the leaf surface interested); 4 = high (more than half leaf surface interested) • Necrosis (N): presence of extended necrotic areas on the leaf (surfaces larger than spots and with different shapes). Scores: 0 = absent; 1 = present • Holes (H): presence of holes due to missing parts of tissue far from the edges. Scores: 0 = absent; 1 = present • Damaged Edge (DE): presence of altered leaf profile due to missing parts of tissue at the edges; Scores: 1 = absent; 2 = low (less than half leaf edge interested); 3 = medium (half of the leaf edge interested); 4 = high (more than half leaf edge interested) The damage level of the leaf was always inferred by visual inspection. Prior to the beginning of the sampling, 15–25 leaves per focal branch were selected and monitored for the duration of the experiment recording for each sampling, damage category and score. Figure 3. Pear leaves showing the main characteristics of the four damage categories used to evaluate their condition: Scab—S (a), Necrosis—N (b), Holes—H (c), Damaged Edge—DE (d). Leaf in picture (b) also shows signs of Holes and Damaged Edge. 2.4.4. Fruit Damages In order to describe fruit damage, four hypothetical damage categories were established: 1. Damage caused by sucking or chewing phytophagous insects (e.g., see [108] for H. halys) 2. Holes produced by codling moth Cydia pomonella (Linnaeus, 1758) caterpillars (Lepidoptera, Tortricidae) 3. Damage caused by fungi (e.g., V. pyrina) 4. Other damage Immediately after being harvested, all fruits of each tree were counted and their statuses labeled as damaged or not damaged for all categories. 2.5. Statistical Analyses If not specified, data were analyzed using IBM SPSS Statistics software (Italian version 25). Analyses and results are presented according to the nomenclature reported in [109]. Figure 3. Pear leaves showing the main characteristics of the four damage categories used to evaluate their condition: Scab—S ( a ), Necrosis—N ( b ), Holes—H ( c ), Damaged Edge—DE ( d ). Leaf in picture (b) also shows signs of Holes and Damaged Edge. The damage level of the leaf was always inferred by visual inspection. Prior to the beginning of the sampling, 15–25 leaves per focal branch were selected and monitored for the duration of the experiment recording for each sampling, damage category and score. 2.4.4. Fruit Damages In order to describe fruit damage, four hypothetical damage categories were established: 1. Damage caused by sucking or chewing phytophagous insects (e.g., see [108] for H. halys) 2. Holes produced by codling moth Cydia pomonella (Linnaeus, 1758) caterpillars (Lepidoptera, Tortricidae) 3. Damage caused by fungi (e.g., V. pyrina) 4. Other damage 20 Insects 2020,11, 129 7 of 21 Immediately after being harvested, all fruits of each tree were counted and their statuses labeled as damaged or not damaged for all categories. 2.5. Statistical Analyses If not specified, data were analyzed using IBM SPSS Statistics software (Italian version 25). Analyses and results are presented according to the nomenclature reported in [109]. In order to test the effect of treatments and time on ant activity, a mean ant flux (n=5) was calculated for each tree and week and was used as the dependent variable in a repeated measures ANOVA (n=180: 5 trees × 4 treatments × 9 weeks). Weeks 1–9 were considered a within-subjects factor (repeated measures design) and treatments (ANs+/Ants+; ANs−/Ants+; ANs−/Ants−; ANs+/Ants−) were used as a between-subjects factor (independent design). For the repeated measures, if assumption for sphericity was violated (Mauchly’s test), Greenhouse-Geisser correction was used. A repeated contrasts procedure was used to compare weeks and Tukey post hoc tests were used for treatments. To test the effect of treatments on arthropods, and because of a data distribution clearly deviating from normality, a non-parametric approach was chosen. For each recorded taxon, a Kruskal-Wallis test on abundances was run (n=360: 2 branches × 5 trees × 4 treatments × 9 weeks). All possible pairwise comparisons (n=6) with adjusted p-values (Bonferroni) were run only in cases of statistically significant results. Taxa with a total number of less than 10 specimens were excluded from the analysis. Data on leaf damage were treated as follows: for each leaf damage category (S, N, DE and H) a mean score (n=15–25) was calculated for each branch and week and used as a dependent variable in a repeated measures ANOVA (n=360: 2 branches × 5 trees × 4 treatments × 9 weeks). Weeks 1–9 were considered as a within-subjects factor (repeated measures) and treatments (ANs+/Ants+; ANs − /Ants+; ANs − /Ants − ; ANs+/Ants − ) were used as a between-subjects factor (independent design). For the repeated measures, if the assumption for sphericity was violated (Mauchly’s test), Greenhouse-Geisser correction was used. A repeated contrasts procedure was used to compare weeks and Tukey post hoc tests were used for treatments. To test the effect of the treatments on fruit damage, two separate analyses were run: one on data from focal branches, and the other on data from the rest of the whole tree. In the case of focal branches, a generalized linear model (GLM) with binomial error structure (logistic regression) was run on the status of the fruit (damaged vs not damaged) (n=271 fruits with an average of 13.55 fruits per tree ± 1.92 SE). Treatments (ANs+/Ants+; ANs − /Ants+; ANs − /Ants − ; ANs+/Ants − ) were considered as a fixed factor. For these analyses, the glm() function of the R statistical package was used [ 110 ]. The analysis was repeated to include a random factor for Tree-id using the glmer() function of the lme4 R package. The same logistic regression model was used for the analysis of fruit damage on the rest of the whole tree (n=7699 fruits with an average of 384.95 fruits per tree ± 81.97 SE). We found no evidence of over-dispersion in either analysis. 3. Results 3.1. Ant Activity A total of 19 ant species were recorded during the preliminary assessment (Table S1). Among these species, 10 were observed on the experimental trees (Table 1). As concerns the total number of trees where ants were allowed to climb (n=10), Formica cunicularia, Camponotus piceus and Plagiolepis pygmaea were respectively recorded in 100%, 90% and 70% of the trees. As concerns the ant flux observations (n=450, trunk +branches), the most frequently observed species were Lasius paralienus,Lasius niger and F. cunicularia, which were observed in 22.44%, 16.44% and 12.89% of records, respectively. The most widespread species were F. cunicularia, C. piceus and P. pygmaea, which were recorded in 100%, 90% and 70% of the trees where ants were allowed to climb (n=10), respectively. The most abundant species were L. paralienus,P. pygmaea and L. niger, with 553, 368 and 335 individuals were counted 21 Insects 2020,11, 129 8 of 21 ( n=450 ), respectively. Only L. paralienus and P. pygmaea were sporadically able to climb trees despite the sticky barriers (Figure 4). Data on the “ant flux” revealed a significant main effect of weeks (F (3.77–60.28) =4.24, p=0.005). Repeated contrasts revealed a general decrease from week 1 to week 9. There was also a significant main effect of treatments (F (3–16) =11.83, p<0.001). Tukey post hoc tests revealed a decreasing gradient of ant flux from ANs+/Ants+to ANs+/Ants−treatments. Results according to treatments and weeks are presented in Figure 4, where differences among treatments are shown, including a decreasing trend from week 1 to week 9 that was mainly evident for the treatments with no ant exclusion (ANs+/Ants+; ANs−/Ants+). Table 1. List of the ant species most frequently observed on the trees during the experiment. For each species, the percentages of visited trees and of presence records are provided for Ants+treatments. Taxon Subfamily, Tribe Trees (n=10) Records (n=450) Counted Individuals (n=450) Camponotus piceus (Leach, 1825) Formicinae, Camponotini 90% 11.3% 79 Camponotus vagus (Scopoli, 1763) Formicinae, Camponotini 30% 0.9% 4 Dolichoderus quadripunctatus (Linnaeus, 1771) Dolichoderinae, Dolichoderini 10% 6.4% 113 Formica cunicularia Latreille, 1798 Formicinae, Formicini 100% 12.9% 105 Lasius niger (Linnaeus, 1758) Formicinae, Lasiini 40% 16.4% 335 Lasius paralienus Seifert, 1992 Formicinae, Lasiini 60% 22.4% 553 Myrmica sabuleti Meinert, 1861 Myrmicinae, Myrmicini 50% 2.0% 21 Plagiolepis pygmaea (Latreille, 1798) Formicinae, Plagiolepidini 70% 9.1% 368 Tapinoma subboreale Seifert, 2012 Dolichoderinae, Tapinomini 10% 0.2% 1 Temnothorax italicus (Consani, 1952) Myrmicinae, Crematogastrini 10% 0.2% 1 Insects 2020, 11, 129 8 of 21 Results according to treatments and weeks are presented in Figure 4, where differences among treatments are shown, including a decreasing trend from week 1 to week 9 that was mainly evident for the treatments with no ant exclusion (ANs+/Ants+; ANs−/Ants+). Table 1. List of the ant species most frequently observed on the trees during the experiment. For each species, the percentages of visited trees and of presence records are provided for Ants+ treatments. Taxon Subfamily, Tribe Trees (n = 10) Records (n = 450) Counted Individuals (n = 450) Camponotus piceus (Leach, 1825) Formicinae, Camponotini 90% 11.3% 79 Camponotus vagus (Scopoli, 1763) Formicinae, Camponotini 30% 0.9% 4 Dolichoderus quadripunctatus (Linnaeus, 1771) Dolichoderinae, Dolichoderini 10% 6.4% 113 Formica cunicularia Latreille, 1798 Formicinae, Formicini 100% 12.9% 105 Lasius niger (Linnaeus, 1758) Formicinae, Lasiini 40% 16.4% 335 Lasius paralienus Seifert, 1992 Formicinae, Lasiini 60% 22.4% 553 Myrmica sabuleti Meinert, 1861 Myrmicinae, Myrmicini 50% 2.0% 21 Plagiolepis pygmaea (Latreille, 1798) Formicinae, Plagiolepidini 70% 9.1% 368 Tapinoma subboreale Seifert, 2012 Dolichoderinae, Tapinomini 10% 0.2% 1 Temnothorax italicus (Consani, 1952) Myrmicinae, Crematogastrini 10% 0.2% 1 Figure 4. Effects of treatments and weeks on ant flux. Bars represent the mean number of ants crossing (both directions) an imaginary circumference of the trunk/branch at each spot during a single minute. For each treatment, bars correspond to the nine weeks of the experiment (left to right). For each bar, the SE interval is provided. Bars are lumped according to treatment and treatments with the same letter are not statistically different (mixed-design ANOVA and Tukey post hoc tests were used on treatments, see text for further details). 3.2. Arthropods Abundance Arthropods found on the trees during the whole experiment were classified as belonging to 70 taxa: 23 species, 16 genera, 21 subfamilies/families and 10 orders (Table S2). The analysis of arthropod abundance on focal branches revealed the presence of spiders (Arachnida, Aranea) and seven orders of insects: Coleoptera, Dermaptera, Hemiptera, Hymenoptera, Lepidoptera and Neuroptera (Table 2). The most abundant species were Hyphantria cunea (Drury, 1773) (Lepidoptera, Erebidae) and Stephanitis pyri (Fabricius, 1775) (Hemiptera, Tingidae), but they were only recorded in high numbers during two single events and on trees where ants were not allowed to climb. Over 330 caterpillars of the fall webworm H. cunea eliminated almost all the leaves of one focal branch, while about 100 individuals of the pear lace bug S. pyri were counted on another. Figure 4. Effects of treatments and weeks on ant flux. Bars represent the mean number of ants crossing (both directions) an imaginary circumference of the trunk/branch at each spot during a single minute. For each treatment, bars correspond to the nine weeks of the experiment (left to right). For each bar, the SE interval is provided. Bars are lumped according to treatment and treatments with the same letter are not statistically different (mixed-design ANOVA and Tukey post hoc tests were used on treatments, see text for further details). 3.2. Arthropods Abundance Arthropods found on the trees during the whole experiment were classified as belonging to 70 taxa: 23 species, 16 genera, 21 subfamilies/families and 10 orders (Table S2). The analysis of arthropod abundance on focal branches revealed the presence of spiders (Arachnida, Aranea) and seven orders of insects: Coleoptera, Dermaptera, Hemiptera, Hymenoptera, Lepidoptera and Neuroptera (Table 2). The most abundant species were Hyphantria cunea (Drury, 1773) (Lepidoptera, Erebidae) and Stephanitis 22 Insects 2020,11, 129 9 of 21 pyri (Fabricius, 1775) (Hemiptera, Tingidae), but they were only recorded in high numbers during two single events and on trees where ants were not allowed to climb. Over 330 caterpillars of the fall webworm H. cunea eliminated almost all the leaves of one focal branch, while about 100 individuals of the pear lace bug S. pyri were counted on another. Table 2. List of the arthropods scanned weekly on the two focal branches from the beginning of July until the end of August 2018 (nine weeks). “Total” represents the total number of individuals recorded during the whole sampling period. In p, statistically significant results are highlighted in bold. In “Treatment”, treatments with the same letter are not statistically different. In “Mean” and “SE”, results refer to n=90. Class Order Family/Species Total H(3) pTreatment Mean SE Arachnida Araneae 29 6.268 0.099 ANs+/Ants+0.04 0.02 ANs−/Ants+0.03 0.02 ANs+/Ants−0.14 0.05 ANs−/Ants−0.10 0.03 Insecta Coleoptera 33 1.759 0.624 ANs+/Ants+0.07 0.03 ANs−/Ants+0.09 0.07 ANs+/Ants−0.13 0.05 ANs−/Ants−0.08 0.03 Dermaptera 1 - - - - - Diptera 35 0.727 0.867 ANs+/Ants+0.07 0.03 ANs−/Ants+0.13 0.05 ANs+/Ants−0.09 0.03 ANs−/Ants−0.10 0.04 Hemiptera M. pruinosa 65 3.402 0.334 ANs+/Ants+0.03 0.02 ANs−/Ants+0.19 0.08 ANs+/Ants−0.17 0.06 ANs−/Ants−0.33 0.22 S. pyri 192 9.094 0.028 ANs+/Ants+0.00 0.00 ANs−/Ants+0.40 0.20 ANs+/Ants−1.73 1.19 ANs−/Ants−0.00 0.00 others 32 2.348 0.503 ANs+/Ants+0.08 0.03 ANs−/Ants+0.08 0.03 ANs+/Ants−0.03 0.01 ANs−/Ants−0.08 0.03 Hymenoptera Vespula sp. 37 37.749 <0.001 (A)ANs+/Ants+0.14 0.04 (B)ANs−/Ants+0.00 0.00 (A)ANs+/Ants−0.27 0.06 (B)ANs−/Ants−0.00 0.00 others 4 - - - - - Lepidoptera H. cunea 631 9.050 0.029 ANs+/Ants+0.00 0.00 ANs−/Ants+0.00 0.00 ANs+/Ants−0.00 0.00 ANs−/Ants−7.01 4.92 others 4 - - - - - Neuroptera Chrysopidae 7 - - - - - Chrysopidae (eggs) 40 6.642 0.503 ANs+/Ants+0.02 0.02 ANs−/Ants+0.10 0.04 ANs+/Ants−0.11 0.04 As concerns the effect of treatments on arthropod abundances, a Kruskal-Wallis test was performed on the following taxa: Araneae, Chrysopidae eggs, Coleoptera, Diptera, Vespula spp. (Hymenoptera, Vespidae), Metcalfa pruinosa (Say, 1830) (Hemiptera, Flatidae), Stephanitis pyri (Fabricius, 1775) (Hemiptera, Tingidae), other Hemiptera and Hyphanthria cunea (Drury, 1773) (Lepidoptera, Erebidae). The remaining taxa were excluded from the analysis due to their low abundances (Table 2). The Kruskal-Wallis test found statistically significant differences in Vespula spp. (H (3) =37.749, p< 0.001), S. piryi (H (3) =9.094, p=0.028) and H. cunaea (H (3) =9.050, p=0.029). For Vespula spp., pairwise comparisons showed the presence of two groups that differed according to the presence of ANs, with a higher abundance in treatments where ANs were present (Table 2). For S. piryi and H. cunaea, pairwise 23 Insects 2020,11, 129 10 of 21 comparisons showed no significant differences between groups in all the possible comparisons. Finally, the Kruskal-Wallis tests found no statistically significant differences in Coleoptera (H (3) =1.759, p= 0.624), Araneae (H (3) =6.268, p=0.099), Diptera (H (3) =0.727, p=0.867), M. pruinosa (H (3) =3.402; p=0.334), other Hemiptera (H (3) =2.348, p=0.503) and Chrysopidae eggs (H (3) =6.642, p=0.084) (Table 2). 3.3. Leaf Damage As concerns Scab, there was a significant main effect of weeks on damage scores (F (3.58–121.82) = 51.61, p<0.001). Repeated contrasts revealed a general increase of scores from week 1 to week 9. There was also a significant main effect of treatments (F (3–34) =4.36, p=0.011). Tukey post hoc tests revealed that ANs+/Ants+treatment had lower scores than both treatments with ant exclusion (ANs+/Ants − and ANs − /Ants − ), which did not differ from one another. ANs − /Ants+treatment was associated with scores in-between the two previous groups (Figure 5). Insects 2020, 11, 129 10 of 21 p = 0.867), M. pruinosa (H (3) = 3.402; p = 0.334), other Hemiptera (H (3) = 2.348, p = 0.503) and Chrysopidae eggs (H (3) = 6.642, p = 0.084) (Table 2). 3.3. Leaf Damage As concerns Scab, there was a significant main effect of weeks on damage scores (F (3.58–121.82) = 51.61, p < 0.001). Repeated contrasts revealed a general increase of scores from week 1 to week 9. There was also a significant main effect of treatments (F (3–34) = 4.36, p = 0.011). Tukey post hoc tests revealed that ANs+/Ants+ treatment had lower scores than both treatments with ant exclusion (ANs+/Ants− and ANs−/Ants−), which did not differ from one another. ANs−/Ants+ treatment was associated with scores in-between the two previous groups (Figure 5). Figure 5. Effects of treatments and weeks on leaf damage for the Scab category. Points represent the mean score per week and treatment. For each point, whiskers show the SE interval. Lines with the same letter are not statistically different (mixed ANOVA and Tukey post hoc tests, see text for further details). The analysis on the Necrosis category showed that there was a significant main effect of weeks on damage scores (F (5.04–272) = 49.00, p < 0.001). Repeated contrasts revealed a general increase of scores from week 1 to week 9. There was also a significant main effect of treatments (F (3–34) = 4.39, p = 0.010). Tukey post hoc tests revealed that ANs+/Ants+ treatment had lower scores than ANs−/Ants− treatment. ANs−/Ants+ and ANs+/Ants− treatments did not differ from one another and had scores in-between the two previous groups (Figure 6). As concerns the Damaged Edge category, scores were generally very low with very few records encompassing a score of 2, meaning that damage was absent for less than half the leaf margin. There was a significant main effect of weeks on damage scores (F (5.89–200.20) = 4.48, p < 0.001). Repeated contrasts revealed no differences between week 2 and week 8, lower scores for week 1 compared to week 2 and higher scores in week 9 as compared to week 8. No significant main effect of treatments was found (F (3–34) = 0.23, p = 0.87). The analysis on the Holes category showed generally very low scores with very few records encompassing a score of 0.4, meaning that holes were usually absent. No significant main effects were found on the damage scores of weeks (F (4.18–142.19) = 1.66, p = 0.16) and treatments (F (3–34) = 1.12, p = 0.36). Figure 5. Effects of treatments and weeks on leaf damage for the Scab category. Points represent the mean score per week and treatment. For each point, whiskers show the SE interval. Lines with the same letter are not statistically different (mixed ANOVA and Tukey post hoc tests, see text for further details). The analysis on the Necrosis category showed that there was a significant main effect of weeks on damage scores (F (5.04–272) =49.00, p<0.001). Repeated contrasts revealed a general increase of scores from week 1 to week 9. There was also a significant main effect of treatments (F (3–34) =4.39, p= 0.010). Tukey post hoc tests revealed that ANs+/Ants+treatment had lower scores than ANs − /Ants − treatment. ANs−/Ants+and ANs+/Ants−treatments did not differ from one another and had scores in-between the two previous groups (Figure 6). As concerns the Damaged Edge category, scores were generally very low with very few records encompassing a score of 2, meaning that damage was absent for less than half the leaf margin. There was a significant main effect of weeks on damage scores (F (5.89–200.20) =4.48, p<0.001). Repeated contrasts revealed no differences between week 2 and week 8, lower scores for week 1 compared to week 2 and higher scores in week 9 as compared to week 8. No significant main effect of treatments was found (F(3–34) =0.23, p=0.87). The analysis on the Holes category showed generally very low scores with very few records encompassing a score of 0.4, meaning that holes were usually absent. No significant main effects were found on the damage scores of weeks (F (4.18–142.19) =1.66, p=0.16) and treatments (F (3–34) =1.12, p=0.36). 24 Insects 2020,11, 129 17 of 21 30. Kost, C.; Heil, M. The defensive role of volatile emission and extrafloral nectar secretion for lima bean in nature. J. Chem. Ecol. 2008,34, 2–13. [CrossRef] [PubMed] 31. Vittecoq, M.; Djieto-Lordon, C.; Buatois, B.; Dormont, L.; McKey, D.; Blatrix, R. The evolution of communication in two ant-plant mutualisms. Evol. Biol. 2011,38, 360–369. [CrossRef] 32. Schettino, M.; Grasso, D.A.; Weldegergis, B.T.; Castracani, C.; Mori, A.; Dicke, M.; Van Lenteren, J.C.; Van Loon, J.J. Response of a predatory ant to volatiles emitted by aphid-and caterpillar-infested cucumber and potato plants. J. Chem. Ecol. 2017,43, 1007–1022. [CrossRef] [PubMed] 33. Quimby, P.C.; King, L.R.; Grey, W.E. Biological control as a means of enhancing the sustainability of crop/land management systems. Agricult. Ecosys. Environ. 2002,88, 147–152. [CrossRef] 34. Gurr, G.M.; Wratten, S.D.; Luna, J.M. Multi-function agricultural biodiversity: Pest management and other benefits. Basic Appl. Ecol. 2003,4, 107–116. [CrossRef] 35. Bianchi, F.J.; Booij, C.J.H.; Tscharntke, T. Sustainable pest regulation in agricultural landscapes: A review on landscape composition, biodiversity and natural pest control. Proc. Biol. Sci. 2006 ,273, 1715–1727. [CrossRef] 36. Van Mele, P. A historical review of research on the weaver ant Oecophylla in biological control. Agric. Forest Entomol. 2008,10, 13–22. [CrossRef] 37. Peng, R.; Christian, K. Box 7.2 Ants as biological-control agents in the horticultural industry. In Ant Ecology; Lach, L., Lori, L., Parr, L., Catherine, L., Abbot, K., Eds.; Oxford University Press: Oxford, UK, 2010. 38. Offenberg, J. Ants as tools in sustainable agriculture. J. Appl. Ecol. 2015,52, 1197–1205. [CrossRef] 39. Peck, S.L.; Mcquaid, B.; Campbell, C.L. Using ant species (Hymenoptera: Formicidae) as a biological indicator of agroecosystem condition. Environ. Entomol. 1998,27, 1102–1110. [CrossRef] 40. De Bruyn, L.A.L. Ants as bioindicators of soil function in rural environments. Agric. Ecosyst. Environ. 1999 , 74, 425–441. [CrossRef] 41. Risch, S.J.; Carroll, C.R. The ecological role of ants in two Mexican agroecosystems. Oecologia 1982 ,55, 114–119. [CrossRef] [PubMed] 42. Way, M.J.; Khoo, K.C. Role of ants in pest management. Annu. Rev. Entomol. 1992,37, 479–503. [CrossRef] 43. Benckiser, G. Ants and sustainable agriculture. A review. Agron. Sustain. Dev. 2010 ,30, 191–199. [CrossRef] 44. Choate, B.; Drummond, F. Ants as biological control agents in agricultural cropping systems. Terr. Arthropod Rev. 2011,4, 157–180. [CrossRef] 45. Seguni, Z.S.K.; Way, M.J.; Van Mele, P. The effect of ground vegetation management on competition between the ants Oecophylla longinoda and Pheidole megacephala and implications for conservation biological control. Crop Prot. 2011,30, 713–717. [CrossRef] 46. Offenberg, J. The use of artificial nests by weaver ants: A preliminary field observation. Asian Myrmecol. 2014,6, 119–128. [CrossRef] 47. Philpott, S.M.; Foster, P.F. Nest-site limitation in coffee agroecosystems: Artificial nests maintain diversity of arboreal ants. Ecol. Appl. 2005,15, 1478–1485. [CrossRef] 48. Offenberg, J.; Nielsen, J.S.; Damgaard, C. Wood Ant (Formica polyctena) Services and Disservices in a Danish Apple Plantation. Sociobiology 2019,66, 247–256. [CrossRef] 49. Jones, I.M.; Koptur, S.; von Wettberg, E.J. The use of extrafloral nectar in pest management: Overcoming context dependence. J. Appl. Ecol. 2017,54, 489–499. [CrossRef] 50. James, D.G.; Stevens, M.; O’Malley, K.J.; Faulder, R.J. Ant foraging reduces the abundance of beneficial and incidental arthropods in citrus canopies. Biol. Control 1999,14, 121–126. [CrossRef] 51. Daane, K.M.; Sime, K.R.; Fallon, J.; Cooper, M.L. Impacts of Argentine ants on mealybugs and their natural enemies in California’s coastal vineyards. Ecol. Entomol. 2007,32, 583–596. [CrossRef] 52. Pekas, A.; Tena, A.; Aguilar, A.; Garcia-Mar í , F. Effect of Mediterranean ants (Hymenoptera: Formicidae) on California red scale (Hemiptera: Diaspididae) populations in citrus orchards. Environ. Entomol. 2010 ,39, 827–834. [CrossRef] [PubMed] 53. Pekas, A.; Tena, A.; Aguilar, A.; Garcia-Mar í , F. Spatio-temporal patterns and interactions with honeydew producing hemiptera of ants in a Mediterranean citrus orchard. Agric. Forest. Entomol. 2011 ,13, 89–97. [CrossRef] 54. Calabuig, A.; Garcia-Mar í , F.; Pekas, A. Ants affect the infestation levels but not the parasitism of honeydew and non-honeydew producing pests in citrus. Bull. Entomol. Res. 2013,104, 405–417. [CrossRef] 55. Yoo, H.J.S.; Kizner, M.C.; Holway, D.A. Ecological effects of multi-species, ant hemipteran mutualisms in citrus. Ecol. Entomol. 2013,38, 505–514. [CrossRef] 31 Insects 2020,11, 129 18 of 21 56. Dao, H.T.; Meats, A.; Beattie, G.A.C.; Spooner-Hart, R. Ant-coccid mutualism in citrus canopies and its effect on natural enemies of red scale, Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae). Bull. Entomol. Res. 2014,104, 137–142. [CrossRef] 57. Klotz, J.H.; Rust, M.K.; Gonzalez, D.; Greenberg, L.; Costa, H.; Phillips, P.; Gispert, C.; Reierson, D.A.; Kido, K. Directed Sprays and Liquid Baits to Manage Ants in Vineyards and Citrus Groves. J. Agric. Urban Entomol. 2003,20, 31–40. 58. Tollerup, K.E.; Rust, M.K.; Dorschner, K.; Phillips, P.; Klotz, J.H. Low-toxicity baits control ants in citrus orchards and grape vineyards. Calif. Agric. 2004,58, 213–217. [CrossRef] 59. Tollerup, K.; Rust, M.K.; Klotz, J.H. Formica perpilosa, an Emerging Pest in Vineyards. J. Agric. Urban Entomol. 2007,24, 147–158. [CrossRef] 60. Greenberg, L.; Tollerup, K.E.; Rust, M.K. Control of Argentine Ants (Hymenoptera: Formicidae) in Citrus using Methoprene and Imidacloprid Delivered in Liquid Bait Stations. Fla. Entomol. 2013 ,96, 1023–1029. [CrossRef] 61. Daane, K.M.; Sime, K.R.; Hogg, B.N.; Bianchi, M.L.; Cooper, M.L.; Rust, M.K.; Klotz, J.H. Effects of liquid insecticide baits on Argentine ants in California’s coastal vineyards. Crop Prot. 2006 ,25, 592–603. [CrossRef] 62. Nondillo, A.; Andzeiewski, S.; Bello Fialho, F.; Bueno, O.C.; Botton, M. Control of Linepithema micans (Hymenoptera: Formicidae) and Eurhizococcus brasiliensis (Hemiptera: Margarodidae) in vineyards using toxic baits. J. Econ. Entomol. 2016,109, 1660–1666. [CrossRef] 63. Westermann, F.L.; Bell, V.A.; Suckling, D.M.; Lester, P.J. Synthetic pheromones as a management technique—Dispensers reduce Linepithema humile activity in a commercial vineyard. Pest. Manag. Sci. 2016 , 72, 719–724. [CrossRef] 64. Cooper, M.L.; Hobbs, M.B.; Boser, C.L.; Varela, L.G. Argentine Ant Management: Using Toxin-Laced Polyacrylamide Crystals to Target Ant Colonies in Vineyards. Catal. Discov. Pract. 2019. [CrossRef] 65. Chong, C.S.; D’Alberto, C.F.; Thomson, L.J.; Hoffmann, A.A. Influence of native ants on arthropod communities in a vineyard. Agric. Forest Entomol. 2010,12, 223–232. [CrossRef] 66. Samways, M.J.; Nel, M.; Prins, A.J. Ants (Hymenoptera: Formicidae) foraging in citrus trees and attending honeydew-producing Homoptera. Phytophylactica 1982,14, 155–157. 67. Samways, M.J.; Grout, T.J.; Prins, A.J. Ants as Citrus Pests. In Citrus Pests in the Republic of South Africa; Bedford, E.C.G., Van Den Berg, M.A., Villiers, E.A., Eds.; Institute for Tropical and Subtropical Crops: Nelspruit, South Africa, 1998. 68. Pavan, M. Attivit á Italiana per la Lotta Biologica Con Formiche del Gruppo Formica rufa Contro Gli Insetti Dannosi Alle Foreste; Ministero dell’Agricoltura e delle Foreste, Collana verde: Rome, Italy, 1959; Volume 4, pp. 1–80. 69. Adlung, K.G. A critical evaluation of the European research on use of red wood ants (Formica rufa group) for the protection of forests against harmful insects. Z. Angew. Entomol. 1966,57, 167–189. [CrossRef] 70. Nielsen, J.S.; Nielsen, M.G.; Offenberg, J. Experiences in Transplanting Wood Ants into Plantations for Integrated Pest Management. Sociobiology 2018,65, 403–414. [CrossRef] 71. Castracani, C.; Mori, A. The role of permanent grasslands on ant community structure: Ants (Hymenoptera: Formicidae) as ecological indicators in the agro-ecosystems of the Taro River Regional Park (Italy). Myrmecol. Nachr. 2006,9, 47–54. 72. La Pergola, A.; Alicata, A.; Longo, S. Survey of the ants (Hymenoptera Formicidae) in citrus orchards with different types of crop management in Sicily. IOBC/WPRS Bull. 2008,38, 233–237. 73. Ottonetti, L.; Tucci, L.; Chelazzi, G.; Santini, G. Stable isotopes analysis to assess the trophic role of ants in a Mediterranean agroecosystem. Agric. Forest Entomol. 2008,10, 29–36. [CrossRef] 74. Santini, G.; Ramsay, P.M.; Tucci, L.; Ottonetti, L.; Frizzi, F. Spatial patterns of the ant Crematogaster scutellaris in a model ecosystem. Ecol. Entomol. 2011,36, 625–634. [CrossRef] 75. Campolo, O.; Palmeri, V.; Malacrin ò , A.; Laudani, F.; Castracani, C.; Mori, A.; Grasso, D.A. Interaction between ants and the Mediterranean fruit fly: New insights for biological control. Biol. Control 2015 ,90, 120–127. [CrossRef] 76. Castracani, C.; Maienza, A.; Grasso, D.A.; Genesio, L.; Malcevschi, A.; Miglietta, F.; Vaccari, F.P.; Mori, A. Biochar–macrofauna interplay: Searching for new bioindicators. Sci. Total Environ. 2015 ,536, 449–456. [CrossRef] [PubMed] 32 Insects 2020,11, 129 19 of 21 77. Masoni, A.; Frizzi, F.; Brühl, C.; Zocchi, N.; Palchetti, E.; Chelazzi, G.; Santini, G. Management matters: A comparison of ant assemblages in organic and conventional vineyards. Agric. Ecosyst. Environ. 2017 ,246, 175–183. [CrossRef] 78. Castracani, C.; Bulgarini, G.; Giannetti, D.; Spotti, F.A.; Maistrello, L.; Mori, A.; Grasso, D.A. Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys.J. Pest Sci. 2017 ,90, 1181–1190. [CrossRef] 79. Giannetti, D.; Castracani, C.; Spotti, F.A.; Mori, A.; Grasso, D.A. Gall-colonizing ants and their role as plant defenders: From ‘bad job’ to ‘useful service’. Insects 2019,10, 392. [CrossRef] 80. Baraibar, B.; Carri ó n, E.; Recasens, J.; Westerman, P.R. Unravelling the process of weed seed predation: Developing options for betterweed control. Biol. Control 2011,56, 85–90. [CrossRef] 81. Le Moli, F.; Mori, A.; Parmigiani, S. Agonistic behaviour of Formica rufa L. (Hymenoptera Formicidae). Ital. J. Zool. 1982,16, 325–331. 82. Grasso, D.A.; Mori, A.; Le Moli, F. Chemical communication during foraging in the harvesting ant Messor capitatus (Hymenoptera, Formicidae). Insectes Soc. 1998,45, 85–96. [CrossRef] 83. Grasso, D.A.; Mori, A.; Le Moli, F. Recruitment and trail communication in two species of Messor ants (Hymenoptera, Formicidae). Ital. J. Zool. 1999,66, 373–378. [CrossRef] 84. Mori, A.; Grasso, D.A.; Le Moli, F. Raiding and foraging behavior of the blood-red ant, Formica sanguinea Latr. (Hymenoptera, Formicidae). J. Insect Behav. 2000,13, 421–430. [CrossRef] 85. Barbosa, P.A. Conservation Biological Control; Academic Press: San Diego, CA, USA, 1998. 86. Jonsson, M.; Wratten, S.D.; Landis, D.A.; Gurr, G.M. Recent advances in conservation biological control of arthropods by arthropods. Biol. Control 2008,45, 172–175. [CrossRef] 87. Bell, R.L. Pears (Pyrus). Acta Hortic. 1991,290, 657–700. [CrossRef] 88. Marino, P.; Schicchi, R.; Barone, E.; Raimondo, F.M.; Domina, G. First results on the phenotypic analysis of wild and cultivated species of Pyrus in Sicily. Fl. Medit. 2013,23, 237–243. [CrossRef] 89. Husain, M.; Rathore, J.P.; Sharma, A.; Raja, A.; Qadri, I.; Wani, A.W. Description and management strategies of important pests of pear: A review. J. Entomol. Zool. Stud. 2018,6, 677–683. 90. Folkina, M.Y. A case of mass extermination of the San Jose scale (Quadraspidiotus perniciosus) by the ant Crematogaster subdentata.Zool. Zhurnal 1978,57, 301. 91. Paulson, G.S.; Akre, D.K. Introducing Ants (Hymenoptera: Formicidae) Into Pear Orchards for the Control of Pear Psylla, Cacopsylla pyricola (Foerster) (Homoptera: Psyllidae). J. Agric. Entomol. 1992,9, 37–39. 92. Paulson, G.S.; Akre, D.K. Role of Predaceous Ants in Pear Psylla (Homoptera: Psyllidae) Management: Estimating Colony Size and Foraging Range of Formica neoclara (Hymenoptera: Formicidae) through a Mark-Recapture Technique. J. Econ. Entomol. 1991,84, 1437–1440. [CrossRef] 93. Paulson, G.S.; Akre, D.K. Evaluating the Effectiveness of Ants as Biological Control Agents of Pear Psylla (Homoptera: Psyllidae). J. Econ. Entomol. 1992,85, 70–73. [CrossRef] 94. Erler, F. Natural enemies of the pear psylla Cacopsylla pyri in treated vs. untreated pear orchards in Antalya, Turkey. Phytoparasitica 2004,32, 295–304. [CrossRef] 95. Sanchez, J.; Ort í n-Angulo, M.C. Abundance and population dynamics of Cacopsylla pyri (Hemiptera: Psyllidae) and its potential natural enemies in pear orchards in southern Spain. Crop Prot. 2007,32, 24–29. [CrossRef] 96. Novak, H. The influence of ant attendance on larval parasitism in hawthorn psyllids (Homoptera: Psyllidae). Oecologia 1994,99, 72–78. [CrossRef] 97. Offenberg, J. Balancing between mutualism and exploitation: The symbiotic interaction between Lasius ants and aphids. Behav. Ecol. Sociobiol. 2001,49, 304–310. [CrossRef] 98. Carabal í -Banguero, D.J.; Wyckhuys, K.A.G.; Montoya-Lerma, J.; Kondo, T.; Lundgren, J.G. Do additional sugar sources affect the degree of attendance of Dysmicoccus brevipes by the fire ant Solenopsis geminata? Entomol. Exp. Appl. 2013,148, 65–73. [CrossRef] 99. Wäckers, F.L.; Alberola, J.S.; Garcia-Mar í , F.; Pekas, A. Attract and distract: Manipulation of a food-mediated protective mutualism enhances natural pest control. Agric. Ecosyst. Environ. 2017 ,246, 168–174. [CrossRef] 100. Pacelhe, F.T.; Costa, F.V.; Neves, F.S.; Bronstein, J.; Mello, M.A. Nectar quality affects ant aggressiveness and biotic defense provided to plants. Biotropica 2019,51, 196–204. [CrossRef] 101. Piñol, J.; Espadaler, X.; Cañellas, N.; Mart í nez-Vilalta, J.; Barrientos, J.A.; Sol, D. Ant versus bird exclusion effects on the arthropod assemblage of an organic citrus grove. Ecol. Entomol. 2010 ,35, 367–376. [CrossRef] 33 Insects 2020,11, 129 20 of 21 102. Piñol, J.; Espadaler, X.; Cañellas, N. Eight years of ant-exclusion from citrus canopies: Effects on the arthropod assemblage and on fruit yield. Agric. Forest Entomol. 2012,14, 49–57. [CrossRef] 103. Radchenko, A.G.; Elmes, G.W. Myrmica ants of the Old World. Fauna Mundi 2010,3, 89–93. 104. Seifert, B. The Ants of Central and North Europe; Lutra Verlagsund Vertriebsgesellschaft: Tauer, Germany, 2018. 105. Chinery, M. Guide to the Insects of Britain and Western Europe; Harper-Collins: London, UK, 1986. 106. Leraut, P. Le Guide entomologique; Delachaux et Niestlé: Paris, France, 2003. 107. Araneae—Spiders of Europe. Available online: https://www.araneae.nmbe.ch (accessed on 31 January 2019). 108. Bariselli, M.; Bugiani, R.; Maistrello, L. Distribution and damage caused by Halyomorpha halys in Italy. EPPO Bull. 2016,46, 332–334. [CrossRef] 109. Field, A. Discovering Statistics Using IBM SPSS Statistics, 4th ed.; Sage Publications Ltd.: London, UK, 2018. 110. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2019; Available online: https://www.R-project.org/(accessed on 31 January 2019). 111. Arnan, X.; Andersen, A.N.; Gibb, H.; Parr, C.L.; Sanders, N.J.; Dunn, R.R.; Angulo, E.; Baccaro, F.B.; Bishop, T.R.; Boulay, R.; et al. Dominance–diversity relationships in ant communities differ with invasion. Glob. Chang. Biol. 2018,24, 4614–4625. [CrossRef] [PubMed] 112. Baroni Urbani, C. Catalogo delle specie di Formicidae d’Italia. Mem. Soc. Entom. Ital. 1971,50, 5–287. 113. Lachaud, J.P. Foraging activity and diet in some neotropical ponerine ants. I. Ectatomma ruidum Roger (Hymenoptera, Formicidae). Folia Entomol. Mex. 1990,78, 241–256. 114. Cornelius, M.L.; Grace, J.K. Influence of Brood on the Nutritional Preferences of the Tropical Ant Species, Pheidole megacephala (F.) and Ochetellus glaber (Mayr). J. Entomol. Sci. 1997,32, 421–429. [CrossRef] 115. Piñol, J.; Ribes, E.; Ribes, J.; Espadaler, X. Longterm changes and ant-exclusion effects on the true bugs (Hemiptera: Heteroptera) of an organic citrus grove. Agric. Ecosyst. Environ. 2012 ,158, 127–131. [CrossRef] 116. Del-Claro, K.; Oliveira, P.S. Ant-Homoptera interaction: Do alternative sugar sources distract tending ants? Oikos 1993,2, 202–206. [CrossRef] 117. L ó pez, R.; Potter, D.A. Ant Predation on Eggs and Larvae of the Black Cutworm (Lepidoptera: Noctuidae) and Japanese Beetle (Coleoptera: Scarabaeidae) in Turfgrass. Environ. Entomol. 2000 ,29, 116–125. [CrossRef] 118. Vandermeer, J.; Perfecto, I.; Nuñez, G.I.; Phillpott, S.; Ballinas, A.G. Ants (Azteca sp.) as potential biological control agents in shade coffee production in Chiapas, Mexico. Agrofor. Syst. 2002,56, 271–276. [CrossRef] 119. Nay, J.E.; Perring, T.M. Impact of Ant Predation and Heat on Carob Moth (Lepidoptera: Pyralidae) Mortality in California Date Gardens. J. Econ. Entomol. 2005,98, 725–731. [CrossRef] [PubMed] 120. Eskafi, F.M.; Kolbe, M.M. Predation on larval and pupal Ceratitis capitata (Diptera: Tephritidae) by the ant Solenopsis geminata (Hymenoptera: Formicidae) and other predators in Guatemala. Environ. Entom. 1990 ,19, 148–153. [CrossRef] 121. Van Mele, P.; Vayssi è res, J.F.; Van Tellingen, E.; Vrolijks, J. Effects of an African weaver ant, Oecophylla longinoda, in controlling mango fruit flies (Diptera: Tephritidae) in Benin. J. Econ. Entomol. 2007 ,100, 695–701. [CrossRef] 122. El Keroumi, A.; Naamani, K.; Dahbi, A.; Luque, I.; Carvajal, A.; Cerda, X.; Boulay, R. Effect of ant predation and abiotic factors on the mortality of medfly larvae, Ceratitis capitata, in the Argan forest of Western Morocco. Biocontrol Sci. Technol. 2010,20, 751–762. [CrossRef] 123. Van Mele, P.; Vayssieres, J.F.; Adandonon, A.; Sinzogan, A. Ant cues affect the oviposition behaviour of fruit flies (Diptera: Tephritidae) in Africa. Physiol. Entomol. 2009,34, 256–261. [CrossRef] 124. Urbaneja, A.; Mar í , F.G.; Tortosa, D.; Navarro, C.; Vanaclocha, P.; Bargues, L.; Castañera, P. Influence of ground predators on the survival of the Mediterranean fruit fly pupae, Ceratitis capitata, in Spanish citrus orchards. BioControl 2006,51, 611–626. [CrossRef] 125. Unruh, T.R.; Lacey, L.A. Control of codling moth, Cydia pomonella (Lepidoptera: Tortricidae), with Steinernema carpocapsae: Effects of supplemental wetting and pupation site on infection rate. Biol. Control 2001 ,20, 48–56. [CrossRef] 126. Mathews, C.R.; Bottrell, D.G.; Brown, M.W. Habitat manipulation of the apple orchard floor to increase ground-dwelling predators and predation of Cydia pomonella (L.) (Lepidoptera: Tortricidae). Biol. Control 2004,30, 265–273. [CrossRef] 127. Lacey, L.A.; Unruh, T.R. Biological control of codling moth (Cydia pomonella, Lepidoptera: Tortricidae) and its role in integrated pest management, with emphasis on entomopathogens. Vedalia 2005 ,12, 33–60. [CrossRef] 34 Insects 2020,11, 129 21 of 21 128. Reyes, M.; Franck, P.; Charmillot, P.J.; Ioriatti, C.; Olivares, J.; Pasqualini, E.; Sauphanor, B. Diversity of insecticide resistance mechanisms and spectrum in European populations of the codling moth, Cydia pomonella.Pest Manag. Sci. 2007,63, 890–902. [CrossRef] [PubMed] 129. Reyes, M.; Franck, P.; Olivares, J.; Margaritopoulos, J.; Knight, A.; Sauphanor, B. Worldwide variability of insecticide resistance mechanisms in the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). Bull. Entomol. Res. 2009,99, 359–369. [CrossRef] [PubMed] 130. Pickett, C.H.; Clark, W.D. The function of extrafloral nectaries in Opuntia acanthocarpa (Cactaceae). Am. J. Bot. 1979,66, 618–625. [CrossRef] 131. Passos, F.; Leal, L.C. Protein matters: Ants remove herbivores more frequently from extrafloral nectary-bearing plants when habitats are protein poor. Biol. J. Linnean Soc. 2019, blz033. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 35 Section I - Ants as biocontrol agents of insect pests Chapter 2 Predatory abilities of two Mediterranean ants on the eggs and larvae of the codling moth Cydia pomonella _______________________________________________________________________________ Schifani, E., Giannetti, D., & Grasso, D.A. (2023). Predatory abilities of two Mediterranean ants on the eggs and larvae of the codling moth Cydia pomonella. Insects, 14, 97. https://doi.org/10.3390/insects14020097 36 Citation: Schifani, E.; Giannetti, D.; Grasso, D.A. Predatory Abilities of Two Mediterranean Ants on the Eggs and Larvae of the Codling Moth Cydia pomonella.Insects 2023,14, 97. https://doi.org/10.3390/ insects14020097 Academic Editor: Kazuki Tsuji Received: 25 December 2022 Revised: 13 January 2023 Accepted: 15 January 2023 Published: 17 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). insects Communication Predatory Abilities of Two Mediterranean Ants on the Eggs and Larvae of the Codling Moth Cydia pomonella Enrico Schifani * , Daniele Giannetti and Donato A. Grasso Department of Chemistry, Life Sciences & Environmental Sustainability, University of Parma, Parco Area delle Scienze, 11/a, 43124 Parma, Italy *Correspondence: [email protected] Simple Summary: Ants are widespread across terrestrial ecosystems, including agroecosystems, where they take part in several important processes. They often can act as predators of a wide range of insect pests in agricultural fields, which should be considered by management programs, and can sometimes be actively exploited to promote sustainable biological control strategies. In a recent experiment conducted in Europe, pear trees visited by larger numbers of ants suffered fewer attacks to their fruits by the codling moth, a small lepidopteran, which is a significant economic pest worldwide, especially in apple, pear, and walnut orchards. However, the exact form of the interaction between the ants and codling moths remained unclear. While ants were already known to prey upon mature larvae or pupae in the soil, this new evidence suggested they could also control the eggs or newly hatched larvae that had not yet attacked the fruits, which are the two stages whose removal would directly prevent fruit damage. We conducted laboratory experiments to determine whether two common European ants could prey upon these stages. Our results suggest that these ants are effectively able to kill newly hatched larvae, while the eggs do not appear directly vulnerable to predation. Further investigation under field conditions would be needed to assess whether ants may also interfere with the oviposition by adult moths. Abstract: The predatory ability of ants (Hymenoptera, Formicidae) against insect pests can offer an important service to agricultural activities and may sometimes be directly exploited in biological control strategies. The codling moth Cydia pomonella (Lepidoptera, Tortricidae) is a major agricultural pest of fruit orchards, whose biological control is complicated by the fact that the larvae spend most of their life protected within the fruits they damage. In a recent experiment in Europe, pear trees in which ant activity was artificially increased by the addition of sugary liquid dispensers (artificial nectaries) suffered less damage caused by the larvae to their fruits. While some ants were already known to prey upon the mature larvae or pupae of C. pomonella in the soil, prevention of fruit damage would require predation upon eggs or newly hatched larvae, which have not yet excavated into the fruits. We verified whether two different Mediterranean ants frequently observed in fruit orchards, Crematogaster scutellaris and Tapinoma magnum, were able to prey upon C. pomonella eggs and larvae in laboratory conditions. Our experiments demonstrated that both species similarly attacked and killed young C. pomonella larvae. On the other hand, the eggs mostly attracted the attention of T. magnum but were never damaged. Further field assessments are required to understand whether ants may also interfere with oviposition by adults or whether larger ant species, although generally rarer in orchards, may also prey upon eggs. Keywords: biological control; pest management; Formicidae; Tortricidae; Lepidoptera; Crematogaster scutellaris;Tapinoma magnum 1. Introduction Ants (Hymenoptera, Formicidae) are among the most successful insect groups, and their widespread presence in terrestrial habitats has significant ecological consequences [1,2] . Insects 2023,14, 97. https://doi.org/10.3390/insects14020097 https://www.mdpi.com/journal/insects 37 Insects 2023,14, 97 2 of 7 Their relationship with plants is of particular interest from both an evolutionary and an applied perspective [ 2 , 3 ]. One of the most important services that ants may provide to plants in these relationships is protection from a range of different herbivore insects that ants may prey upon or at least displace [ 4 , 5 ]. In addition, ants in agriculture may also play important roles in soil enrichment and bioturbation, as well as control of weeds and certain plant pathogens [ 6 – 8 ]. Ants’ ability to protect certain honeydew insect pests must be acknowledged; at the same time, their generalist predatory ability against several phytophagous arthropods promotes their recognition as biological control agents across different agricultural contexts [ 4 , 5 , 9 ]. This is especially well known in the tropics and comparatively less studied in temperate regions [4]. The codling moth, Cydia pomonella (Linnaeus, 1758) (Lepidoptera, Tortricidae), is a key polyphagous fruit pest whose economic relevance is particularly significant in apple, pear, and walnut orchards [ 10 – 12 ]. Its control is complicated by the development of resistance against pesticides and baculoviruses [ 12 – 14 ], while pesticide usage may disrupt the control of secondary pests [ 15 ]. Biological control strategies normally focus on last instar larvae that seek a shelter to pupate, on pupae, or on adults, using predators, parasitoids, and viruses [ 16 – 21 ]. In addition, pheromones or the sterile insect technique can be used in mating disruption strategies [ 22 – 24 ]. However, few biological control agents are known to target eggs or younger larvae, which spend almost their entire life protected inside the fruit they consume except for a short window after hatching (usually within 24 h), during which they may travel for up to a few meters searching for some fruit to dig into [ 10 , 25 ]. Predatory heteropterans and earwigs are the only known predators of eggs [ 26 – 28 ], which are very small (1–1.2 mm long), may be laid directly on the surface of fruits or on nearby areas of the plants, and hatch in about 5–12 days [10,25]. Among the different generalist predators that may play a role in the control of C. pomonella [ 29 ], ground-dwelling ants can prey upon last instar larvae and pupae [ 18 ]. More recently, field data suggested that trees visited more intensively by ants may suffer less damage to their fruits by the moths [30]. While this result suggested an effect of ants on the activity of C. pomonella before its larvae dig into the fruits, it remained unclear whether ants affected the eggs and/or the newly hatched larvae [ 30 ]. We aimed to test whether two Mediterranean ants that are common in fruit orchards and agroecosystems, Crematogaster scutellaris (Olivier, 1792) and Tapinoma magnum Mayr, 1861 [ 31 , 32 ], may act as predators of C. pomonella eggs and/or newly hatched larvae by documenting their behavioral interactions in laboratory experiments. 2. Materials and Methods All experiments were conducted during June 2022. Four days before the experiments, fragments of C. scutellaris and T. magnum of at least 500 workers each [ 33 ] were taken from Parma University Campus (northern Italy) and temporarily reared under laboratory conditions (T: 25 ± 1 ◦ C, RH: 60 ± 0.5%, photoperiod 12:12 L:D; honey provided as food). Commercially available C. pomonella eggs were obtained from Andermatt Biocontrol (Grossdietwil, Switzerland) and kept under the same laboratory conditions. Cydia pomonella eggs and first-instar larvae (hatched in the previous 2–8 h) were used in the experiments alongside ant workers randomly selected from the colony fragments. In each trial, we introduced into a petri dish ( ∅ = 9 cm) either an ant and a group of 6 C. pomonella eggs laid on a 1 cm × 1.5 cm paper or an ant and a single C. pomonella larva. The C. pomonella eggs or larva were initially put at the center of the petri dish and the ant was introduced one minute later. When the ant was introduced, the petri dish was filmed for 10 min using a camera to record the behavioral interactions. Insects used for an experimental trial were not reused in any following trial. A total of 12 trials were conducted for each ant species to study its interaction with C. pomonella eggs (n = 24), while 15 trials were conducted to study the interaction of each ant species with C. pomonella larvae (n = 30). 38 Insects 2023,14, 97 3 of 7 The videos were subsequently analyzed using the software, Solomon Coder 19.08.02, to evaluate behavioral interactions. We recorded the following behaviors performed by the ants towards the larvae: (i) Antennation: the ant touches the eggs/larva with its antennae while slowing or stopping nearby. (ii) Mandible opening: the ant opens its mandibles in front of the eggs/larva without biting. (iii) Biting: the ant bites the eggs/larva with its mandibles. (iv) Chemical attack: the ant uses its chemical repellent to the eggs/larvae (this behavior is performed by applying the venom topically, using the spatulate stinger in C. scutellaris and by short distance spraying in T. magnum). (v) Transportation/feeding: the ant starts to feed on the eggs/larva or transport them with its mandibles—this is considered as predation and/or as a proxy of food retrieval to the nest. (vi) Walking over: the ant walks over eggs/larva. The frequency of each behavior was recorded in each experimental trial. Biting and transportation/feeding were always displayed together in our observations and were therefore treated as a single behavior for the purpose of statistical analyses. Furthermore, at the end of each trial, we inspected under a stereoscopic microscope whether the eggs appeared damaged and whether the larvae were dead or injured. We used a generalized linear model (GLM) followed by Tukey’s post hoc tests to analyze the frequency of the behavioral interactions between the ants and the eggs, according to the identity of the ant species and of the behavior, considering their possible interactions. Differences between the two ant species concerning single behaviors performed were analyzed using Mann–Whitney U tests. We used a GLM with binomial distribution followed by Tukey’s post hoc tests to analyze the frequency of the behavioral interactions between the ants and the larvae, according to the identity of the ant species and of the behavior, considering their possible interactions. Differences between the two ant species for single behaviors performed were then analyzed using the chi-square test. The data were analyzed using the software R 4.2.2 and RStudio [34,35]. 3. Results In the interactions between the ants and the eggs, only three behaviors were observed: antennation, mandible opening, and walking over (see Supplementary File S1). We found no significant difference in the frequency of the different behaviors (0.864 ≤ p ≤ 0.997), while T. magnum interacted more frequently with the eggs as compared with C. scutellaris (p= 0.009). All three behaviors were more frequently expressed by T. magnum as compared to C. scutellaris (Antennation: W = 37, p= 0.038; Mandible opening: W = 39.5, p= 0.021; Walking over: W = 29.5, p= 0.009; Figure 1, Supplementary File S2). No eggs were harmed by the ants during the trials. In the interactions between the ants and the larvae, four behaviors were observed: antennation, biting and transportation/feeding, and mandible opening (see Supplementary File S1). Each behavior was observed only once per experiment. Mandible opening was performed significantly less frequently than antennation (p= 0.023), while no significant differences were detected between the frequency of the interaction by the two ant species (p= 0.705) nor between the frequency of individual behaviors (Antennation: χ2 = 0.14, p= 0.705; Biting and Transportation/feeding: χ2 = 0.13, p= 0.712; Mandible opening: χ2= 2.16 ,p= 0.142; Figure 2, Supplementary File S2). Biting and transportation/feeding always implied that the larvae were dead by the end of the experiment; so, 43% of the larvae were killed during the 10-min trials. 39 Insects 2023,14, 97 4 of 7 Insects 2023, 14, x FOR PEER REVIEW 4 of 7 Figure 1. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella eggs. Asterisks represent the significance level of the differences between the two species (*, p ≤ 0.05; **, 0.001 < p ≤ 0.01). In the interactions between the ants and the larvae, four behaviors were observed: antennation, biting and transportation/feeding, and mandible opening (see Supplementary File S1). Each behavior was observed only once per experiment. Mandible opening was performed significantly less frequently than antennation (p = 0.023), while no significant differences were detected between the frequency of the interaction by the two ant species (p = 0.705) nor between the frequency of individual behaviors (Antennation: χ 2 = 0.14, p = 0.705; Biting and Transportation/feeding: χ 2 = 0.13, p = 0.712; Mandible opening: χ 2 = 2.16, p = 0.142; Figure 2, Supplementary File S2). Biting and transportation/feeding always implied that the larvae were dead by the end of the experiment; so, 43% of the larvae were killed during the 10-min trials. Figure 2. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella larvae. No statistically significant differences (n.s.) between the two ant species were detected. 4. Discussion Our data revealed that common Mediterranean ants may act as predators of newly hatched C. pomonella larvae. Newly hatched larvae are particularly vulnerable to predators, as well as temperature variation and rainfall, until they can locate and excavate into Figure 1. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella eggs. Asterisks represent the significance level of the differences between the two species (*, p≤0.05; **, 0.001 < p≤0.01). Insects 2023, 14, x FOR PEER REVIEW 4 of 7 Figure 1. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella eggs. Asterisks represent the significance level of the differences between the two species (*, p ≤ 0.05; **, 0.001 < p ≤ 0.01). In the interactions between the ants and the larvae, four behaviors were observed: antennation, biting and transportation/feeding, and mandible opening (see Supplementary File S1). Each behavior was observed only once per experiment. Mandible opening was performed significantly less frequently than antennation (p = 0.023), while no significant differences were detected between the frequency of the interaction by the two ant species (p = 0.705) nor between the frequency of individual behaviors (Antennation: χ 2 = 0.14, p = 0.705; Biting and Transportation/feeding: χ 2 = 0.13, p = 0.712; Mandible opening: χ 2 = 2.16, p = 0.142; Figure 2, Supplementary File S2). Biting and transportation/feeding always implied that the larvae were dead by the end of the experiment; so, 43% of the larvae were killed during the 10-min trials. Figure 2. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella larvae. No statistically significant differences (n.s.) between the two ant species were detected. 4. Discussion Our data revealed that common Mediterranean ants may act as predators of newly hatched C. pomonella larvae. Newly hatched larvae are particularly vulnerable to predators, as well as temperature variation and rainfall, until they can locate and excavate into Figure 2. The interactions observed between the ants (Crematogaster scutellaris and Tapinoma magnum) and the Cydia pomonella larvae. No statistically significant differences (n.s.) between the two ant species were detected. 4. Discussion Our data revealed that common Mediterranean ants may act as predators of newly hatched C. pomonella larvae. Newly hatched larvae are particularly vulnerable to predators, as well as temperature variation and rainfall, until they can locate and excavate into fruit, which may take from 10 min to a few hours to accomplish [ 25 , 36 ]. In our experiments, both C. scutellaris and T. magnum behaved similarly towards the larvae, killing them in approximately half of the short trials by repeatedly biting their soft parts and then immediately feeding on them or transporting them with their mandibles. Detection through antennation was typically followed by attacks, while in most trials in which no attacks were recorded, the larvae remained undetected. We can speculate that very small newly hatched larvae may be a more attractive and more easily encountered item for smaller ants. While both species did not attack the eggs, these attracted the attention of T. magnum significantly, as the workers were repeatedly observed performing stereotyped mandible threats and often kept antennating or walking over them several times. Eggs may offer little foothold to the ants’ mandibles and can adhere strongly to the substratum of leaves and fruits, thus becoming physically invulnerable at least to the species we tested [ 37 , 38 ]. Larger ants with stronger and larger mandibles may be more capable of damaging or feeding on the eggs, 40 Biological Control 174 (2022) 105032 3 the beetle; iii) bites directed against the beetle; iv) aggressive interactions performed with the gaster (gaster flexing/extension, stinger use or chemical spraying). A beetle was considered preyed by the ant if the latter carried it away in its mandibles. 2.4. Exposure of beetle nests to ant colonies The experimental apparatus consisted in a 21 ×15 ×7 cm arena. Except for the control treatment, the arena was occupied by a group of ants (C. scutellaris or T. magnum, 60 workers) or a colony (Temnothorax spp., 60 workers and one queen). In each arena, we placed a twig colonized 15 days earlier by 6 female beetles because, at the rearing conditions used in the experiments, 15 days were enough to ensure the presence of developing progeny inside infested galleries (Antonio Gugliuzzo unpublished data). The experiment started after the twig was put in the arena and lasted 35 days. To observe ants’ behavior, after the start, we performed a 2-hr long continuous sampling followed by 10 min continuous sampling 6-hr later, and by two scan samplings per day (at 10:00 and 16:00) on each of the following days. During each observation, we recorded the number of ants in a 1 cm range from each of the beetles’ nests, the number of ants entering the nests and the number of X. compactus individuals outside the nests (dead or alive). Finally, we also quantified the number of beetle offspring reaching the adult stage within each nest. We conducted a number of 6 replicates per each ant species and each of the two plant species (using T. affinis in trials with C. sativa and T. mediterraneus in trials with L. nobilis, N =6 ×3 ×2 =36). In addition, 18 control groups, with no ant for each of the two plant species (N =18 ×2 =36), were carried out. 2.5. Chemical attractiveness of infested twigs to ants We tested the olfactory response of T. mediterraneus, C. scutellaris and T. magnum to different volatiles related to X. compactus infestation via dual-choice experiments using a Y-tube olfactometer with the characteristics illustrated by Naselli et al. (2017). Considering that its performance was extremely similar to T. mediterraneus in the other experiments, T. affinis was not tested in this experiment. Each ant worker was individually introduced into the entry arm of the Y-tube and observed until it reached the end of one of the two arms (making a choice) or until five minutes passed (no choice made). For each of the three ant species and three beetle stages, different worker individuals belonging to at least six different colonies were tested, and a minimum of 30 choices were recorded. The bioassays were conducted between 08:00 and 20:00, at 23 ±2 ◦C and 60 ±10 % R.H.. Three combinations of odor sources were tested: i) laurel twig section predominantly containing beetle larvae vs control (not infested); ii) laurel twig section predominantly containing pupae vs control; iii) laurel twig section predominantly containing adults vs control. Twig sections were prepared as described in the rearing section above, but they were 5 cm long and infested by a single female. Based on data collected under the same rearing conditions, we used twigs infested 18–21 days before for larvae, 27–30 days before for pupae and 36–39 days before for adults. After each trial, twigs were replaced with new ones, and were dissected to verify their content: if this did not coincide with the expected, the trial was repeated. 2.6. Effects of chemical residuals of ant patrolling on twig colonization by beetles The experimental apparatus consisted in a small arena (21 ×15 ×7 cm) where two twigs were put on the two opposite sides while a group of four beetle foundresses was placed in between (10 cm away from either twig). One of the twigs was previously exposed to patrolling ants, while the other was not (control), and the experiment lasted 24 hrs starting when the beetles were introduced. Then, the number of beetles who bored either twig was recorded. Twigs were prepared according to the same procedure described in the rearing section. Tests were conducted for all the four ant species and with both C. sativa and L. nobilis twigs, but T. affinis was only tested with C. sativa and T. mediterraneus only with L. nobilis (N =12 ×3 ×2 =72). To expose them to patrolling ants, they were put in an arena with an ant colony for 48 hrs. During this time, we assessed the number of ants walking on each twig with scan samplings (one every hour for 16 hrs and then one after 24 hrs and one after 48 hrs). As a result, we quantified this number as following (given as mean ±sd): on C. sativa twigs, 143 ±12 for C. scutellaris, 227 ±9 for T. magnum, and 24 ±14 for T. affinis; on L. nobilis twigs, 90 ±10 for C. scutellaris, 118 ±9 for T. magnum, and 28 ±5 for T. mediterraneus. 2.7. Data analysis For each behavior (concerning the behavioral interactions during 1vs1 encounters, section 2.3.), differences in its frequency between different ant species were analyzed using Kruskal-Wallis tests followed by Dunn post hoc tests whenever the firsts indicated significant differences. Concerning the exposure of beetle nests to ant colonies (section 2.4), statistical analyses were carried out separately for C. sativa and L. nobilis. Data concerning the number of X. compactus foundresses found dead in the arenas in treatments with different ant species were compared through Kruskal-Wallis tests followed by Dunn post hoc tests (each value corresponded to an experimental arena). On the other hand, differences in the number of ant workers near the entrance of the beetle nests and in the number of beetle offspring per nest were compared using a linear mixed model in which the experimental arena where data were taken was set as the random factor (each value corresponded to an individual nest, with multiple nests per arena). These were followed by Tukey post hoc tests whenever significant differences between treatments with different ant species were detected. Data concerning the chemical attractiveness of infested twigs to ants (section 2.5) were analyzed to evaluate the possible preference showed by the three ant species toward different X. compactus biological stages during Y-tube olfactometer bioassays. In particular, Chi-squared goodness-of fit tests were conducted in order to assess whether the ant response to different volatile sources was significantly different from a 50:50 distribution. Ant workers that did not make a choice were not considered in the analyses. Similarly, data concerning the effects of chemical residuals of ant patrolling on twig colonization by beetles (section 2.6.) were analyzed by running separate Chi-squared tests comparing beetles’ choice between ant-exposed and control twigs for each combination of plant and ant species utilized. Beetles who did not made a choice were not considered in the analyses. Analyses were carried out using the software R v4.1.1 (R Core Team, 2021) and RStudio v2021.09.0 351 (RStudio Team, 2021). 3. Results 3.1. Behavioral interactions during 1vs1 encounters Interactions between ants and beetle larvae and pupae resulted in the latter being immediately taken and carried away as prey without any complex behavioral display on at least 90 % of tests. Crematogaster scutellaris preyed upon 90 % of both larvae and pupae, T. magnum 93 % of larvae and 97 % of pupae, T. affinis 90 % of both larvae and pupae, and T. mediterraneus 100 % of both larvae and pupae. On the other hand, interactions between ants and adult beetles were characterized by a more complex behavioral pattern. Antennation, open-mandibles threatening and biting were the most frequently recorded behaviors (respectively recorded during 88 %, 69 % and 39 % of the trials). The frequency of exhibition of the three behaviors was statistically different among the four tested ant species (antennation: H 3 =25.92, p <0.001; open-mandibles threatening: H 3 =27.02, p <0.001; biting: H 3 =14.78, p =0.002), with C. scutellaris and T. magnum D. Giannetti et al. 47 Biological Control 174 (2022) 105032 4 performing them more frequently. In particular, pairwise comparisons revealed that each species differed from the others for antennation frequency (p <0.001). On the other hand, concerning open-mandibles threatening, no statistically significant differences were detected between T. affinis and T. mediterraneus (p =0.945), nor between C. scutellaris and T. magnum (p =0.710). However, differences were significant between the two groups (0.006 <p <0.001). Finally, significant differences were detected for biting between C. scutellaris and T. affinis (p =0.046), between C. scutellaris and T. mediterraenus (p = 0.011), and between T. mediterraneus and T. magnum (p =0.023), but not in the other pairwise comparisons (1.000 <p <0.072) (Fig. 1). 3.2. Exposure of beetle nests to ant colonies Beetle nest entrances attracted workers of all ant species, and their attractiveness to different ant species differed significantly (C. sativa: F 2,15 =98.24, p <0.001; L. nobilis: F 2,15 =26.54, p <0.001). In particular, in both the C. sativa and the L. nobilis trials, Temnothorax spp. workers were more numerous around the nest entrances compared to those of C. scutellaris and T. magnum (p <0.001), while C. scutellaris and T. magnum differed one from the other in C. sativa trials (p <0.001) but not in L. nobilis trials (p =0.929). Nests showed significantly different mortality rates of beetle foundresses according to treatment (C. sativa: H 3 =27.77, p <0.001; L. nobilis: H 3 =31.57, p <0.001). The highest mortality scores were always recorded in the presence of Temnothorax spp., intermediate scores characterized tests with C. scutellaris, further lower scores were recorded in the presence of T. magnum, and the lowest mortality scores were recorded in the absence of ants (Fig. 2). The beetles’ reproductive success was on average greater in L. nobilis trials than in C. sativa trials (as mean ±sd: 20.5 ±9.5 in L. nobilis; 7.1 ± 4.4 in C. sativa), but in both cases it was significantly affected by treatment (C. sativa: F 3,32 =29.05, p <0.001; L. nobilis: F 3,32 =78.42, p <0.001). In both, it was lowest in the presence of Temnothorax spp. and C. scutellaris (not different one from the other, 0.521 <p < 1.000, and different from all the other treatments, p <0.001), and in almost all cases highest in the absence of ants (p <0.001). However, in treatments with T. magnum, the recorded reproductive success was either intermediate between that of treatments with other ants and the control group (in L. nobilis trials, p <0.001), or not statistically different from the control group (in C. sativa trials, p =0.998) (Fig. 2). Finally, on two occasions during scan samplings, T. mediterraneus was observed entering the beetles’ nests, its workers carrying away pieces of dead adult beetles and the whole colony transferring inside the nest in one case (Fig. 3) (Supplementary Video). 3.3. Chemical attractiveness of infested twigs to ants Crematogaster scutellaris and T. magnum showed no significant preference for volatiles cues emitted by healthy stem sections nor those infested by the beetle (Fig. 4a). In particular, workers of C. scutellaris did not show any preference for volatiles emitted by stem sections infested by X. compactus larvae ( χ 2 =0.937, p =0.333), pupae ( χ 2 =0, p =1) or adults ( χ 2 =0.137, p =0.711) when compared with those of not infested stem sections. Similarly, volatiles related to the X. compactus infestation were not significantly attractive compared to those emitted by healthy laurel stem sections for T. magnum workers (larvae: χ 2 =2.041, p = 0.153, pupae: χ 2 =0.082, p =0.775, adults: χ 2 =0.657, p =0.417). By contrast, T. mediterraneus workers were significantly attracted by the volatiles emitted by laurel stem sections infested by beetle larvae compared to those of not infested stem sections ( χ 2 =9.766, p =0.002) (Fig. 4a). However, when the dual choice was between healthy stem sections and stem sections infested by beetle pupae, workers of this ant species did not show any preference ( χ 2 =0, p =1). Lastly, T. mediterraneus workers showed a significant preference for volatiles emitted by not infested stem sections when compared with those of stem sections infested by X. compactus adults ( χ 2 =14.063, p <0.001) (Fig. 4a). 3.4. Effects of previous ant patrolling on twig colonization by beetles From 4 to 39 % of the beetle foundresses did not make any choice. Analyses of the choice data revealed a clear foundresses preference for twigs that were not previously exposed to ants (p <0001) (Fig. 4b). 4. Discussion The lack of coevolved natural predators can be a major facilitating factor in biological invasion processes (Pyˇ sek et al., 2020; Yousuf et al., 2021; Desneux et al. 2022). Ubiquitous generalist predators, such as ants, may offer an important service in this regard, and their study as biocontrol agents in Europe is still little explored (Campolo et al., 2015; Castracani et al., 2017; Nielsen et al., 2018; Schifani et al. 2020; Bulgarini et al., 2021). Invasive exotic ambrosia beetles may cause alteration of native tree communities, reduced yields, and increased control costs (Grousset et al., 2020, Hulcr et al., 2021; Marchioro and Faccoli, 2021, Mendel et al., 2021). This is particularly true for some invasive Fig. 1. Most frequently recorded behavioral interactions of ant workers in front of X. compactus adults. Groups with the same latter are not statistically different. D. Giannetti et al. 48 Biological Control 174 (2022) 105032 5 Xylosandrus spp. which biological invasions has the potential to cause a cascade of impacts both at ecological and economic level (Gugliuzzo et al., 2021). Moreover, the peculiar biology and cryptic nature (i.e., completing their lifecycle within galleries inside the wood) of this ecological group of insects typically protects them from many potential predators. However, the relationship between Scolytinae beetles and predators, such as ants, has been rarely investigated, mostly with the coffee berry borer Hypothenemus hampei (Ferrari, 1867) rather than Xyleborini (e.g. Larsen and Philpott, 2010; Gonthier et al., 2013). All the ant species we tested in 1vs1 encounters showed a high interest in predating immature stages of X. compactus (larvae and pupae), yet aggression against adults was very limited and only rarely damaging for the beetles, with the larger species, C. scutellaris and T. magnum, performing more attacks. Larvae and pupae are normally hidden deep inside X. compactus galleries, which are narrow and guarded by an adult, i.e., the foundress mother. Nonetheless, when beetle nests were exposed to ant colonies, all ant species had a meaningful impact, with ant workers always surrounding the nests’ entrances and significantly increasing the mortality of the foundress mother. Albeit weaker in the case of T. magnum, this result was associated with a much reduced reproductive success of the beetles. The premature death of the foundress when beetle nests were exposed to ant colonies likely deprived the offspring and the nest of the necessary cares that the mother usually performs (controlling the hygienic conditions and taking care of the mutualistic fungus), greatly limiting their ability to develop into adults. This result appears coherent to what reported by Ogogol et al., (2017), who witnessed a control effect on X. compactus populations by P. megacephala despite the latter’s inability to enter the beetles’ nests. However, one of the two smaller ant species used in our tests, T. mediterraneus was able to enter the nest galleries, dragging outside larvae, pupae and dismembered adults. Our data suggest that volatiles may have very little influence over ants’ ability to locate X. compactus nests. Indeed, results of olfactometer bioassays show that volatiles related to the beetle infestation do not affect the orientation behavior of C. scutellaris and T. magnum workers. On the other hand, T. mediterraneus workers exhibited significant attraction for volatiles released by laurel stem sections infested by X. compactus larvae, but not for those emitted by stems infested by pupae. Furthermore, volatiles emitted by stems which galleries were infested by beetle adults were not attractive for workers of T. mediterraneus that preferred healthy stem sections. This result could be related to volatile substances potentially involved in the beetle adult aggregation inside galleries that may have affect the orientation of ant workers. Aggregation pheromones are used by many herbivorous insects Fig. 2. Attraction of ants to X. compactus nest entrances, effect of ants on the mortality of X. compactus foundresses, and reproductive success of X. compactus nests exposed to ant colonies. Groups with the same latter are not statistically different. D. Giannetti et al. 49 Biological Control 174 (2022) 105032 6 exhibiting a pheromone-based colonization behavior, including some bark beetle species (Meurisse et al. 2021). On the other hand, these semiochemicals can act as prey-finding kairomones by predators or parasitoids (Scala et al. 2022). However, to the best of our knowledge, there is no evidence of sex or species-specific aggregation pheromone occurrence for most ambrosia beetle species, and despite they could not be ecologically relevant for other species of this group of insects (Kirkendall et al., 1997), it could be worthy investigating this aspect in massaggregating Xylosandrus species. Moreover, it is possible to hypothesize that other volatile sources, e.g., those produced by mutualistic fungi, related to the wood infestation by these fungus-farming insects (Egonyu and Torto 2018; Ranger et al. 2021), could be involved in the predator–prey interactions. At the same time, our results show that X. compactus females would strongly avoid building their nests in twigs that retain the chemical trace of ant activity. Ant trails may be widespread and stable chemical signposts (e.g. H¨ olldobler and Wilson, 1990; Grasso et al., 1998, 1999) offering the beetles useful cues to avoid unsuitable nesting places, and ant cues may have a strong deterring effect against phytophagous insects (Offenberg et al., 2004; Adandonon et al., 2009; Vayssi` eres et al., 2013). The results of this study indicate a potential role of native ant species to limit the success of this invasive pest in Europe and in the Mediterranean. It is worth noting that the only ant species so far clearly identified as an effective predator of X. compactus was the Afrotropical bigheaded ant P. megacephala (Ogogol et al., 2017), an invasive species of serious ecological concern in much of the world (Sarnat et al., 2015). In the study by Egonyu et al. (2015) Plagiolepis sp. showed to be a potential efficient predator of X. compactus in Uganda; however, the ant pictures published in the article seems to be related to ants of the genus Cardiocondyla. European Plagiolepis species are considered mostly gliciphagous rather than predatory, while Cardiocondyla species in Europe do not forage on trees (Seifert, 2018). On the other hand, Crematogaster Fig. 3. Interactions between T. mediterraneus and X. compactus. a,b T. mediterraneus workers drag out dead and partly dismembered X. compactus adults out of their galleries. c,d T. mediterraneus colony occupying a X. compactus nest after eliminating the beetles: workers (c) and queen (d) shot after cutting a section of the twig. Fig. 4. a) Choice by ant workers among twigs infested by X. compactus or not. b) Choice by Xylosandrus compactus among twigs previously exposed or unexposed to ants. Asterisks indicate statistical significance of the differences (*: p <0.05; **: p <0.005; ***: p <0.0001). D. Giannetti et al. 50 Biological Control 174 (2022) 105032 7 species of the scutellaris group and arboreal-nesting Temnothorax species are both widespread in the Holarctic region (Prebus, 2017; Ward and Blaimer, 2022). In Italy, C. scutellaris, T. affinis, and T. mediterraneus are commonly observed foraging on C. sativa and/or L. nobilis (authors unpublished data). Our data encourage to further investigate the interactions between ants and ambrosia beetles and to extend this investigation to other ant species and genera, especially considering that we obtained comparable results from ants belonging to rather different evolutionary lineages. In the case of X. compactus, such investigations should now continue in the field, to verify the potential use of ants as biocontrol agents or the actual ecological consequences of these interactions. In addition, the possible roles of the mutualistic fungi associated to Xyleborini in the relations between the beetles and other arthropods such as ants deserve further investigations. In conclusion, the pervasive ecological role of ants as generalist predators across terrestrial habitats may have had an evolutionary impact on ambrosia beetles too (Parker and Kronauer, 2021). The results of our study, which is the first to investigate potential natural enemies of X. compactus in the Euro-Mediterranean region, encourage to take into account the predatory role of ants in management strategies aimed at controlling this invasive pest. CRediT authorship contribution statement Daniele Giannetti: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Enrico Schifani: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Antonio Gugliuzzo: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Lucia Zappal` a: Conceptualization, Resources, Writing – review & editing. Antonio Biondi: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. Donato A. Grasso: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements Authors are thankful to the University of Catania (Project “Emergent Pests and Pathogens and Relative Sustainable Strategies” (5A722192113) and PhD grant to AG). We thank also Paola Grazia Lo Faro for the help with the olfactory bioassays and the beetle colony maintenance as a part of her MSc thesis. Appendix A. Supplementary material Video on Temnothorax mediterraneus predation on Xylosandrus compactus infested twig. Supplementary data to this article can be found online at https://doi. org/10.1016/j.biocontrol.2022.105032. References Adandonon, A., Vayssieres, J.F., Sinzogan, A., Van Mele, P., 2009. Density of pheromone sources of the weaver ant Oecophylla longinoda affects oviposition behaviour and damage by mango fruit flies (Diptera: Tephritidae). Int J. Pest Manag. 55, 285–292. Biedermann, P.H., Vega, F.E., 2020. Ecology and evolution of insect-fungus mutualisms. Annu. Rev. Entomol. 65, 431–455. Brill, E., Follett, P.A., Kawabata, A.M., 2021. Feeding habits, movement, and reproduction of the predatory flat bark beetles Cathartus quadricollis (Coleoptera: Silvanidae) and Leptophloeus sp. (Coleoptera: Laemophloeidae) in Hawaii coffee and macadamia nut. Int. J. of Trop. Insect Sci. 41, 285–294. Bulgarini, G., Castracani, C., Mori, A., Grasso, D.A., Maistrello, L., 2021. Searching for new predators of the invasive Halyomorpha halys: the role of the black garden ant Lasius niger. Entomol. Exp. Appl. 169, 799–806. Campolo, O., Palmeri, V., Malacrin` o, A., Laudani, F., Castracani, C., Mori, A., Grasso, D. A., 2015. Interaction between ants and the Mediterranean fruit fly: New insights for biological control. Biol. Control 90, 120–127. Castracani, C., Bulgarini, G., Giannetti, D., Spotti, F.A., Maistrello, L., Mori, A., Grasso, D. A., 2017. Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys. J. Pest. Sci. 90, 1181–1190. Castrillo, L.A., Griggs, M.H., Vandenberg, J.D., 2016. Competition between biological control fungi and fungal symbionts of ambrosia beetles Xylosandrus crassiusculus and X. germanus (Coleoptera: Curculionidae): mycelial interactions and impact on beetle brood production. Biol. Control 103, 138–146. Choate, B., Drummond, F., 2011. Ants as biological control agents in agricultural cropping systems. Terr. Arthropod. Rev. 4, 157–180. Desneux, N., Han, P., Mansour, R., Arn´ o, J., et al., 2022. Integrated pest management of Tuta absoluta: practical implementations across different world regions. J. Pest. Sci. 95, 17–39. Dzurenko, M., Ranger, C.M., Hulcr, J., Galko, J., Kaˇ nuch, P., 2021. Origin of non-native Xylosandrus germanus, an invasive pest ambrosia beetle in Europe and North America. J. Pest. Sci. 94, 553–562. Egonyu, J.P., Baguma, J., Ogari, I., Ahumuza, G., Kyamanywa, S., Kucel, P., Kagezi, H.G., Erbaugh, M., Phiri, N., Ritchie, B.J., Wagoire, W.W., 2015. The formicid ant, Plagiolepis sp., as a predator of the coffee twig borer, Xylosandrus compactus. Biol. Control 91, 42–46. Egonyu, J.P., Torto, B., 2018. Responses of the ambrosia beetle Xylosandrus compactus (Coleoptera: Curculionidea: Scolytinae) to volatile constituents of its symbiotic fungus Fusarium solani (Hypocreales: Nectriaceae). Arthropod.-Plant Interact. 12, 9–20. Galkowski, C., Cagniant, H., 2017. Contribution ` a la connaissance des fourmis du groupe angustulus dans le genre Temnothorax (Hymenoptera, Formicidae). Revue de l’Association Roussillonnaise d’Entomologie 26, 180–191. Gonthier, D.J., Ennis, K.K., Philpott, S.M., Vandermeer, J., Perfecto, I., 2013. Ants defend coffee from berry borer colonization. Biocontrol 58, 815–820. Grasso, D.A., Mori, A., Le Moli, F., 1998. Chemical communication during foraging in the harvesting ant Messor capitatus (Hymenoptera, Formicidae). Insectes Soc. 45, 85–96. Grasso, D.A., Mori, A., Le Moli, F., 1999. Recruitment and trail communication in two species of Messor ants (Hymenoptera, Formicidae). The Ital. J. Zool. 66, 373–378. Greco, E.B., Wright, M.G., 2015. Ecology, biology, and management of Xylosandrus compactus (Coleoptera: Curculionidae: Scolytinae) with emphasis on coffee in Hawaii. J. Integr. Pest. Manag. 6, 7. Grousset, F., Gr´ egoire, J.C., Jactel, H., Battisti, A., Benko Beloglavec, A., et al., 2020. The risk of bark and ambrosia beetles associated with imported non-coniferous wood and potential horizontal phytosanitary measures. Forests 11, 342. Gugliuzzo, A., Mazzeo, G., Mansour, R., Tropea, Garzia G., 2019a. Carob pests in the Mediterranean region: bio-ecology, natural enemies and management options. Phytoparasitica 47, 605–628. Gugliuzzo, A., Criscione, G., Siscaro, G., Russo, A., Tropea Garzia, G., 2019b. First data on the flight activity and distribution of the ambrosia beetle Xylosandrus compactus (Eichhoff) on carob trees in Sicily. EPPO Bull. 49, 340–351. Gugliuzzo, A., Criscione, G., Tropea Garzia, G., 2019c. Unusual behavior of Xylosandrus compactus (Coleoptera: Scolytinae) on carob trees in a Mediterranean environment. Insects 10, 82. Gugliuzzo, A., Criscione, G., Biondi, A., Aiello, D., Vitale, A., Polizzi, G., Tropea Garzia, G., 2020. Seasonal changes in population structure of the ambrosia beetle Xylosandrus compactus and its associated fungi in a southern Mediterranean environment. PLoS ONE 15, 1–13. Gugliuzzo, A., Biedermann, P.H., Carrillo, D., Castrillo, L.A., Egonyu, J.P., et al., 2021. Recent advances toward the sustainable management of invasive Xylosandrus ambrosia beetles. J. Pest. Sci. 94, 615–637. Gugliuzzo, A., Aiello, D., Biondi, A., Giurdanella, G., Siscaro, G., Zappal` a, L., Vitale, A., Tropea Garzia, G., Polizzi, G., 2022. Microbial mutualism suppression by Trichoderma and Bacillus species for controlling the invasive ambrosia beetle Xylosandrus compactus. Biol. Control 170. Hara, A.H., Beardsley, J.W., 1979. The biology of the black twig borer, Xylosandrus compactus (Eichhoff). Hawaii. Proc. Hawaii. Entomol. Soc. 23, 55–70. H¨ olldobler, B., Wilson, E.O., 1990. The Ants. Harvard University Press. Hulcr, J., Gomez, D.F., Skelton, J., Johnson, A.J., Adams, S., Li, Y., Jusino, M.A., Smith, M.E., 2021. Invasion of an inconspicuous ambrosia beetle and fungus may affect wood decay in Southeastern North America. Biol. Invasions 23, 1339–1347. Kirkendall, L.R., Kent D.S., Raffa, K.F., 1997. Interactions among males, females, and offspring in bark and ambrosia beetles: the significance of living in tunnels for the evolution of social behavior. In: Choe, J.C., Crespi, B.J. (Eds.), Evolution of Social D. Giannetti et al. 51 Biological Control 174 (2022) 105032 8 Behavior in Insects and Arachnids, vol. 561. Cambridge Univ. Press, Cambridge, UK, 181–215. Lantschner, M.V., Corley, J.C., Liebhold, A.M., 2020. Drivers of global Scolytinae invasion patterns. Ecol. Appl. 30. Larsen, A., Philpott, S.M., 2010. Twig-nesting ants: the hidden predators of the coffee berry borer in Chiapas, Mexico. Biotropica 42, 342–347. Marchioro, M., Faccoli, M., 2021. Dispersal and colonization risk of the Walnut Twig Beetle, Pityophthorus juglandis, in southern Europe. J. Pest. Sci. 95, 303–313. Mendel, Z., Lynch, S.C., Eskalen, A., Protasov, A., Maymon, M., Freeman, S., 2021. What determines host range and reproductive performance of an invasive ambrosia beetle Euwallacea fornicatus; lessons from Israel and California. Front. For. Glob. Change 4, 29. Meurisse, N., Pawson, S.M., Somchit, C., 2021. Bark beetles on pine logs: forecasting winter colonisation dynamics based on trap catches and temperature records. J. Pest. Sci. 94, 1357–1373. Naselli, M., Zappala, L., Gugliuzzo, A., Tropea Garzia, G., Biondi, A., Rapisarda, C., Cincotta, F., Condurso, C., Verzera, A., Siscaro, G., 2017. Olfactory response of the zoophytophagous mirid Nesidiocoris tenuis to tomato and alternative host plants. Arthropod. Plant Interact. 11, 121–131. Ngoan, N.D., Wilkinson, R.C., Short, D.E., Moses, C.S., Mangold, J.R., 1976. Biology of an introduced ambrosia beetle, Xylosandrus compactus. Florida. Ann. Entomol. Soc. Am. 69, 872–876. Nielsen, J.S., Nielsen, M.G., Offenberg, J., 2018. Experiences in transplanting wood ants into plantations for integrated pest management. Sociobiology 65, 403–414. Offenberg, J., 2015. Ants as tools in sustainable agriculture. J. Appl. Ecol. 52, 1197–1205. Offenberg, J., Nielsen, M.G., MacIntosh, D.J., Havanon, S., Aksornkoae, S., 2004. Evidence that insect herbivores are deterred by ant pheromones. Proc. R. Soc. B: Biol. Sci. 271, S433–S435. Ogogol, R., Egonyu, J.P., Bwogi, G., Kyamanywa, S., Erbaugh, M., 2017. Interaction of the predatory ant Pheidole megacephala (Hymenoptera: Formicidae) with the polyphagus pest Xylosandrus compactus (Coleoptera: Curculionidea). Biol. Control 104, 66–70. Parker, J., Kronauer, D.J., 2021. How ants shape biodiversity. Curr. Biol. 31, R1208–R1214. Peng, R., Christian, K., 2010. Box 7.2 Ants as biological-control agents in the horticultural industry. In: Lach, L., Lori, L., Parr, L., Catherine, L., Abbot, K. (Eds.), Ant Ecology. Oxford University Press, pp. 123–125. Prebus, M., 2017. Insights into the evolution, biogeography and natural history of the acorn ants, genus Temnothorax Mayr (Hymenoptera: Formicidae). BMC Evol. Biol. 17, 1–22. Pureswaran, D.S., Meurisse, N., Rassati, D., Liebhold, A.M., Faccoli, M., 2022. Climate change and invasions by nonnative bark and ambrosia beetles. In: Bark Beetle Management, Ecology, and Climate Change. Academic Press, pp. 3–30. Pyˇ sek, P., Hulme, P.E., Simberloff, D., Bacher, S., Blackburn, T.M., et al., 2020. Scientists’ warning on invasive alien species. Biol. Rev. 95, 1511–1534. R Core Team, 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project. org/. Ranger, C.M., Dzurenko, M., Barnett, J., Geedi, R., Castrillo, L., Ethington, M., et al., 2021. Electrophysiological and behavioral responses of an ambrosia beetle to volatiles of its nutritional fungal symbiont. J. Chem. Ecol. 47, 463–475. RStudio Team, 2020. RStudio: Integrated Development for R. RStudio, PBC, Boston, MA, USA. URL http://www.rstudio.com/. Sarnat, E.M., Fischer, G., Gu´ enard, B., Economo, E.P., 2015. Introduced Pheidole of the world: taxonomy, biology and distribution. ZooKeys 543, 1–109. Scala, M., Fouani, J.M., Zapponi, L., Mazzoni, V., Wells, K.E., et al., 2022. Attraction of egg parasitoids Trissolcus mitsukurii and Trissolcus japonicus to the chemical cues of Halyomorpha halys and Nezara viridula. Insects 13, 439. Schifani, E., Castracani, C., Giannetti, D., Spotti, F.A., Reggiani, R., Leonardi, S., Mori, A., Grasso, D.A., 2020. New tools for conservation biological control: testing antattracting artificial nectaries to employ ants as plant defenders. Insects 11, 129. Seifert, B., D’Eustacchio, D., Kaufmann, B., Centorame, M., Lorite, P., Modica, M., 2017. Four species within the supercolonial ants of the Tapinoma nigerrimum complex revealed by integrative taxonomy (Hymenoptera: Formicidae). Myrmecol. News 24, 123–144. Seifert, B., 2018. The ants of central and north Europe. Lutra Verlags-und Vertriebsgesellschaft. Sreedharan, K., Balakrishnman, M.M., Bhat, P.K., 1992. Callimerus sp. (Coleoptera; Cleridae), a predator of the shot-hole borer, Xylosandrus compactus (Eichh.). J. Coffee Res. 22, 139–142. Urvois, T., Auger-Rozenberg, M.A., Roques, A., Rossi, J.P., Kerdelhue, C., 2021a. Climate change impact on the potential geographical distribution of two invading Xylosandrus ambrosia beetles. Sci. Rep. 11, 1339. Urvois, T., Perrier, C., Roques, A., Saun´ e, L., Courtin, C., Li, Y., Johnson, A.J., Kerdelhu´ e, C., 2021b. A first inference of the phylogeography of the worldwide invader Xylosandrus compactus. J. Pest. Sci. 95, 1217–1231. Vannini, A., Contarini, M., Faccoli, M., Valle, M.D., Rodriguez, C.M., Mazzetto, T., Guarneri, D., Vettraino, A.M., Speranza, S., 2017. First report of the ambrosia beetle Xylosandrus compactus and associated fungi in the Mediterranean maquis in Italy, and new host–pest associations. EPPO Bull. 47, 100–103. Vayssi` eres, J.F., Sinzogan, A., Adandonon, A., Van Mele, P., Korie, S., 2013. Ovipositional behaviour of two mango fruit fly species (Diptera Tephritidae) in relation to Oecophylla cues (Hymenoptera: Formicidae) as compared to natural conditions without ant cues. Int. J. Biol. Chem. Sci. 7, 447–456. Wang, Y., Lu, J., Sun, R., Gomez, D.F., Hulcr, J., Li, Y., Li, Y., Gao, L., 2022. Uncovering hidden diversity within the Euwallacea fornicatus species complex in China. Entomol. Gen. 42, 631–639. Ward, P.S., Blaimer, B.B., 2022. Taxonomy in the phylogenomic era: species boundaries and phylogenetic relationships among North American ants of the Crematogaster scutellaris group (Formicidae: Hymenoptera). Zool. J. Linn. Soc. 194, 893–937. Way, M.J., Khoo, K.C., 1992. Role of ants in pest management. Ann. Rev. Entomol. 37, 479–503. Yousuf, F., Follett, P.A., Gillett, C.P.D.T., Honsberger, D., Chamorro, L., et al., 2021. Limited host range in the idiobiont parasitoid Phymastichus coffea, a prospective biological control agent of the coffee pest Hypothenemus hampei in Hawaii. J. Pest. Sci. 94, 1183–1195. D. Giannetti et al. 52 Section I - Ants as biocontrol agents of insect pests Chapter 4 Interactions between egg parasitoids and predatory ants for the biocontrol of the invasive brown marmorated stink bug Halyomorpha halys _______________________________________________________________________________ Schifani, E., Giannetti, D., Costi, E., Franconi, G., Campostrini, A., Maistrello, L., & Grasso, D.A. (2023). Interactions between egg parasitoids and predatory ants for the biocontrol of the invasive brown marmorated stink bug Halyomorpha halys. Journal of Applied Entomology. https://doi.org/10.1111/jen.13179 53 J Appl Entomol. 2023;00:1–7. | 1wileyonlinelibrary.com/journal/jen 1 | INTRODUCTION The brown marmorated stink bug Halyomorpha halys (Stål), native to Eastern Asia and with invasive populations throughout the European, northern and southern American continents, is currently one of the most dangerous pests of fruit and seed crops (Leskey & Nielsen, 2018). Its high invasive capacity is facilitated by human activities and trade (Maistrello et al., 2018) and by high polyphagy (Rice et al., 2014), high mobility of the adults (Lee & Leskey, 2015), and high reproductive potential (Costi et al 2017). In Italy, where it Received: 29 April 2023 | Revised: 2 August 2023 | Accepted: 7 August 2023 DOI: 10.1111/jen.13179 ORIGINAL ARTICLE Interactions between egg parasitoids and predatory ants for the biocontrol of the invasive brown marmorated stink bug Halyomorpha halys Enrico Schifani1 | Daniele Giannetti1,2 | Elena Costi2 | Giulia Franconi1 | Arianna Campostrini2 | Lara Maistrello2 | Donato A. Grasso1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2023 The Authors. Journal of Applied Entomology published by Wiley-VCH GmbH. Enrico Schifani and Daniele Giannetti contributed equally to this study. 1Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy 2Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy Correspondence Enrico Schifani, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma 40123, Italy. Email: [email protected] Lara Maistrello, Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia 42122, Italy. Email: [email protected] Abstract The brown marmorated stink bug Halyomorpha halys is an Asian species that has become a major agricultural pest in North America and Europe. Ants from the genus Crematogaster are predators of H. halys nymphs in Asia, as well as in the Mediterranean, where known native predators are still few. At the same time, ants usually do not harm H. halys eggs, which are the target of the main biological control agents, the scelionid parasitoids of the genus Trissolcus. However, ants, as generalist predators and territorial organisms, may kill or displace a variety of other insects, potentially interfering with parasitoids and biological control programmes. We conducted laboratory experiments to investigate the interactions between the Mediterranean ant Crematogaster scutellaris and the parasitoids T. japonicus and T. mitsukurii, evaluating the possibility that the ants could damage the parasitized eggs, attack the parasitoids during emergence or interfere with the egglaying behaviour of female parasitoids. Our results demonstrate that C. scutellaris is not able to damage parasitized eggs and is not aggressive towards adult parasitoids at any stage. The presence of ants can slow down the parasitization rate in T. mitsukurii females in the smallest laboratory setups; however, this has not been observed in a more natural setting. We suggest that ants may play a complementary role together with eggparasitoids in the control of H. halys without interfering with each other. KEYWORDS Crematogaster scutellaris, intraguild relationships, Trissolcus japonicus, Trissolcus mitsukurii 54 2 | SCHIFANI et al. was first officially detected in 2012 (Maistrello et al., 2016), H. halys quickly became a key pest of fruit orchards (Maistrello et al., 2017); and in 2019, the estimated damage to fruit production in northern Italy was € 588 million, with yield losses of up to 80%– 100% in orchards (CSO Italy, 2020). To counter this invasive pest, the use of broadspectrum insecticides has increased dramatically, resulting in a major disruption to previous integrated pest management (IPM) programmes with negative consequences on the environment (Maistrello et al., 2017). Longterm and more sustainable management strategies include conservation and classical biological control. In native Asia, H. halys egg masses are attacked by different species of egg parasitoids, among which the Scelionidae Trissolcus japonicus (Ashmead) and T. mitsukurii (Ashmead) have the highest specificity and parasitization efficiency, ranging between 50% and 90% (Qiu, 2010; Yang et al., 2009; Zhang et al., 2017). In Northern Italy, adventive populations of T. mitsukurii and T. japonicus were first detected in 2016 (Scaccini et al., 2020) and 2018 (Sabbatini Peverieri et al., 2018), respectively. A largescale survey conducted throughout northern Italy and Switzerland in 2019, showed that both species had rapidly spread into all types of habitats where H. halys is present, with a wide distribution, continuous expansion and high levels of parasitism (Zapponi et al., 2021). Furthermore, in 2020, T. japonicus was selected by the Italian Ministry of Environment and the Protection of the Land and Sea as a candidate for classical biocontrol of the invasive pest (MATTM, 2020) and thousands of these parasitoids were released in the northern Italian regions for 3 years, leading to one of the largest biocontrol projects ever attempted in Italy and Europe. Meanwhile, laboratory studies conducted to verify the potential of generalist antagonists showed that ants are among the most efficient predators of H. halys (Bulgarini, Badra, et al., 2021; Bulgarini, Castracani et al., 2021; Castracani et al., 2017). Specifically, experiments with the two European ants most frequently encountered in agroecosystems, Crematogaster scutellaris (Olivier) and Lasius niger (Linnaeus), demonstrated their ability to kill H. halys nymphs without damaging eggs or adult stink bugs (Bulgarini, Castracani, et al., 2021; Castracani et al., 2017). Further studies conducted with the Japanese ant Crematogaster matsumurai Forel, 1901 and C. osakensis Forel, 1900 as well the cosmopolite invasive Argentine ant Linepithema humile (Mayr, 1868), had a similar outcome (Kamiyama et al., 2021). This study aims to investigate the interactions between the native European ant C. scutellaris and the exotic egg parasitoids T. japonicus and T. mitsukurii in terms of the outcome on the efficiency of biological control of H. halys. Ants are ubiquitous across most terrestrial ecosystems of the world, where they often play a significant ecological role as generalist predators (Hölldobler & Wilson, 1990; Lach et al., 2010; Parker & Kronauer, 2021). Their predatory abilities against insect pests can not only make them good key biological control agents (Choate & Drummond, 2011; Offenberg, 2015) but also may negatively affect other important biocontrol agents, including both parasitoids and predators (e.g. Appiah et al., 2014; Jiggins et al., 1993; Mgocheki & Addison, 2009). We hypothesized that ants may attack H. halys egg parasitoids as they do with H. halys nymphs (Bulgarini, Castracani, et al., 2021; Castracani et al., 2017). In particular, we investigated two moments in the life of adult parasitoids in which they could be particularly vulnerable: the moment in which the female parasitoid lays her eggs, which requires her to stand still on the stink bug egg mass for an extended time, and the moment of emergence of the newly metamorphosed individuals, as they need time to break an opening in the stink bug egg to free themselves. We also tested whether parasitized eggs might be more susceptible to ant attack than nonparasitized eggs, which are usually not attacked, and whether their attractiveness to ants could vary over time. 2 | MATERIALS AND METHODS 2.1 | Insect rearing and equipment Adults of Halyomorpha halys were collected during the spring and summer of 2020– 2021 from urban parks in Modena and Reggio Emilia provinces (EmiliaRomagna, Italy) using the treebeating technique. Stink bugs were set in BugDorm cages (17.5 × 17.5 × 17.5 cm) and placed in climatic chambers at 26°C and L16: D8. Each cage contained up to 50 adults with a sex ratio of 50:50. The stink bugs were fed twice a week with fresh organic fruits and peanuts. Sheets of filter paper were placed in the cage as egglaying substrates. Freshly laid egg masses of H. halys (<24 h old) with 27– 28 eggs were used for the experiments. Rarer egg masses with different numbers of eggs were excluded. Trissolcus japonicus and T. mitsukurii adults were obtained from fieldcollected H. halys egg masses and were reared in BugDorm cages (12 × 12 × 12 cm) in climatic chambers at 23°C and L16: D8, and fed with drops of a honeywater solution (70% organic honey solution). Every 3 days, freshly laid egg masses of H. halys (<24 h old) were offered to the parasitoids. The parasitized egg masses were individually transferred to empty vials and stored at 26°C and L16: D8 pending the emergence of the parasitoids. The newly emerged parasitoids of each species were mated (one female and one male) for 1 week in vials (Falcon 50 mL, the lid of which was replaced by a piece of pantyhose fixed with an elastic band) and supplied with drops of the honeywater solution. After the mating period, the females of each species were used in the experiments. Colony fragments consisting in several hundred workers of Crematogaster scutellaris (Olivier, 1792) were collected in the wild from Parma (Italy) and reared in plastic cages under the following conditions: T: 25° ± 1 C, RH: 55 ± 10%, L16: D 8. They were fed with the same honeywater solution used for parasitoids and with Tenebrio molitor Linnaeus larvae. Ants endured a 48 h starvation period prior to the experiments. All tests were conducted in a climatic chamber at 26°C and L16:D8 in the Laboratory of Applied Entomology of the University of Modena and Reggio Emilia. All video recordings were performed using an HCV380 Panasonic camera. A binocular microscope Zeiss Stemi 508 was used to verify if ants and/or parasitoids were alive after the experiments. 14390418, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jen.13179 by CochraneItalia, Wiley Online Library on [23/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 55 | 3 SCHIFANI et al. 2.2 | Experimental procedure We carried out three experiments. Experiment I aimed at verifying whether parasitized eggs and emerging parasitoids can be attacked and damaged by ants. Experiments II and III aimed at evaluating whether ants and adult parasitoids behave aggressively in a simplified context (onetoone interactions in a Petri dish) and a more complex system (a parasitoid couple, a larger number of ants, and a plant) respectively. In the simplified context of Petri dishes, single workers of C. scutellaris retain their basic foraging behaviours, killing and carrying away prey insects (e.g. Giannetti et al., 2022; Schifani, Giannetti, & Grasso, 2023; Schifani, Peri, Giannetti, Alınç, et al., 2023; Schifani, Peri, Giannetti, Colazza, & Grasso, 2023). In all experiments, we counted the number of sting bugs and parasitoids that emerged from the eggs, and the number of surviving parasitoids. 2.3 | Experiment I: Interactions between ants and parasitized eggs or emerging parasitoids To verify whether parasitized eggs attracted the interest of ants, we prepared egg masses in which parasitization of all eggs by T. japonicus or T. mitsukurii was established during preliminary observations. Specifically, after introducing a parasitoid female to each egg mass, its activities were videorecorded and the number of markings was checked. The following behaviours have been observed: probing the host, inserting the ovipositor and performing headpumping movements and body vibrations associated with eggrelease, partially extracting the ovipositor and sweeping it over the surface of the host egg with ‘figure 8’- shaped movements, as described by Field (1998). Each egg mass was transferred in the centre of a Petri dish (⌀ = 9 cm), which was followed by the introduction of a single ant worker. The petri dish was then filmed for 40 min to collect behavioural data, after which the ant was removed. Egg masses were exposed to ants after either 0, 2, 4, 6 or 9 days after parasitization, to test the behaviour of ants towards parasitized eggs at different development stages, or during parasitoid emergence, to test ant behaviour towards emerging adults. Six replicates were performed for each developmental stage of each parasitoid species, both for the treatment (presence of the ant) and for the control (no ant). 2.4 | Experiment II: 1 versus 1 interactions in petri dishes (40 min) Tests were conducted by placing a nonparasitized egg mass in the centre of a Petri dish (⌀ = 9 cm) and introducing a single female parasitoid. As soon as the parasitoid made its first contact with the egg mass, we introduced an ant worker. Once the ant was introduced, we filmed the petri dish for 40 min to collect behavioural data. No ants were introduced into the control replicates, and filming started as soon as the parasitoid made its first contact with the egg mass. At the end of each test, we checked under the microscope whether the ant and the parasitoid were still alive and if any of them had suffered visible injuries. We conducted 10 treatment replicates with ants and 10 control replicates (no ants) for each of the two parasitoid species. 2.5 | Experiment III: Interactions in insect cages (24 h) Tests were conducted using a 30 × 30 × 30 cm insect cage. At the centre of each cage, we placed the following items: (i) a Capsicum annuum L. plant (approximately 15 cm tall); (ii) a Falcon vial containing a female and a male parasitoid of either T. japonicus or T. mitsukurii; (iii) a plastic jar (⌀ = 4 cm, height = 7 cm) containing a group of 50 ant workers, partially filled with small wood pieces, and with the inner upper edge covered with an ant repellent substance (50% glycerine oil, 50% petroleum jelly) to prevent their escape. To start the experiments, we performed the following steps: (i) on an apical leaf of each plant we clipped a 1 × 3 cm filter paper with a single egg mass previously attached with a glue stick; (ii) we placed a 12 cm wooden stick to connect the plant on one hand and the wood pieces in the plastic jar on the other, allowing the ants to get out of the jar and visit the plant; (iii) we opened the lid of the vial, allowing the two parasitoids to move freely inside the cage. Each experimental test lasted 24 h, after which we removed the egg masses and the parasitoids and checked whether the latter were alive or dead. The egg masses were incubated until they hatched, or parasitoids emerged. We conducted 24 replicates per parasitoid species (T. japonicus or T. mitsukurii), equally divided between replicates with ants and control replicates without ants. 2.6 | Behavioural data The behaviour of ants and parasitoids was analysed by videorecording the experiments and analysing the resulting videos with the software Solomon Coder (https://solom on.andra speter.com/). Concerning ants, we recorded the time between their entry into the experimental arena and their first contact with the eggs or parasitoids (contact latency), and the number of times the following six behaviours, directed towards the eggs or the parasitoids as targets, were observed: (i) antennation (making contact with the antennae); (ii) biting with mandibles; (iii) licking; (iv) walking over the female parasitoid; (v) threatening with open mandibles (assuming a motionless posture with open mandibles); (vi) threatening with the stinger by directing it in the direction of the target at close range, as typical of the spatulate stinger of Crematogaster ants; (vii) gaster rising, consisting in an alarm posture typical of Crematogaster ants in which the gaster is raised above in a position perpendicular to the body plane. Concerning parasitoids, we recorded the number of times the following three behaviours were observed: (i) oviposition (including marking), which consists of probing the host, inserting the ovipositor, and making headpumping movements and body vibrations 14390418, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jen.13179 by CochraneItalia, Wiley Online Library on [23/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 56 2 | SCHIFANI et al. Stink bugs (Hemiptera: Pentatomidae) are a large group of insects which includes several agricultural pests, of which ants have proved to be effective predators in some cases (e.g., Kryspin & Todd, 1982; Yang,1984; Van Den Berg et al., 1995; Jones et al., 2001; Hosetti & Rudresh,2012; Castracani et al.,2017; Bulgarini et al.,2021; Kamiyama et al., 2021). The southern green stink bug, Nezara viridula (L.), is a cosmopolitan pest species whose geographic origin may reside in the Mediterranean region and/or the African continent (Jones,1988). This highly polyphagous insect is considered one of the most important pentatomid pests worldwide (Conti et al.,2021). The egg parasitoid Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) is a key natural enemy of N. viridula and it is used in biocontrol programs across the world (Colazza & Bin,1995; Esquivel et al.,2018). Ant activity may interfere with the action of parasitoids under various circumstances: this is mostly observed for those parasitoids that attack antmutualist aphids or coccids (MartinezFerrer et al., 2003; Chen et al., 2014), but parasitoids of insect species that are prey for ants can also be repelled (Appiah et al.,2014). Three tropical ants are known as important predators of N. viridula, attacking both eggs and nymphs: the Asian weaver ant, Oecophylla smaragdina (Fabricius), and two invasive species, the fire ant Solenopsis invicta Buren and the bigheaded ant, Pheidole megacephala (Fabricius) (Krispyn & Todd,1982; Yang,1984; Van Den Berg et al.,1995; Jones et al.,2001). Furthermore, Monomorium minimum (Buckley), Pheidole dentata (Mayr), and Tetramorium guineense (Bernard) are known to be nymph predators (Lockwood & Story,1986). However, it is unknown whether ants from temperate regions can act as predators of N. viridula eggs or nymphs. Moreover, the interactions among ant species, N. viridula, and parasitoids like T. basalis were never investigated. To fill these gaps, we conducted laboratory experiments investigating the interactions between two Mediterranean ant species, eggs and nymphs of N. viridula, and females of the egg parasitoid T. basalis. The two ant species we chose as models are widespread in agroecosystems (Campolo et al.,2015; Giannetti et al.,2021; Bazzato et al.,2022; Schifani et al.,2022): Crematogaster scutellaris (Olivier) and Tapinoma magnum Mayr. The two ants are both very disturbancetolerant species with large colonies, and their workers actively forage on a wide variety of plants, feeding on honeydew and acting as generalist predators (Campolo et al.,2015; Castracani et al.,2017; Seifert,2018; Giannetti et al., 2022). At the same time, they are representatives of very different ant lineages with distinct behavioral and morphological adaptations. Crematogaster scutellaris is an arborealnesting species with monomorphic workers that apply their venom topically with a spatulate stinger, whereas T. magnum is a groundnesting species, characterized by highly polymorphic but on average smaller workers which at short range can spray a toxic secretion produced by their anal glands (Seifert, 2018). We tested whether these ants would increase the mortality of the stink bug nymphs, damage their eggs, or interfere with the parasitoids. MATERIALS AND METHODS Plant and insects rearing Seeds of broad bean plants, Vicia faba L. cv. ‘Aguadulce Supersimonia’ (Fabaceae), were immersed in a slurry of water and soil (1:4 vol/vol) for 24 h, to promote root nodulation. The seeds were then individually seeded in plastic pots (9 × 9 × 13 cm) that were filled with a mixture of agriperlite (Superlite; Gyproc SaintGobain, Milan, Italy), vermiculite (Silver; Gyproc SaintGobain), and sand (1:1:1 vol/vol/vol). The seeds were germinated, and the plants were grown in a climatecontrolled chamber (24 ± 2 °C, 55 ± 10% r.h., L12:D12 h, with light intensity 400 μmol photons m−2 s−1). The plants were watered daily and 1 week after germination they were fertilized with an aqueous solution of fertilizer (1.4 g L−1; NPK = 5– 1545; Plantfol, Valagro, Italy). Plants of 20– 25 cm tall were used for the experiments. Nezara viridula was reared in wooden cages (50 × 30 × 35 cm), ventilated with meshcovered holes (5 cm diameter), in an environmental room (24 ± 1 °C, 70 ± 5% r.h., L16:D8). Stink bugs were fed with a diet of seasonal fresh vegetables and sunflower seeds. Food was changed every 2– 3 days, and separate cages were used for nymphs and adults. Paper towels were placed inside each adult cage as an ovipositional substrate. Egg masses collected daily were used to maintain the stink bug colony, which was from time to time refreshed with fieldcollected bugs. The T. basalis colony was established from wasps emerging from naturally laid N. viridula egg masses collected from cultivated fields and surrounding uncultivated areas near Palermo (Sicily, Italy). Adult parasitoids were reared in 16mL glass tubes (density = 50– 60 wasps per tube), fed with a solution of honey and water, and kept in an incubator at the same environmental conditions described for the stink bugs. Egg masses of N. viridula collected from the colony were exposed to parasitoids for 48 h, then the wasps were removed, and the parasitized eggs were stored for incubation. Crematogaster scutellaris and T. magnum laboratory colonies were established from samples of about 2000 workers from colonies collected near Palermo. Both colonies were temporarily reared in plastic cages (75 × 35 × 20 cm) for the duration of the experiment under the same environmental conditions described for the stink bugs. They were fed with a honey– water solution. Ants used during each experimental trial were not reused in following tests. Experimental setup Experiments were conducted under controlled conditions of 24 ± 1 °C, 66 ± 1.4% r.h., and L16:D8 photoperiod, 15707458, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/eea.13357 by Papau New Guinea HINARI REGIONAL, Wiley Online Library on [10/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 63 | 3 MEDITERRANEAN ANTS, NEZARA VIRIDULA, AND TRISSOLCUS BASALIS using 30 × 30 cm insect cages. In the center of each cage, we placed a V. faba plant with its plastic pot. On one corner of the cage, we placed a plastic cup (8 cm high, 6.5 cm diameter) filled for 1/3 with broken twig pieces and, at its upper internal margins, covered for 1 cm with an antrepelling substance (50% glycerin oil, 50% petroleum jelly) to prevent ants from escaping. In each plastic cup, we also inserted a 10cmlong wooden stick, whose upper end was leaning on the plant. The plastic cup was meant to offer a shelter to the ants during the experiments, allowing them to move towards the plant along the wooden stick, without dispersing in all other directions thanks to the repellent substance. After each experiment, the insect cages and plastic cups were washed with clean water, and the plants, the content of the plastic cups, and the wooden sticks were replaced with new ones. We conducted three tests for each experiment, running 15 replicates per test: one with C. scutellaris, one with T. magnum, and a control test with no ants. Experiment 1: effects of ant activity on stink bug nymph mortality On an apical leaf of each plant, we placed with a clip a 1 × 3 cm filter paper with an artificially made egg mass of 15 eggs attached with a glue stick (Pritt; Henkel, Hemel Hempstead, UK). We then waited for 6– 10 h after the eggs hatched and counted the N. viridula nymphs (mean ± SD = 12.4 ± 2.8). The experiment then started with the introduction of 50 ants in each plastic cup, and lasted 24 h, during which the cage was kept closed. At the end, we collected the stink bug nymphs, checking how many of them were still alive. Experiment 2: effects of ant activity on stink bug eggs and parasitoid oviposition and survival We first introduced on each plant three N. viridula virgin females for 24 h to allow them to walk over the plant to contaminate it with chemical footprints that are relevant cues exploited by T. basalis in its searching behavior (Colazza et al.,1999). We used a glue stick to attach N. viridula egg masses (50– 100 eggs each) on 1 × 3 cm filter papers and used a clip to put one of them on the apical leaf of each plant. To start the experiment, we introduced 50 ants into each plastic cup, and three T. basalis females (24– 48 h old). Each experiment lasted 24 h, during which the cages were kept closed, after which we removed the egg masses and the parasitoids and checked whether the latter were alive or dead. The egg masses were incubated until the eggs hatched, or parasitoids emerged. We discriminated between eggs from which parasitoids emerged and the rest from which stink bugs emerged or that did not hatch. Statistical analysis The normality of the data and equality of their variance between treatments were tested by means of Shapiro– Wilk and Levene's tests, respectively. To evaluate the differences between treatments concerning stink bug nymph mortality and the egg parasitization rate, we relied on KruskalWallis tests followed by Dunn's post hoc tests for pairwise comparisons if significant differences between treatments had been detected. As no more than one parasitoid died in each trial, data on parasitoid mortality were binomial and differences between treatments were analyzed using a generalized linear model (GLM). All statistical analyses were conducted with the software R v.4.2.0 and RStudio2022.02.2– 485 (R Core Team,2022). RESULTS Experiment 1: effects of ant activity on stink bug nymph mortality Stink bug mortality rate differed between treatments (H = 12.24, d.f. = 2, P = 0.002). In particular, the treatment with no ants was different from the other two (no ants vs. C. scutellaris: P = 0.023; no ants vs. T. magnum: P = 0.003), whereas there were no differences between the treatment with C. scutellaris vs. T. magnum (P = 0.49). Stink bug mortality was 0 ± 7% in the treatment with no ants, and 17 ± 32% in the treatments with ants (median ± interquartile range; Figure1AC). Experiment 2: effects of ant activity on stink bug eggs and parasitoid oviposition and survival Stink bug eggs were not removed by ants and their shell was not damaged after the experiments in which they were exposed to ants. There was no effect of treatment on the parasitization rate and the consequent number of stink bugs that hatched (H = 0.27, d.f. = 2, P = 0.87) (Figure1D, E). Furthermore, there was also no treatment effect on the number of dead parasitoids (0.24 < P < 0.70). DISCUSSION Our results show that native Mediterranean ants may act as antagonists of N. viridula nymphs, significantly increasing their mortality. Furthermore, we found no evidence of interference between ants and the parasitization activity of T. basalis on N. viridula eggs. As these ants do not appear to prey upon stink bug eggs, they may be even less likely to interact with parasitoids directly. Although we cannot rule out that increasing the number of ants per plant even more would eventually have posed some problems to the 15707458, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/eea.13357 by Papau New Guinea HINARI REGIONAL, Wiley Online Library on [10/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 64 4 | SCHIFANI et al. parasitoids, and perhaps further increase stink bug mortality, we would not expect such high concentrations to occur under natural conditions. The fact that we did not record egg predation by the ants, unlike what was observed for O. smaragdina (Yang,1984; Hosetti & Rudresh,2012), may be due to the relatively smaller size of the ant species we used for the experiments. Crematogaster scutellaris, as well as the native European ant Lasius niger (L.), are similarly unable to attack the eggs of the brown marmorated stink bug, Halyomorpha halys Stål, whereas they do prey upon its nymphs (Castracani et al., 2017; Bulgarini et al., 2022), highlighting a similar pattern among Mediterranean ants interacting with various stink bug species. Larger Mediterranean ant species may be able to prey upon N. viridula eggs, but they are seemingly less frequent in agroecosystems than eggs of species such as C. scutellaris and T. magnum (Mansour et al., 2012; Campolo et al., 2015; Giannetti et al., 2021; Bazzato et al., 2022; Schifani et al.,2022). On the other hand, ants may increase nymphal mortality through different mechanisms, which require further investigation. In addition to suffering from direct attacks (Castracani et al.,2017), disturbance may prompt young nymphs to break aggregations, which leads to a higher risk of death by desiccation and may even expose them to other predators (Lockwood & Story,1986). Ants are often reported to either displace or favor parasitoids, often due to specialized myrmecophilic adaptations by either the hosts or the parasitoids themselves (Pierce & Mead,1981; Völkl,1992), whereas in our experiments we witnessed a substantial neutrality between the two actors. The lack of direct interference with T. basalis and the lack of interest for N. viridula eggs by C. scutellaris and T. magnum opens the possibility of using these ants as complementary tools along with T. basalis in the control programs against N. viridula. In this perspective, it is notable that the activity of both ants and T. basalis can be enhanced or manipulated by using natural and artificial nectars, favoring their presence on target plants, increasing their survivability, and, in the case of ants, even distracting them from tending coccids or aphids (Offenberg,2001; Rahat et al.,2005; Schifani et al.,2020). Habitat characteristics may significantly contribute to determining whether ants or parasitoids play a more significant role in controlling N. viridula and other stink bugs (Wright & Diez,2011). Crematogaster scutellaris and T. magnum are already known to play an interesting role in the control of other insect pests, including brown marmorated stink bug, H. halys, but also horsechestnut leaf miner, Cameraria FIGURE 1 Interactions between the ants (A) Crematogaster scutellaris and (B) Tapinoma magnum, the stink bug Nezara viridula, and its egg parasitoid Trissolcus basalis. Mortality (%) of (C) stink bug nymphs and (D) the parasitoids. (E) Parasitization rate (%) of stink bug eggs. Asterisks indicate significant treatment effects (KruskalWallis tests followed by Dunn posthoc tests for pairwise comparisons: P < 0.001; ns, P > 0.05). Note that the ants in A and B are attacking stink bug nymphs. 15707458, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/eea.13357 by Papau New Guinea HINARI REGIONAL, Wiley Online Library on [10/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 65 | 5 MEDITERRANEAN ANTS, NEZARA VIRIDULA, AND TRISSOLCUS BASALIS ohridella Deschka & Dimić, Mediterranean fruit fly, Ceratitis capitata (Wiedemann), codling moth, Cydia pomonella (L.), and the ambrosia beetle Xylosandrus compactus (Eichhoff) (Radeghieri, 2004; Campolo et al., 2015; Castracani et al., 2017; Giannetti et al., 2022; Schifani et al., 2023). Considering the significant behavioral, morphological, and phylogenetic differences between the ant species known to prey upon N. viridula nymphs so far, we expect that several other ants may play a similar role (Offenberg,2015). However, even superficially similar species may still differ markedly in their attitude towards both stink bugs and their parasitoids (Chen et al.,2014). Further assessments are required to quantify the predatory role of Mediterranean ants on stink bug nymphs in agricultural fields, where generalist species such as C. scutellaris and T. magnum are expected to cooccur frequently with N. viridula. AUTHOR CONTRIBUTIONS Enrico Schifani: Conceptualization (equal); data curation (lead); formal analysis (lead); investigation (lead); methodology (equal); resources (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Ezio Peri: Conceptualization (equal); funding acquisition (equal); methodology (equal); project administration (equal); resources (equal); supervision (equal); validation (equal); writing – review and editing (lead). Daniele Giannetti: Conceptualization (equal); methodology (equal); resources (supporting); writing – review and editing (supporting). Tuğcan Alınç: Investigation (supporting); resources (lead); writing – review and editing (supporting). Stefano Colazza: Conceptualization (equal); funding acquisition (equal); methodology (equal); project administration (equal); resources (equal); supervision (equal); validation (equal); writing – review and editing (lead). Donato Antonio Grasso: Conceptualization (lead); funding acquisition (equal); methodology (equal); project administration (lead); supervision (lead); validation (equal); writing – review and editing (supporting). ACKNOWLEDGMENTS We thank Rihem Moujahed (University of Palermo) for assisting us with plant and insect rearing. FUNDING INFORMATION The work benefited from the equipment and framework of the COMPHUB Initiative, funded by the ‘Departments of Excellence’ program of the Italian Ministry for Education, University and Research (MIUR, 2018– 2022). DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Enrico Schifani https://orcid.org/0000-0003-0684-6229 Ezio Peri https://orcid.org/0000-0002-1095-7258 Daniele Giannetti https://orcid.org/0000-0003-1622-1329 Tuğcan Alınç https://orcid.org/0000-0002-1146-2863 Stefano Colazza https://orcid.org/0000-0001-8023-7814 Donato A. Grasso https://orcid.org/0000-0001-9334-4280 REFERENCES Adandonon A, Vayssieres JF, Sinzogan A & Van Mele P (2009) Density of pheromone sources of the weaver ant Oecophylla longinoda affects oviposition behaviour and damage by mango fruit flies (Diptera: Tephritidae). International Journal of Pest Management 55: 285– 292. Appiah EF, Ekesi S, AfrehNuamah K, ObengOfori D & Mohamed SA (2014) African weaver antproduced semiochemicals impact on foraging behaviour and parasitism by the opiine parasitoid, Fopius arisanus on Bactrocera invadens (Diptera: Tephritidae). Biological Control 79: 49– 57. Baraibar B, Carrión E, Recasens J & Westerman PR (2011) Unravelling the process of weed seed predation: developing options for better weed control. Biological Control 56: 85– 90. Bazzato E, Lallai E, Caria M, Schifani E, Cillo D et al. (2022) Landuse intensification reduces multitaxa diversity patterns of small woodlots outside forests in a Mediterranean area. Agriculture, Ecosystems and Environment 340: 108149. Bulgarini G, Castracani C, Mori A, Grasso DA & Maistrello L (2021) Searching for new predators of the invasive Halyomorpha halys: the role of the black garden ant Lasius niger. Entomologia Experimentalis et Applicata 169: 799– 806. Campolo O, Palmeri V, Malacrinò A, Laudani F, Castracani C et al. (2015) Interaction between ants and the Mediterranean fruit fly: new insights for biological control. Biological Control 90: 120– 127. Castracani C, Bulgarini G, Giannetti D, Spotti FA, Maistrello L et al. (2017) Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys. Journal of Pest Science 90: 1181– 1190. Chen Y, Lu Z, Li Q, Hoffmann BD & Zhang W (2014) Multiple ant species tending lac insect Kerria yunnanensis (Hemiptera: Kerriidae) provide asymmetric protection against parasitoids. PLoS One 9: e98975. Colazza S & Bin F (1995) Efficiency of Trissolcus basalis (Hymenoptera: Scelionidae) as an egg parasitoid of Nezara viridula (Heteroptera: Pentatomidae) in central Italy. Environmental Entomology 24: 1703– 1707. Colazza S, Salerno G & Wajnberg E (1999) Volatile and contact chemicals released by Nezara viridula (Heteroptera: Pentatomidae) have a kairomonal effect on the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae). Biological Control 16: 310– 317. Conti E, Avila G, Barratt B, Cingolani F, Colazza S et al. (2021) Biological control of invasive stink bugs: review of global state and future prospects. Entomologia Experimentalis et Applicata 169: 28– 51. Dejean A, Grangier J, Leroy C & Orivel J (2009) Predation and aggressiveness in host plant protection: a generalization using ants from the genus Azteca. Naturwissenschaften 96: 57– 63. Esquivel JF, Musolin DL, Jones WA, Rabitsch W, Greene JK et al. (2018) Nezara viridula (L.). In: Invasive Stink Bugs and Related Species (Pentatomoidea) – Biology, Higher Systematics, Semiochemistry and Management (ed. by JE McPherson), pp. 1– 74. CRC Press, Boca Raton, FL, USA. Giannetti D, Schifani E, Castracani C, Ghizzoni M, Delaiti M et al. (2021) Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley. Redia 104: 97– 109. Giannetti D, Schifani E, Gugliuzzo A, Zappalà L, Biondi A & Grasso DA (2022) Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus. Biological Control 174: 105032. Grasso DA, Mori A & Le Moli F (1998) Chemical communication during foraging in the harvesting ant Messor capitatus (Hymenoptera, Formicidae). Insectes Sociaux 45: 85– 96. 15707458, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/eea.13357 by Papau New Guinea HINARI REGIONAL, Wiley Online Library on [10/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 66 6 | SCHIFANI et al. Grasso DA, Mori A & Le Moli F (1999) Recruitment and trail communication in two species of Messor ants (Hymenoptera, Formicidae). Italian Journal of Zoology 66: 373– 378. Grasso DA, Sledge MF, Le Moli F, Mori A & Turillazzi S (2005) Nestarea marking with faeces: a chemical signature that allows colonylevel recognition in seed harvesting ants (Hymenoptera, Formicidae). Insectes Sociaux 52: 36– 44. Hölldobler B & Wilson EO (1990) The Ants. Harvard University Press, Cambridge, MA, USA. Hosetti BB & Rudresh BS (2012) Studies on Oecophylla smaragdina as a biocontrol agent against pentatomid bug infesting on Pongamia tree. Journal of Environmental Biology 33: 1103. Jones WA (1988) World review of the parasitoids of the southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae). Annals of the Entomological Society of America 81: 262– 273. Jones VP, Westcott DM, Finson NN & Nishimoto RK (2001) Relationship between community structure and southern green stink bug (Heteroptera: Pentatomidae) damage in macadamia nuts. Environmental Entomology 30: 1028– 1035. Kamiyama MT, Matsuura K, Yoshimura T & Yang CCS (2021) Predation of the brown marmorated stink bug, Halyomorpha halys by the Japanese acrobat ants, Crematogaster matsumurai and Crematogaster osakensis. Biological Control 157: 104570. Katayama N & Suzuki N (2005) The importance of the encounter rate between ants and herbivores and of ant aggressiveness against herbivores in herbivore exclusion by ants on Vicia angustifolia L. (Leguminosae) with extrafloral nectaries. Applied Entomology and Zoology 40: 69– 76. Krispyn JW & Todd JW (1982) The red imported fire ant as a predator of the southern green stink bug on soybean in Georgia. Journal of the Georgia Entomological Society 17: 19– 26. Lach L, Parr C & Abbott K (2010) Ant Ecology. Oxford University Press, Oxford, UK. Le Moli F, Grasso DA, Mori A & Ugolini A (1994) Ecoethological factors affecting the scouting and raiding behaviour of the slavemaking ant, Polyergus rufescens Latr. (Hymenoptera, Formicidae). Ethology 96: 289– 302. Lockwood JA & Story RN (1986) Adaptive functions of nymphal aggregation in the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). Environmental Entomology 15: 739– 749. Mansour R, Suma P, Mazzeo G, La Pergola A, Pappalardo V et al. (2012) Interactions between the ant Tapinoma nigerrimum (Hymenoptera: Formicidae) and the main natural enemies of the vine and citrus mealybugs (Hemiptera: Pseudococcidae). Biocontrol Science and Technology 22: 527– 537. MartinezFerrer MT, GraftonCardwell EE & Shorey HH (2003) Disruption of parasitism of the California red scale (Homoptera: Diaspididae) by three ant species (Hymenoptera: Formicidae). Biological Control 26: 279– 286. Offenberg J (2001) Balancing between mutualism and exploitation: the symbiotic interaction between Lasius ants and aphids. Behavioral Ecology and Sociobiology 49: 304– 310. Offenberg J (2015) Ants as tools in sustainable agriculture. Journal of Applied Ecology 52: 1197– 1205. Offenberg J & Damgaard C (2019) Ants suppressing plant pathogens: a review. Oikos 128: 1691– 1703. Parker J & Kronauer DJ (2021) How ants shape biodiversity. Current Biology 31: R1208R1214. Pierce NE & Mead PS (1981) Parasitoids as selective agents in the symbiosis between lycaenid butterfly larvae and ants. Science 211: 1185– 1187. R Core Team (2022) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Radeghieri P (2004) Cameraria ohridella (Lepidoptera Gracillariidae) predation by Crematogaster scutellaris (Hymenoptera Formicidae) in Northern Italy (Preliminary note). Bulletin of Insectology 57: 63– 64. Rahat S, Gurr GM, Wratten SD, Mo J & Neeson R (2005) Effect of plant nectars on adult longevity of the stinkbug parasitoid, Trissolcus basalis. International Journal of Pest Management 51: 321– 324. RicoGrey V & Oliveira PS (2007) The Ecology and Evolution of AntPlant Interactions. University of Chicago Press, Chicago, IL, USA. Schifani E, Castracani C, Giannetti D, Spotti FA, Reggiani R et al. (2020) New tools for conservation biological control: testing antattracting artificial nectaries to employ ants as plant defenders. Insects 11: 129. Schifani E, Giannetti D, Castracani C, Spotti FA, Mori A & Grasso DA (2022) Trunk size influences species richness and functional composition of biogeographically different treevisiting ant communities in pear orchards. Redia 105: 163– 168. Schifani E, Giannetti D & Grasso DA (2023) Predatory abilities of two Mediterranean ants on the eggs and larvae of the codling moth Cydia pomonella. Insects 14: 97. Seifert B (2018) The Ants of Northern and Central Europe. Lutra Verlagsund Vertriebsgesellschaft, Boxberg, Germany. Swanson AC, Schwendenmann L, Allen MF, Aronson EL, ArtaviaLeón A et al. (2019) Welcome to the Atta world: a framework for understanding the effects of leafcutter ants on ecosystem functions. Functional Ecology 33: 1386– 1399. Taylor AR, Lenoir L, Vegerfors B & Persson T (2019) Ant and earthworm bioturbation in coldtemperate ecosystems. Ecosystems 22: 981– 994. Van Den Berg H, Bagus A, Hassan K, Muhammad A & Zega S (1995) Predation and parasitism on eggs of two podsucking bugs, Nezara viridula and Piezodorus hybneri in soybean. International Journal of Pest Management 41: 134– 142. Van Mele P, Vayssieres JF, Adandonon A & Sinzogan A (2009) Ant cues affect the oviposition behaviour of fruit flies (Diptera: Tephritidae) in Africa. Physiological Entomology 34: 256– 261. Völkl W (1992) Aphids or their parasitoids: who actually benefits from antattendance? Journal of Animal Ecology 61: 273– 281. Way MJ (1963) Mutualism between ants and honeydewproducing Homoptera. Annual Review of Entomology 8: 307– 344. Way MJ & Khoo KC (1992) Role of ants in pest management. Annual Review of Entomology 37: 479– 503. Wright MG & Diez JM (2011) Egg parasitism by Trissolcus basalis (Hymenoptera: Scelionidae) in architecturally varied habitats, and observations on parasitism in macadamia nut orchards and other habitats following augmentative release. Proceedings of the Hawaiian Entomological Society 43: 23– 31. Yang P (1984) The application of Oecophylla smaragdina Fabr. in South Fujian. Fujian Agricultural Science and Technology 5: 23– 25. How to cite this article: Schifani E, Peri E, Giannetti D, Alınç T, Colazza S & Grasso DA (2023) Mediterranean ants can increase nymph mortality in the stink bug Nezara viridula without interfering with its egg parasitoid Trissolcus basalis. Entomologia Experimentalis et Applicata 00: 1–6. https://doi. org/10.1111/eea.13357 15707458, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/eea.13357 by Papau New Guinea HINARI REGIONAL, Wiley Online Library on [10/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 67 3. SECTION II: ANTS AS MUTUALIST PARTNERS OF INSECT PESTS 68 This second section is composed of three chapters. Chapter 6 illustrated an ecological experiment carried out in Sicily, in which ant-exclusion through the application of sticky barriers was used on fava bean plants to study the impact of the local ant community on mutualist and non-mutualist aphid species attacking the plants, and the relationship between the presence of these ants and the presence of aphid natural enemies (parasitoids and predators). According to the results, the three ant species visiting the plants (one exotic and two natives) had no role in deterring aphid parasitoids and predators of the mutualist Aphis fabae Scopoli, 1763. Aphids were still more abundant in plants without sticky barriers, but it was unclear whether this was caused by ants or by sticky barriers themselves. In any case, while other ant species are known to protect A. fabae from parasitoids and predators, our experiment demonstrates how the identity of the ant species involved matters, and the associations between attending ants and aphids does not automatically imply protection from aphid natural enemies. Chapter 7 digs deeper into the topic, this time using as a model system the association between the walnut aphid Panaphis juglandis (Goeze, 1778), an ant-mutualist, and different ant species that attend it. We carried out field experiments aimed at comparing the behavioral responses of different ants associated with P. juglandis to the approach of different ladybeetle species. We detected interesting patterns of behaviors, again emphasizing strong differences between different ants in their behavioral responses and effectiveness at protecting their aphid partners from the ladybeetles, with a gradient from very strong protection to no protection at all. Finally, in Chapter 8 we expand on a review of management tactics for ant-hemipteran associations that can be employed whenever these associations are demonstrated to produce significant economic damage to cultivated plants. Four main tactics are identified, some of which can be integrated with one another, and their pros and cons are evaluated: i) poisoning ant colonies with low-toxicity baits; ii) preventing ant access to the plants using physical or chemical barriers; iii) diverting ant attention from mutualist hemipterans by offering alternative food sources; iv) utilizing 69 parasitoids or predators that are capable of circumventing the defensive abilities of the locally prevailing ant species. Future research directions are also commented upon. 70 Section II - Ants as mutualist partners of insect pests Chapter 6 Ant attendance does not necessarily imply protection of aphids from their arthropod natural enemies _______________________________________________________________________________ Schifani, E., Peri, E., Giannetti, D., Colazza, S., & Grasso, D.A. (2023). Ant attendance does not necessarily imply protection of aphids from their arthropod natural enemies. Ecological Entomology, 48(3), 384-388. https://doi.org/10.1111/een.13226 71 SHORT COMMUNICATION Ant attendance does not necessarily imply protection of aphids from their arthropod natural enemies Enrico Schifani 1 | Ezio Peri 2 | Daniele Giannetti 1 | Stefano Colazza 2 | Donato A. Grasso 1 1 Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy 2 Department of Agricultural, Food and Forest Sciences, University of Palermo, Palermo, Italy Correspondence Enrico Schifani, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy. Email: [email protected] Funding information Italian Ministry for Education, University and Research Associate Editor: Simon Hodge Abstract 1. Many ants and hemipterans are bound by a mutualistic relationship (trophobiosis) which represents an ecological keystone: ants receive food (honeydew) providing different kinds of protection. Partner protection against arthropod natural enemies is considered to be frequent and to interfere with biological control strategies of hemipteran pests. 2. We carried out an ant-exclusion field experiment in a fava bean (Vicia faba) plantation in Italy to monitor the abundance and behaviour of the ants (Plagiolepis pygmaea,Tetramorium semilaeve and the exotic Nylanderia jaegerskioeldi) and their relationship with facultative mutualist (Aphis fabae) and non-mutualist (Megoura viciae) aphids, the arthropod natural enemies of the aphids, and extrafloral nectaries. 3. Ants concentrated their activity on the attendance of facultative mutualist aphids much more than on the extrafloral nectaries. The ant-exclusion treatment had no effect on the abundance of M. viciae and on the parasitization rate of A. fabae, while it reduced the abundance of A. fabae, aphid predators, and aphid parasitoids. 4. Our results demonstrate that ant attendance does not imply the protection of aphids from arthropod natural enemies and suggest that the identity of the ant species involved is important for the outcome. As relatively few species have been studied in this regard, extending our knowledge to the role of more ant species is desirable to understand the ecology and evolution of ant-aphid mutualisms and to refine integrated control strategies of aphid pests. KEYWORDS ant-aphid mutualism, aphid parasitoids, aphid predators, biological control, extrafloral nectaries, honeydew, integrated pest management, trophobiosis INTRODUCTION Ants play an important ecological role across terrestrial ecosystems, in both natural and human-managed environments (Hölldobler & Wilson, 1990; Parker & Kronauer, 2021). Trophobiotic mutualism between ants and hemipterans (mostly aphids or coccids) ranges from facultative to obligate and represents a widespread ecological keystone: ants provide their partners with different kinds of protection in exchange for honeydew (Delabie, 2001; Depa, Kaszyca-Taszakowska, Taszakowski, & Kanturski, 2020; Hölldobler & Wilson, 1990; Parker & Kronauer, 2021). Ants may protect mutualist hemipterans from pathogens (Nielsen, Agrawal, & Hajek, 2010), offer them shelter from Received: 2 November 2022 Accepted: 21 December 2022 DOI: 10.1111/een.13226 Ecological Entomology. 2023;1–5. wileyonlinelibrary.com/journal/een © 2023 Royal Entomological Society. 1 72 phytophagous insects (Styrsky and Eubanks, 2007). Some efficient predatory ants may also be strongly protective of their hemipteran partners (Styrsky and Eubanks, 2007; Offenberg et al., 2019), but little is still known about this behaviour for most European ants that may act as pest predators (Campolo et al., 2015; Schifani et al., 2020). Lady beetles are key predators of ant-mutualist hemipterans in natural environments and are frequently employed as biological control agents in agroecosystems (Obrycki and Kring, 1998). We carried out a field experiment to study the behavioural interactions between five European ant species associated with the walnut aphid Panaphis juglandis and two lady beetle species. These lady beetles are important predators and biological control agents of the walnut aphid (Kök et al., 2018; Czechowski et al., 2019; Gao et al., 2020): the smaller and native Adalia bipuntata and the larger allochthonous Asian species Harmonia axyridis (Brown et al., 2007;Liet al., 2021). Our experiment aimed at describing the behaviours of the associated ant species and comparing their differences in relationship with the protective ability they were able to grant to the aphids against the two lady beetle species. Materials and methods Data collection was carried out at the Parma University Campus, Italy (44.7684, 10.3140), from 6 to 17 June 2022 (and daily from 11:00 to 15:00), when P. juglandis was abundant. A total of 12 walnut trees (Juglans regia L.) colonised by P. juglandis were selected based on the presence of five different ant species associated: Camponotus piceus (Leach, 1825), Ca. vagus (Scopoli, 1763), Crematogaster scutellaris (Olivier, 1792), Dolichoderus quadripunctatus (Linnaeus, 1771) and Lasius emarginatus (Olivier, 1792). The identity of these ants was ascertained before the beginning of the experiment by sampling two workers per each species and each tree, identifying them using a ZEISS Stemi 508 stereoscopic microscope and the keys provided by Seifert (2018). Adults of H. axyridis were collected in the field, while A. bipunctata adults were acquired from Bioplanet (Italy). Both species were temporarily reared in plastic cages under laboratory conditions (T: 25 ± 1°C, R.H.: 55 ± 10%) and fed with field-collected P. juglandis aphids. In each experimental trial, we carefully introduced one adult lady beetle of either species to the dorsal surface of a leaf containing at least 15 P. juglandis aphids attended by ant workers. The introduction of the lady beetle was conducted slowly to avoid causing any reaction in the ants or aphids by creating vibrations on the plant –experimental trials in which the introduction itself accidentally caused visible vibrations and reactions by aphids or ants were aborted and another leaf was taken. In all cases, only one ant species was present on each leaf, and the leaves were located at a height of about 1.5 m from the ground. Once the lady beetle was introduced, we filmed the insects on the leaf until at least one of the following events occurred: (i) the lady beetle left the dorsal surface of the leaf; (ii) all aphids were either killed by the lady beetle or left the dorsal surface of the leaf; (iii) five minutes have passed since the introduction of the ladybug. Videos were analysed with the software Solomon Coder (solomon.andraspeter.com) to collect data on the interactions of ants, lady beetles and aphids. We recorded three parameters as continuous variables: ➢Ant workers on the leaf. The number of ant workers per leaf at the beginning of each experimental trial. ➢Lady beetle on the leaf (time). The cumulative time (seconds) during which the lady beetle stayed on the leaf since the first encounter with ants. ➢Lady beetle with the aphids (time). The cumulative time (seconds) during which the lady beetle stayed in proximity (<3 mm) of the aphids. Moreover, ten behaviours were recorded as binary variables (presence/absence): ➢Lady beetle biting aphids. The lady beetle bites and/or eats at least one of the aphids. ➢Aphids flee from the leaf. At least one of the aphids stops feeding and walks away. In all our observations, this behaviour was always involving multiple aphids simultaneously. ➢Ants flee from the leaf. At least one of the ants abandons the leaf by walking away to other parts of the plant. ➢Ants opening mandibles (threat). At least one of the ants opens its mandibles towards the ladybug, without a successful bite following; genuine but unsuccessful biting attempts are not distinguished from bite threats. ➢Ants biting the ladybug. At least one of the ants successfully bites the ladybug, grasping any part of its body or appendages. ➢Ants bending their gaster. At least one of the ants directs its gaster towards the lady beetle at a short distance. This behaviour corresponds to threatening the release or releasing toxic chemicals against an enemy, the two being normally indistinguishable in the field. There are slight differences in the movements performed by the examined species: in Camponotus spp. and L. emarginatus, the gaster passes under the body and the head; in Cr. scutellaris the gaster may be directed at almost any possible angle and the spatulate stinger connects with the target to release its venom topically; in D. quadripunctatus, the gaster is oriented laterally, diverging from the body axis. ➢Ants chasing the ladybug. While the lady beetle is far from the aphids (>5 mm distance), at least one of the ants stops attending to the aphid colony to threaten or attack the lady beetle with one of the abovementioned behaviours (ants open mandibles (threat), ants biting the ladybug or ants bending their gaster). ➢Ants falling off the leaf. At least one ant falls off the leaf after approaching the ladybug. Ants were never observed falling off the leaf under any other circumstances. ➢Ants grooming. At least one ant cleans its mouthparts after biting the ladybug. ➢Ants rescuing aphids. After the lady beetle grabs an aphid with its mouth (see above lady beetle biting aphids), at least one ant starts pulling the aphid in the opposite direction. We recorded whether the aphid was freed or not from the lady beetle because of this behaviour. We conducted 12 experimental trials for each combination of ant and lady beetle species for a total of 120 trials (12 replicates × 2 lady beetle species × 5 ant species). Data were analysed using the software R and RStudio (R Core Team, 2022; RStudio Team, 2022). We preliminarily explored the data through generalised linear models considering the role of the lady beetle species (factor with two levels), the ant species (factor with five levels) and their interaction on each of the continuous or binomial variables recorded. The interaction term was never statistically significant, 2 Enrico Schifani et al. https://doi.org/10.1017/S0007485323000500 Published online by Cambridge University Press 79 which led us to analyse the role of the ant or lady beetle species through separate statistical tests. For continuous variables (ant workers on the leaf, lady beetle on the leaf and lady beetle with the aphids), we analysed differences between ant or lady beetle species using Kruskal–Wallis tests, which were followed by Conover’s tests of multiple comparisons from the PMCMRplus R package whenever significant differences were detected. The Bonferroni correction was applied to Conover multiple comparison tests. Binomial variables (lady beetle biting aphids, aphids flee from the leaf, ants flee from the leaf, ants opening mandibles (threat), ants biting the ladybug, ants bending their gaster, ants chasing the ladybug, ants falling off the leaf, ants grooming, ants rescuing aphids) were analysed by using χ 2 tests, which were followed by the analysis of standardised residuals if significant differences were detected. Results Concerning the continuous variables, there were no statistical differences between experiments conducted with the two lady beetle species (ant workers per leaf: H (1) = 0.06, P= 0.80; lady beetle on the leaf (time): H (1) = 3.13, P= 0.07; lady beetle near aphids (time): H (1) = 1.33, P= 0.25), while differences between ant species were always significant (ant workers per leaf: H (4) = 101.25, P< 0.001; lady beetle on the leaf (time): H (4) = 17.80, P= 0.001; lady beetle near aphids (time): H (4) = 25.50, P<0.001). Regarding ant workers per leaf, pairwise comparisons revealed that all ant species differed significantly from each other (0.014 <P< 0.001), except for Ca. piceus and Ca. vagus which shared the lowest numbers of workers (P= 0.998), while D. quadripunctatus had the highest one (fig. 1A). The lady beetle on the leaf Figure 1. Behavioural data collected during the experiments, divided according to the identity of the ant species involved in the trials. Concerning continuous numerical data (A–C), groups marked with the same lowercase letter were not significantly different according to pairwise comparisons. In the case of binomial data (D–L), groups significantly different than the expected are highlighted using asterisks according to the significance level (*P≤0.05; **P≤0.005; ***P≤0.001). Bulletin of Entomological Research 3 https://doi.org/10.1017/S0007485323000500 Published online by Cambridge University Press 80 (time) was significantly different between D. quadripunctatus and Ca. piceus (P= 0.021), D. quadripunctatus and Cr. scutellaris (P= 0.05) and between Cr. scutellaris and L. emarginatus (P= 0.04) (fig. 1B). The lady beetle near aphids (time) differed between Ca. piceus and D. quadripunctatus (P< 0.001) and Ca. piceus and L. emarginatus (P= 0.005) (fig. 1C). For any of the other recorded binary variables, there were no statistically significant differences between experiments conducted with the two lady beetle species, while all of them differed significantly between trials conducted with different ant species except for the ants opening mandibles (threat) behaviour (table 1). The analysis of standardised residuals of χ 2 tests revealed that lady beetle biting aphids, aphids flee from the leaf and ants flee from the leaf behaviours were all observed more frequently than the expected with Ca. piceus (P< 0.001, fig. 1D–F). The frequency of ants bending their gaster was lower than expected in D. quadripunctatus (P= 0.002, fig. 1I). Ants chasing the lady beetle were observed less frequently than expected in Ca. piceus, and more frequently than the expected in D. quadripunctatus and L. emarginatus (P< 0.001; fig. 1J). Finally, ants grooming was observed more frequently than expected in Ca. vagus (P< 0.001; fig. 1L). The ants rescuing aphids behaviour was the only one not to be statistically analysed as it was exhibited only twice (fig. 2). In both cases, this behaviour was performed by Cr. scutellaris, once interacting with A. bipunctata, and once with H. axyridis. In the first case, the ant successfully managed to free the aphid from the ladybug, while in the second case, both the lady beetle and the ant kept pulling the aphid in opposite directions beyond the time duration of the video. Discussion The high diversity of interactions between ants, their hemipteran partners and the arthropod natural enemies of the hemipterans is still undocumented in most cases, but of high interest in evolutionary terms as well as in an applied perspective for pest management and biological control (Oliver et al., 2008; Depa et al., 2020; Parker and Kronauer, 2021; Castracani et al., 2023; Schifani et al., 2023a). Our results highlight how a large proportion of this variation may depend on the identity of the ant species (Völkl et al., 1996; Schifani et al., 2023a). On one hand, D. quadripunctatus and L. emarginatus were the most effective species in the protection of aphids. Workers of D. quadripunctatus were on average more numerous than those of L. emarginatus and used fewer chemical attacks, but the behaviour of both species similarly caused the aphids to stay safe. Both aggressively attacked the lady beetles even if they were not close to the aphids, causing them to abandon the leaves rapidly. These results suggest that D. quadripunctatus can be a pugnacious species (Schifani et al., 2022), dismissing the idea that it does not defend the associated P. juglandis colonies from lady beetles as stated by Czechowski et al.(2019). On the other hand, the attacks of Ca. piceus were mostly ineffective at repelling the lady beetles, and its workers (which were Table 1. Results of χ 2 analyses of differences between lady beetles and among ant species for nine binomial behavioural variables recorded in this study Variable Between lady beetle species Among ant species Lady beetle biting aphids χ 2 1 = 0.54, P= 0.46 χ 2 4 = 29.40, P<0.001 Aphids flee from the leaf χ 2 1 = 0.48, P= 0.48 χ 2 4 = 22.41, P<0.001 Ants flee from the leaf χ 2 1 = 0.73, P= 0.39 χ 2 4 = 28.60, P<0.001 Ants opening mandibles (threat) χ 2 1 = 0.26, P= 0.61 χ 2 4 = 8.79, P= 0.07 Ants biting the ladybug χ 2 1 = 0.73, P= 0.39 χ 2 4 = 12.3, P= 0.015 Ants bending their gaster χ 2 1 = 0.54, P= 0.46 χ 2 4 = 15.80, P= 0.003 Ants chasing the ladybug χ 2 1 = 0.03, P= 0.85 χ 2 4 = 55.40, P<0.001 Ants falling off the leaf χ 2 1 = 0.11, P= 0.74 χ 2 4 = 4.40, P= 0.35 Ant grooming χ 2 1 = 0.00, P= 1.00 χ 2 4 = 20.09, P<0.001 Significant differences are highlighted in bold. Figure 2. Aphid rescue behaviour performed by Cr. scutellaris in filmed interactions with A. bipunctata (left) and H. axyridis (right). In both cases, an ant worker is shown attempting to save a P. juglandis aphid grabbed by the ladybug. 4 Enrico Schifani et al. https://doi.org/10.1017/S0007485323000500 Published online by Cambridge University Press 81 normally few in numbers) rarely attacked the lady beetles if these were not close to the aphids, and often completely abandoned the leaf soon after the lady beetle attacks on the aphids started. This resulted in the lady beetles spending considerably more time on the leaves, and frequently killing aphids, often to the point of causing the whole aphid colony to flee. An intermediate performance in terms of aphid protection was recorded for Ca. vagus and Cr. scutellaris.Ca. vagus workers occurred in small numbers like Ca. piceus but are much larger than any other ant species observed in this study (Seifert, 2018). Stronger biting force because of larger size may have determined their more effective protective service compared to Ca. piceus, and the higher frequency of self-cleaning behaviour after bites. Cr. scutellaris, an aggressive and dominant species of the canopy (Castracani et al.,2017; Seifert, 2018; Giannetti et al., 2019,2022; Schifani et al., 2022,2023b,2023c), was the only species to perform the aphid rescue behaviour. Considering that Cr. scutellaris queens sometimes store in special chambers of their nests living P. juglandis aphids during the earlier stages of colony foundation (Giannetti et al., 2021), our observations on the aphid rescue behaviour reinforce the idea of a special relationship between the two species. Both Ca. vagus and Cr. scutellaris often did not attack lady beetles that were not close to the aphids, and in their presence, lady beetles managed to kill some aphids. However, only in the case of Ca. vagus whole aphid colonies were observed to flee. It was interesting to observe how ants had similar interactions with the two lady beetle species. Ant–lady beetle interactions can sometimes deeply differ based on the specific characteristic of the lady beetle species involved, with some specialised species being completely immune to ant attacks or even taking advantage of ant presence (Liere and Perfecto, 2008). Apart from their geographic origin, A. bipunctata and H. axyridis are not known to possess any highly specific adaptation to cope with ants, but differ in size, with the latter being considerably larger than the former. However, the larger ants examined in our experiment, Ca. piceus and Ca. vagus, performed as good or worse than the remaining smaller species, suggesting that size is not a key determinant in the outcomes of ant–lady beetle encounters. Mutualistic aphids are attacked by several different predators and parasitoids, which often co-occur and may compete for the same trophic resource (Schifani et al., 2023a). While the behavioural response of ants may also vary significantly at the individual level (Novgorodova, 2015), the differences among ant species may be crucial in favouring some arthropod natural enemies over others for pest control (Völkl et al., 1996), and a better understanding of these dynamics may lead to improved biological control of several pest hemipteran species. In our experiment, the worse aphid protector, Ca. piceus, is a species with smallto medium-sized colonies that is generally described as timid, while the remaining, more effective species are characterised by either very large worker size (Ca. vagus) or very large colony size (Cr. scutellaris,D. quadripunctatus,L. emarginatus) (Seifert, 2018). Interestingly, in comparison to the other three, D. quadripunctatus is not traditionally described as an aggressive species. While general aggressiveness has been indicated as important, no conclusive evidence has yet emerged over natural history traits that can be used to predict the role of an ant species as a good or bad defender of its hemipteran partners, making it still important to extend the baseline knowledge to the behaviour of more species (Buckley and Gullan, 1991; Novgorodova and Gavrilyuk, 2012; Wang et al., 2021; Schifani et al., 2023a). Acknowledgements. This work has benefited from the equipment and framework of the COMP-HUB Initiative, funded by the ‘Departments of Excellence’program of the Italian Ministry for Education, University and Research (MIUR, 2018–2022). We thank Carlo Vitali for his support in the fieldwork as a part of his internship at the University of Parma. Competing interests. None. References Brown PMJ, Adriaens T, Bathon H, Cuppen J, Goldarazena A, Hägg T, Kenis M, Klausnitzer BEM, KovářI, Loomans AJM, Majerus MEN, Nedved O, Pedersen J, Rabitsch W, Roy HE, Ternois V, Zakharov IA and Roy DB (2007) The harlequin ladybird, Harmonia axyridis: global perspectives on invasion history and ecology. Biological Invasions 18, 997– 1044. Buckley R and Gullan P (1991) More aggressive ant species (Hymenoptera: Formicidae) provide better protection for soft scales and mealybugs (Homoptera: Coccidae, Pseudococcidae). Biotropica 23, 282–286. Campolo O, Palmeri V, Malacrinò A, Laudani F, Castracani C, Mori A and Grasso DA (2015) Interaction between ants and the Mediterranean fruit fly: new insights for biological control. Biological Control 90, 120–127. Castracani C, Bulgarini G, Giannetti D, Spotti FA, Maistrello L, Mori A and Grasso DA (2017) Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys.Journal of Pest Science 90, 1181–1190. Castracani C, Giannetti D, Spotti FA, Schifani E, Ghizzoni M, Delaiti M, Penner F, Leonardi S, Mori A, Ioriatti C and Grasso DA (2023) Ants as mealybug detectors: a novel tool for monitoring Planococcus ficus infestation based on ant behaviour. Agricultural and Forest Entomology 25, 237– 250. Cocco A, Pacheco da Silva VC, Benelli G, Botton M, Lucchi A and Lentini A(2021) Sustainable management of the vine mealybug in organic vineyards. Journal of Pest Science 94, 153–185. Collins CM and Leather SR (2002) Ant-mediated dispersal of the black willow aphid Pterocomma salicis L.; does the ant Lasius niger L. judge aphid-host quality? Ecological Entomology 27, 238–241. Czechowski W, Trigos-Peral G, Maák I and Vepsäläinen K (2019) Alate gyne of the ant Dolichoderus quadripunctatus (L.) (Hymenoptera, Formicidae) follows foraging trail to aphids. Journal of Hymenoptera Research 71, 241–248. Dao HT, Meats A, Beattie GAC and Spooner-Hart R (2014) Ant-coccid mutualism in citrus canopies and its effect on natural enemies of red scale, Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae). Bulletin of Entomological Research 104, 137–142. Depa Ł, Kaszyca-Taszakowska N, Taszakowski A and Kanturski M (2020) Ant-induced evolutionary patterns in aphids. Biological Reviews 95, 1574– 1589. Franco JC, Suma P, Da Silva EB, Blumberg D and Mendel Z (2004) Management strategies of mealybug pests of citrus in Mediterranean countries. Phytoparasitica 32, 507–522. Gao G, Liu S, Feng L, Wang Y and Lu Z (2020) Effect of temperature on predation by Harmonia axyridis (Pall.) (Coleoptera: Coccinellidae) on the walnut aphids Chromaphis juglandicola Kalt. and Panaphis juglandis (Goeze). Egyptian Journal of Biological Pest Control 30,1–6. Giannetti D, Castracani C, Spotti FA, Mori A and Grasso DA (2019) Gall-colonizing ants and their role as plant defenders: from ‘bad job’to ‘useful service’.Insects 10, 392. Giannetti D, Mandrioli M, Schifani E, Castracani C, Spotti FA, Mori A and Grasso DA (2021) First report on the acrobat ant Crematogaster scutellaris storing live aphids in its oak-gall nests. Insects 12, 108. Giannetti D, Schifani E, Gugliuzzo A, Zappalà L, Biondi A and Grasso DA (2022) Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus. Biological Control 174, 105032. Hölldobler B and Wilson EO (1990) The Ants. Cambridge, USA: Harvard University Press. Bulletin of Entomological Research 5 https://doi.org/10.1017/S0007485323000500 Published online by Cambridge University Press 82 Kök Ş, Aktaç N, Özdemir I and Kasap I (2018) The new association records on ants (Hymenoptera: Formicidae) and aphids (Hemiptera: Aphididae) in the Central Province of Çanakkale. Bitki Koruma Bülteni 58,47–53. Li H, Li B, Lövei GL, Kring TJ and Obrycki JJ (2021) Interactions among native and non-native predatory Coccinellidae influence biological control and biodiversity. Annals of the Entomological Society of America 114, 119–136. Liere H and Perfecto I (2008) Cheating on a mutualism: indirect benefits of ant attendance to a coccidophagous coccinellid. Environmental Entomology 37, 143–149. Majerus ME, Sloggett JJ, Godeau JF and Hemptinne JL (2007) Interactions between ants and aphidophagous and coccidophagous ladybirds. Population Ecology 49,15–27. Navarrete B, McAuslane H, Deyrup M and Peña JE (2013) Ants (Hymenoptera: Formicidae) associated with Diaphorina citri (Hemiptera: Liviidae) and their role in its biological control. Florida Entomologist 96, 590–597. Nielsen C, Agrawal AA and Hajek AE (2010) Ants defend aphids against lethal disease. Biology Letters 6, 205–208. Novgorodova TA (2015) Interaction of ants with aphid enemies: do inexperienced ants specializing in honeydew collection recognize aphidophages at their first contact? Entomological Review 95, 1182–1190. Novgorodova TA and Gavrilyuk AV (2012) The degree of protection different ants (Hymenoptera: Formicidae) provide aphids (Hemiptera: Aphididae) against aphidophages. European Journal of Entomology 109, 187–196. Obrycki JJ and Kring TJ (1998) Predaceous Coccinellidae in biological control. Annual Review of Entomology 43, 295–321. Offenberg J and Damgaard C (2019) Ants suppressing plant pathogens: a review. Oikos 128, 1691–1703. Offenberg J, Nielsen JS and Damgaard C (2019) Wood Ant (Formica polyctena) services and disservices in a danish apple plantation. Sociobiology 66, 247–256. Oliver TH, Leather SR and Cook JM (2008) Macroevolutionary patterns in the origin of mutualisms involving ants. Journal of Evolutionary Biology 21, 1597–1608. Parker J and Kronauer DJC (2021) How ants shape biodiversity. Current Biology 31, R1208–R1214. Queiroz JM and Oliveira PS (2001) Tending ants protect honeydewproducing whiteflies (Homoptera: Aleyrodidae). Environmental Entomology 30, 295–297. R Core Team (2022) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. Available at https://www.R-project.org/. RStudio Team (2022) RStudio: Integrated Development for R. Boston, MA: RStudio, PBC. Available at http://www.rstudio.com/. Schifani E, Castracani C, Giannetti D, Spotti FA, Reggiani R, Leonardi S, Mori A and Grasso DA (2020) New tools for conservation biological control: testing ant-attracting artificial Nectaries to employ ants as plant defenders. Insects 11, 129. Schifani E, Giannetti D, Csősz S, Castellucci F, Luchetti A, Castracani C, Mori A and Grasso DA (2022) Is mimicry a diversification-driver in ants? Biogeography, ecology, ethology, genetics and morphology define a second West-Palaearctic Colobopsis species (Hymenoptera: Formicidae). Zoological Journal of the Linnean Society 194, 1424–1450. Schifani E, Peri E, Giannetti D, Colazza S and Grasso DA (2023a) Ant attendance does not necessarily imply protection of aphids from their arthropod natural enemies. Ecological Entomology 48, 384–388. Schifani E, Giannetti D and Grasso DA (2023b) Predatory abilities of two Mediterranean ants on the eggs and larvae of the codling moth Cydia pomonella.Insects 14, 97. Schifani E, Peri E, Giannetti D, Alınç T, Colazza S and Grasso DA (2023c) Mediterranean ants can increase nymph mortality in the stink bug Nezara viridula without interfering with its egg parasitoid Trissolcus basalis. Entomologia Experimentalis et Applicata 171, 739–744. Seifert B (2018) Ants of Northern and Central Europe. Tauer, Germany: Lutra Verlags-und Vertriebsgesellschaft. Styrsky JD and Eubanks MD (2007) Ecological consequences of interactions between ants and honeydew-producing insects. Proceedings of the Royal Society B: Biological Sciences 274, 151–164. Vanek SJ and Potter DA (2010) Ant-exclusion to promote biological control of soft scales (Hemiptera: Coccidae) on woody landscape plants. Environmental Entomology 39, 1829–1837. Völkl W (1992) Aphids or their parasitoids: who actually benefits from ant-attendance? Journal of Animal Ecology 61, 273–281. Völkl W, Liepert C, Birnbach R, Hübner G and Dettner K (1996) Chemical and tactile communication between the root aphid parasitoid Paralipsis enervis and trophobiotic ants: consequences for parasitoid survival. Experientia 52, 731–738. Wang B, Lu M, Peng Y and Segar ST (2021) Direct and indirect effects of invasive vs. native ant-hemipteran mutualism: a meta-analysis that supports the mutualism intensity hypothesis. Agronomy 11, 2323. 6 Enrico Schifani et al. https://doi.org/10.1017/S0007485323000500 Published online by Cambridge University Press 83 Section II - Ants as mutualist partners of insect pests Chapter 8 Towards sustainable management of ant-hemipteran mutualism in agricultural setting _______________________________________________________________________________ Schifani, E., Giannetti, D., & Grasso, D.A. (2023). Towards sustainable management of anthemipteran mutualism in agricultural setting. Crop Protection, 175, 106468. https://doi.org/10.1016/j.cropro.2023.106468 84 Crop Protection 175 (2024) 106468 Available online 17 October 2023 0261-2194/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Toward sustainable management of ant-hemipteran mutualism in agricultural settings: a comparison of different approaches Enrico Schifani ** , Daniele Giannetti, Donato A. Grasso * Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy ARTICLE INFO Keywords: Aphids Mealybugs Scales Agroecology Biological control Integrated pest management (IPM) Pesticides ABSTRACT Mutualistic associations between ants and honeydew-producing hemipterans have a great ecological and evolutionary significance across terrestrial habitats but can also cause pest outbreaks in agroecosystems. At the same time, ants are often effective predators of several agricultural pests, can improve soil quality, and can control some plant pathogens. Neither ant attendance of hemipteran pests alone, nor a positive correlation between the abundance of ants and hemipterans automatically imply that ants are a worthy target or a key element in hemipteran management strategies. The main tactics in the management of ant-hemipteran associations in agroecosystems include the use of sticky or insecticidal barriers, low-toxicity baits, alternative sugary sources, or ant-adapted biocontrol agents. Barriers can quickly seal ground-nesting ants from the canopy of perennial plants but are unselective towards other arthropods and costly in terms of maintenance. Low-toxicity baits have been particularly tested against invasive and supercolonial ant species and yet are worth considering only when the complete elimination of ant colonies can be desirable. More recently developed and not yet widely available methods based on the provision of alternative sugary sources to manipulate ant behavior can allow to retain or even enhance the contribution of ants to the control of other phytophagous insects or plant pathogens while effective disrupting their mutualism with hemipteran pests. Finally, many parasitoids and predators possess specific adaptations to bypass attending ants, but the existence of species-specific factors complicatesthese networks. Further basic research and longer-term studies are needed to refine and improve the development of sustainable management strategies. 1. Introduction 1.1. The ecological success of ant-hemipteran mutualisms Ants are among the most successful groups of insects across the world, establishing a myriad of interactions within complex multitrophic networks that involve other animals, plants, and fungi (H¨ olldobler and Wilson, 1990; Rico-Gray and Oliveira, 2008; Stadler and Dixon, 2008; Lach et al., 2010; Parker and Kronauer, 2021). Trophobiotic interactions between ants and honeydew-producing hemipterans of the suborder Sternorrhyncha significantly impacted the evolutionary radiations of some of today’s main ant, aphid, scale, psyllid, and whitefly lineages, and became an ecological keystone in many ecosystems (H¨ olldobler and Wilson, 1990; Lach et al., 2010; Parker and Kronauer, 2021). The attractiveness of the honeydew produced by hemipterans usually represents the most important reason ants visit plants apart from the eventual presence of extrafloral nectaries (Blüthgen et al., 2004; Engel et al., 2001; Campos and Camacho, 2014; Grasso et al., 2015). Ant-hemipteran mutualistic associations have offered a remarkable model for evolutionary biologists. In exchange for honeydew, ants may help their partners in several ways: i) displacing or killing their natural enemies, including parasitoids and predators (Jiggins et al., 1993; Kaplan and Eubanks, 2002; Martinez-Ferrer et al., 2003; Majerus et al., 2007; Dao et al., 2014), and reducing their need to invest in protective microbiota (Henry et al., 2015; Mandrioli et al., 2016); ii) reducing the abundance of competing non-mutualist honeydew-producing hemipterans (Engel et al., 2001; Mi˜ narro et al., 2010); iii) controlling the hygienic conditions of their colonies by cleaning excess honeydew and releasing anti-microbial and anti-fungi substances (Lit et al., 1999; Morales, 2000; Queiroz and Oliveira, 2001; Matsuura and Yashiro, 2006; Nielsen et al., 2010); iv) actively dispersing them to new plants (Das, 1959; Collins and Leather, 2002; Giannetti et al., * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (E. Schifani), [email protected] (D.A. Grasso). Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro https://doi.org/10.1016/j.cropro.2023.106468 Received 11 September 2023; Received in revised form 6 October 2023; Accepted 9 October 2023 85 Crop Protection 175 (2024) 106468 2 2021); v) offering them protection from adverse meteorological conditions and offering them shelter inside their nests during adverse seasons (Maschwitz and H¨ anel 1985; Giannetti et al., 2021). Those ant and hemipteran species that participate in these mutualistic relationships developed a variety of morphological and behavioral adaptations based on their level of dependence, spanning from facultative to strictly obligate mutualisms depending on the ant or hemipteran species examined (Delabie, 2001; Stadler and Dixon, 2005; Depa et al., 2020; Parker and Kronauer, 2021). The emergence of mutualism between ants and honeydew-producing hemipterans also caused an evolutionary arms race with the natural enemies of the latter, resulting in many different adaptations to reduce their vulnerability to ants (Stadler and Dixon, 2008). For instance, ladybug larvae of several groups possess specific defensive structure to deter ant attacks (Majerus et al., 2007; Schwartzberg et al., 2010). Myrmecophilous aphid parasitoids even benefit or depend on the presence of certain ants attending their host species (V¨ olkl, 1992, 1994; V¨ olkl and Mackauer, 1993; V¨ olkl et al., 1996), and so do certain coccidophagous lady beetles (Liere and Perfecto, 2014). In any case, the presence of mutualist hemipterans on plants represents a key attracting factor for many ant species to visit the plants (Styrsky and Eubanks, 2007). 1.2. Ant-hemipteran associations as a threat to crop protection Ant-hemipteran mutualism is by far the most frequently reported problematic interaction involving ants in agroecosystems (Way and Khoo, 1992; Anjos et al., 2022) and, unlike plant foraging or seed predation, is not restricted to few specialized ant lineages (leafcutter ants and harvester ants, see Della Lucia et al., 2014; Uhey and Hofstetter, 2022). While many honeydew-producing hemipterans that are important agricultural pests are not ant-mutualists, several species of ant-mutualist hemipterans rank among the top agricultural pests. For instance, out of 16 studies documenting the effect of ants on honeydew-producing citrus pests that are not obligate mutualists, 10 reported a higher abundance of the pest species in the presence of ants (Anjos et al., 2021). On fava bean plantations, Banks and Macaulay (1967) recorded up to a 50% decrease in the number of seeds produced when facultatively mutualist aphids were attended by ants. Examples of Fig. 1. Crematogaster scutellaris ant workers attending an aphid colony (a), defending the aphid colony against the ladybeetle Adalia bipunctata (b), and preying upon a caterpillar (c). E. Schifani et al. 86 Crop Protection 175 (2024) 106468 3 hemipteran pests attended by ants include economically important species of both aphids (e.g., the black bean aphid Aphis fabae Scopoli and the cotton aphid A. gossypii Glover – see Banks and Macaulay, 1967; Kaplan and Eubanks, 2002; Powell and Silverman, 2010; Mirzamohammadi et al., 2019), mealybugs (e.g., the vine mealybug Planococcus ficus Signoret, the citrus mealybug P. citri Risso, or the cotton mealybug Phenacoccus solenopsis Tinsley – see Daane et al., 2007; Marras et al., 2008; Mgocheki and Addison, 2009; Zhou et al., 2012; Cocco et al., 2021; Delabie et al., 2021), and whiteflies (e.g., the wooly whitefly Aleurothrixus floccosus Maskell – see Anjos et al., 2021). Notably, all these hemipteran species are facultative mutualist, not strictly requiring ant attendance to survive, yet often visited by a variety of different ant species. Interference with biological control agents is by far the most frequently documented cause of hemipteran outbreaks in the presence of ants (e.g., Kaplan and Eubanks, 2002; Mgocheki and Addison, 2009; Cheng et al., 2015; McCalla et al., 2023; Plata et al., 2023; Fig. 1a and b), while other mechanisms (such as protection from fungal outbreaks on uncollected honeydew or increased survival of first-instar hemipterans) are more rarely investigated but may play a significant role (Queiroz and Oliveira, 2001; Daane et al., 2007). Moreover, it is important to note that not all ant species associated with hemipteran pests have a significant protective role against their natural enemies (e.g., de Jesus et al., 2016; Schifani et al., 2023a; 2023e). In this context, aggressive ant species (Buckley and Gullan, 1991; Novgorodova and Gavrilyuk, 2012), and more importantly invasive alien species are more likely to efficiently defend hemipterans against parasitoids and predators (Wang et al., 2021). 1.3. Provision of ecosystem services by ants in agricultural settings A large part of the interactions of ants with phytophagous insects are positive for agriculture, resulting in the killing or displacement of these insects due to predation or territorial aggression, with an overall positive effect on pest control (Choate and Drummond, 2011; Anjos et al., 2022; Fig. 1c). Ants are known as the first reported example of the use of a biological control agent in agriculture with the Asian weaver ant Oecophylla smaragdina (Fabricius) used against citrus pests in China since 304 AD (Huang and Yang, 1987; Van Mele, 2008; Offenberg, 2015). The active use of many species as biocontrol agents across several countries continues today (Peng and Christian, 2010; Offenberg, 2015). However, ant services as natural enemies of pest insects are significant even before the employment of active strategies and are globally reported, and in many systems may lead to neat crop yield increases (Anjos et al., 2022). Most literature focused on the tropics, with weaver ants in Asian fruit orchards (Offenberg, 2015) or Azteca ants in Mesoamerican coffee agroforests serving as primary examples, but increasing efforts focused on agroecosystems of temperate regions (Campolo et al., 2015; Schifani et al., 2020; Jensen et al., 2023). The predatory services that ants provide against particularly problematic phytophagous insects may sometimes offer to the plant a much greater benefit compared to the costs caused by sustaining ant-mutualist hemipterans at the same time (Styrsky and Eubanks, 2007, 2010). Apart from aggressive interactions with phytophagous arthropods, by simply visiting plants, ants can also contribute to suppress plant pathogen incidence by passively releasing many effective antibiotics on the surfaces they walk over (Offenberg and Damgaard, 2019; Offenberg et al., 2022). Even though less frequently documented, these interactions appear abundant and of probably high ecological impact (Offenberg and Damgaard, 2019; Giannetti et al., 2019; Offenberg et al., 2022). On the ground, ants also act as very important ecosystem engineers and agents for soil bioturbation and enrichment, with most species excavating their nests in the soil and the nests becoming a hotspot of nutrients due to the amount of food ants concentrate (Altfeld and Stiling, 2009; Lach et al., 2010; Solida et al., 2011; Taylor et al., 2019). Furthermore, ants can also be important seed predators playing a role in the control of weeds, although the same ability may result in losses of cultivated seeds in other contexts (Baraibar et al., 2011). 2. Management tactics for ant-hemipteran associations A number of tactics have been developed to deal with the problematic role of ants as partners of honeydew-producing hemipteran pests (Fig. 2), and their pros and cons are here reviewed and discussed. The application of any strategy should however be ideally preceded by an assessment of the real contribution of the local ants to a possible problem related to these hemipterans, keeping in mind that attendance by ants does not necessarily equals the provision of important services from the ants to their partners (Schifani et al., 2023a,e). While such assessments are infrequent or sometimes limited to correlational evidence, it is important to note that any strategy to reduce ant-hemipteran mutualism bears some costs, in some cases implying a meaningful loss of ecosystem services otherwise provided by ants. Sometimes, the same ant species that provide important pest control services may also cause disservices related to the control of ant-mutualist hemipteran pests, although their extent can greatly vary (Anjos et al., 2022). For instance, Oecophylla tropical weaver ants, while being widely recognized as excellent control agents for countless phytophagous pests, can sometimes still have some negative effect on the biological control of mealybugs (Offenberg, 2015; Forbes and Northfield, 2017), in certain case worth the application of appropriate management tactics (Correa et al., 2023). Elsewhere, ants of the Mediterranean Tapinoma nigerrimum-complex are often associated with mealybugs in citrus orchards and vineyards, and laboratory experiments suggest that they may significantly lower the success of parasitoids and predators of the citrus and vine mealybugs, respectively (Mansour et al., 2012). However, field data demonstrate that they can also significantly lower the survival rate of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Campolo et al., 2015), while laboratory experiments suggest that they can act as an enemy of the ambrosia beetle Xylosandrus compactus (Eichhoff), the codling moth Cydia pomonella (L.), and the green stink bug Nezara viridula (L.) (Giannetti et al., 2022; Schifani et al., 2023b,c,d). In Europe, Formica rufa-group ants have historically attracted significant attention as biocontrol agents of pests in forestry systems, such as the pine processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller) and the western larch case-bearer Coleophora laricella (Hübner) (e.g., Pavan, 1951, 1961; Adlung, 1966). However, more recent studies dealt with their application in north-European apple plantations, where F. rufa-group ants can cause a significant reduction of the winter moth Operophtera brumata (L.) and the apple scab Venturia inaequalis (Cooke), but may also significantly increased aphid infections by Aphis pomi de Geer if not properly managed (Offenberg et al., 2019; Jensen et al., 2023). A common European ant, Lasius niger (L.), can have a negative impact over aphid natural enemies (Jiggins et al., 1993; V¨ olkl and Mackauer, 1993) but also be a predator of the brown marmorated stink bug Halyomorpha halys (Stål) (Bulgarini et al., 2021). Finally, the red imported fire ant Solenopsis invicta Buren, an invasive alien species of global relevance and a threat to agriculture and biodiversity (Menchetti et al., 2023), is at the same time a major predator of phytophagous insects and a promoter of mutualist hemipteran outbreaks in the US (Kaplan and Eubanks, 2002; Hood et al., 2003; Coppler et al., 2007; Rashid et al., 2013). In this context, a careful evaluation of the cost-efficiency of different alternative tactics is important. 2.1. Preventing ants to access plants by applying barriers The use of barriers to prevent ants from climbing on plants probably represents the earliest attempt to deviate from the much damaging unselective wide use of pesticides in the control of ant-hemipteran associations in agroecosystems (Davis and Van Schagen, 1993). The barriers may be either insecticidal or sticky, and their use is normally limited to perennial crops of plants whose trunk is suitable for their applications and in which the prevailing ants are ground-nesting and need to climb E. Schifani et al. 87 Crop Protection 175 (2024) 106468 4 on the plant to access the hemipteran colonies (Juan-Blasco et al., 2011). With a relatively simple functioning, the correct application of barriers immediately stops ants from accessing the plants they are applied to, making them a very rapid method of intervention. Sticky barriers are also often used in research experiments on ant exclusion (e.g., Pi˜ nol et al., 2010, 2012; Nagy et al., 2015; Schifani et al., 2020, 2023a). However, their use can also bear some noteworthy difficulties and costs. Sticky barriers usually need to be replaced at least monthly if not more frequently, since rain and dust, as well as dead arthropods attached on the glue can greatly affect their effectiveness (Schifani et al., 2020; McCalla et al., 2023). Their application is particularly difficult on plants with very irregular surfaces, preventing their complete adherence, since the minimal gaps can often be exploited by ants. For the same reason, their effective use requires a continuous grove micromanagement to Fig. 2. The four main approaches for the management of ant-hemipteran mutualistic relationships in agroecosystems are exemplified: (a) exclusion of ants from the plants with the application of physical or toxic barriers, which can also limit the access to climbing predators; (b) use of selective toxic baits that eliminate ants and their colonies; (c) behavioral manipulation of ants through alternative sugary sources that reduce their dependance on hemipteran colonies; (d) use of selected natural enemies species with the ability to bypass the locally dominant ants as opposed to those that ants are effectively able to repel. Minus and plus symbols indicate the expected abundance increase (+) or decrease (−) of ants and natural enemies based on each of the four tactics. Image created with biorender.com and modified. E. Schifani et al. 88 Section III - First data on ant diversity and distribution in Italian agroecosystems Chapter 9 Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley _______________________________________________________________________________ Giannetti, D., Schifani, E., Castracani, C., Ghizzoni, M., Delaiti, M., Pfenner, F., Spotti, F.A., Mori, A., Ioriatti, C., & Grasso, D.A. (2021). Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley. Redia, 104, 97-109. http://dx.doi.org/10.19263/REDIA104.21.11 95 REDIA, 104, 2021: 97-109 http://dx.doi.org/10.19263/REDIA-104.21.11 - Received 19 March 2021 Accepted 18 May 2021 DANIELE GIANNETTI a§ - ENRICO SCHIFANI a§ - CRISTINA CASTRACANI a* - MARTINA GHIZZONI a - MARCO DELAITI b - FRANCESCO PENNER b - FIORENZA A. SPOTTI a - ALESSANDRA MORI a – CLAUDIO IORIATTI b - DONATO A. GRASSO a ASSESSING ANT DIVERSITY IN AGROECOSYSTEMS: THE CASE OF ITALIAN VINEYARDS OF THE ADIGE VALLEY a Department of Chemistry, Life Sciences & Environmental Sustainability, Parco Area delle Scienze, 11/a, University of Parma, I-43124 Parma, Italy b Technology Transfer Centre, Fondazione Edmund Mach, Via E. Mach, 1, 38010 San Michele all’Adige (TN), Italy *Corresponding Author: [email protected] § These authors contributed equally Giannetti D., Schifani E., Castracani C., Ghizzoni M., Delaiti M., Penner F., Spotti F.A., Mori A., Ioriatti C., Grasso D.A. - Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley Agroecosystems have gained a dominant position on worldwide land-usage, and therefore preserving their biodiversity is crucial for environmental sustainability. Ants are one of the most widespread groups of terrestrial arthropods, and, thanks to their significant diversification, they are considered as a good proxy group for biodiversity monitoring, also in agroecosystems. Vineyards are economically valuable cultures widespread worldwide, and hosting many ant species, that provide meaningful ecosystem services and disservices. Despite the important role that ants play in these agroecosystems, ant biodiversity in vineyards is still poorly studied, especially in Italy. In this context, we present a first detailed quantitative and qualitative assessment of the ant fauna of Italian vineyards from the Adige Valley based on pitfall traps data, and discuss the results in comparison with the few other similar assessments from Europe and other continents. We document an assemblage of 22 species (7-16 per orchard), mostly dominated by three disturbance-tolerant species (including an introduced species). Vineyards’ ant faunas appear to be rather heterogeneous worldwide, mainly following local ecological and biogeographical constraints, and the role that most ant species play in these agroecosystems is presently unknown. KEY WORDS: vines; biodiversity monitoring; myrmecofauna; Prealps. INTRODUCTION Since agriculture has become a dominant category of land usage worldwide, crop management practices have become a decisive factor to preserve the environment (TILMAN et al., 2001; GREEN et al., 2005; TSCHARNTKE et al., 2005; FIRBANK et al., 2008). Overlooked for decades, insect and arthropod decline and its severe potential outcomes on ecosystems functioning recently attained much attention showing the need for a deeper commitment in the development of effective monitoring systems in contexts with different anthropic impacts (BURGIO & SOMMAGGIO, 2007; CAMPANARO et al., 2011; BURGIO et al., 2015; DIRZO et al., 2014; HALLMANN et al., 2017; LEATHER, 2017; PIZZOLOTTO et al., 2018; HOMBURG et al., 2019). While agricultural transformations may play a key in this process, diversity and distribution of the arthropodofauna in cultivated areas is still insufficiently documented. Wine grapes (Vitis vinifera L.) are a widespread cultivated species of important economic value, whose cultivated surface is likely to increase in the future due to climate change (HANNAH et al., 2013; MORIONDO et al., 2013). European vineyards alone cover 3.2 million ha representing 45% of the world’s total areas under vines and 1.8% of the total utilized agricultural area. Over 20% of them is located in Italy (688,000 ha), representing about 5% of the total utilized agricultural surface (SAU) of the country (EUROSTAT, 2017; ISTAT, 2019). Under conventional management practices, establishment of viticulture is often associated with notable negative impacts on soil and local biodiversity, and thus may represent a serious conservation threat in certain contexts (ALTIERI & NICHOLLS, 2002; FAIRBANKS et al., 2004; HILTY & MERENLENDER, 2004; COULOUMA et al., 2006; HILTY et al., 2006; HILDENBRANDT et al., 2008; COLL et al., 2011; LAWRENCE et al., 2011; ROSADO et al., 2013). However, implementing correct agro-ecological practices can be an effective way to address some of these issues (VIERS et al., 2012): for example, organic viticulture may allow richer communities of organisms to thrive, both within the vineyards themselves and in neighboring forested areas (e.g. GAIGHER & SAMWAYS, 2010; COLL et al., 2012; KEHINDE & SAMWAYS, 2014; CAPRIO et al., 2015; MASONI et al., 2017; DAANE et al., 2018). Due to their high diversity and strong ecological impacts, ants are considered an important group for biodiversity monitoring in both natural and anthropic impacted ecosystems, including agroecosystems (e.g. PECK et al., 1998; DE BRUYN et al., 1999; AGOSTI et al., 2000; LACH et al., 2010; GIBB et al., 2017), where they provide impactful services and disservices. For example, they may control other arthropods, fungi or even weeds (e.g. RISCH & CARROL, 1982; BARAIBAR et al., 2011; OFFENBERG & DAMGAARD, 2019) and favor foliar uptake of nitrogen (e.g. PINKALSKI et al., 2018), but may also favor mutualistic pest species (e.g. PEKAS et al., 2010; CALABUIG et al., 2013; DAO et al., 2014). As a result, ants can be employed as biocontrol agents in 96 98 GIANNETTI ET AL. REDIA, Vol. 104, 2021 some cases (e.g. WAY & KHOO, 1992; PENG et al., 2010; CHOATE & DRUMMOND, 2011; OFFENBERG, 2015; CASTRACANI et al., 2017; SCHIFANI et al., 2020), but they can be target of control strategies in other situations (e.g. TOLLERUP et al., 2004; GREENBERG et al., 2013). The balance between negative and positive effects of the ant presence in agroecosystems is variable, and it depends on many factors (e.g. STYRSKY & EUBANKS, 2006). In the last two decades, several studies began to investigate the role of ants in Italian agroecosystems and their possible use as bioindicators (e.g. CASTRACANI & MORI, 2006; OTTONETTI et al., 2008; LA PERGOLA et al., 2008; SANTINI et al., 2011; MASONI et al., 2017; CAMPOLO et al., 2015; CASTRACANI et al., 2015; SCHIFANI et al., 2020). However, most contexts of the highly diversified Mediterranean agriculture remain currently unexplored in this sense. Accounts of the ant fauna inhabiting vineyards are available through scattered checklists from very different geographic regions. For example, ant check-lists in Australian and South American vineyards were provided by CHONG et al. (2011) and ROSADO et al. (2012; 2013), while in Europe some assessments were provided by BELTRÀ et al. (2017) in Spain, GONÇALVES et al. (2017) in Portugal and MASONI et al. (2017) in central Italy (Tuscany region). However, European vineyards are found under several different climatic conditions. Italy offers a great variety of climatic conditions in this sense: on one hand, vineyards can be found under hot temperate and subtropical temperate climate in Sicily, while they are affected by a sub-continental climate in the Prealpine river valleys (FRATIANNI & ACQUAOTTA, 2017). While MASONI et al. (2017) offered a first assessment from an Italian area characterized by a sub coastal temperate climate, we decided to investigate vineyards’ ants at the northernmost latitudes of Italian viticulture, considering that ants colonizing vineyards under a subcontinental climate have never been documented elsewhere in Europe. Therefore, we conducted a first qualitative and quantitative assessment of ant diversity in vineyards from the Prealpine Adige Valley in Italy in order to provide a baseline overview and compare the results to the accounts from other geographic regions. MATERIALS AND METHODS A total of 10 vineyards from the Adige Valley in northern Italy (region: Trentino-Alto Adige; cities: Rovereto and Trento), treated under conventional agriculture, were selected for this study (see Table 1). The vines (Pinot grigio variety) were grown with a straight, single trunk and then trained onto a pergola system (Fig. I). The vineyards ground was permanently grass covered between the rows while chemical weed control was applied on a 50 cm strip under the vines. Grass was periodically mowed and mulched on place. Pest control was performed with repeated applications of fungicides and one or two insecticide treatments. Our monitoring program was conducted from June to September 2016 (which is a good coverage of ants’ activity season in the study area), focusing on two rows of each vineyard (each consisting of 16 vines). To obtain data on the arthropodofauna, we relied on pitfall traps (50-ml polypropylene Falcon vials) filled with 30 ml of propylene glycol. In each row, 12 traps were employed at a time: 4 placed on the vines’ branches (B traps), 4 in the soil between two vines (S2 traps) and 4 at 1 m from the vines, between the rows (S1 traps) (Figs. II, III). Traps were replaced every 15 days, resulting in 7 sampling dates (from 07.06.2016 to 07.09.2016). Therefore, a total of 1680 traps were used (12 traps x 2 rows x 10 vineyards x 7 sampling dates). Table 1List of investigated vineyards. Site name Latitude and longitude Altitude (m) A: La Favorita 45.862860, 11.002325 175 B: De Bellat-Pulito 45.845952, 11.007980 155 C: Serravalle Campanella Alto 45.801827, 11.027747 210 D: Serravalle Campanella Basso 45.796905, 11.020143 135 E: Avio Depuratore 45.732027, 10.946634 130 F: Carnal Avio 45.739156, 10.941774 210 G: Avio Campei Alto 45.753609, 10.984719 175 H: Avio Campei Basso 45.752127, 10.984354 140 I: Marine 46.036315, 11.113790 215 J: Maso Grande Ravina 46.032534, 11.107437 260 Systematic identification was achieved using general dichotomous keys for arthopods and for soil microarthopods (CHINERY, 1986; AA VV, 2005). Specimens were recognized at different systematic levels depending on their taxon, but at least at order level. Ants were sorted and identified to species level and identification was achieved using the information provided by WAGNER et al. (2017) and SEIFERT (2018; 2020). Ants from the cryptic Tetramorium caespitum complex were initially not identified during 2016 as the taxonomy of this complex was still unclear (SCHLICK-STEINER et al., 2006). After WAGNER et al. (2017) eventually provided taxonomic keys, only a part of the initial collection still remained in our possession. Since all available specimens were identified as T. immigrans (see Results), we refer as T. cf. immigrans to all the collected specimens from this group. Species accumulation curves were computed using R 4.0.3 and the specaccum() function of the vegan package (OKSANEN et al., 2017; R CORE TEAM, 2021). 97 ASSESSING ANT DIVERSITY IN AGROECOSYSTEMS: THE CASE OF ITALIAN VINEYARDS OF THE ADIGE… 99 Fig. I - Vines grown with a single straight trunk and trained onto a pergola system in one of the investigated vineyards. Fig. II - Pitfall traps placed on vines’ branches (1) and in the soil (2). 98 100 GIANNETTI ET AL. REDIA, Vol. 104, 2021 Fig. III - Traps placement in the vineyards’ rows: B traps on the vines’ branches, S1 traps between the rows and S2 traps between the vines. Table 2 - Arthropod groups collected during the survey. Class Order Tot. Ind. (n = 20,284) % Ind. Tot. Traps (n = 1,680) % Traps Arachnida Acarina 135 0.7 85 5.5 Araneae 1,210 6.5 526 31.3 Opiliones 171 0.9 111 6.6 Crustacea Isopoda 264 1.4 141 8.4 Hexapoda Collembola 471 2.5 132 7.8 Coleoptera, Adephaga 2,521 13.4 688 41.0 Coleoptera, Polyphaga 1,137 6.1 395 23.5 Coleoptera, larvae 401 2.1 243 14.5 Dermaptera 168 0.9 115 6.8 Diptera 1,036 5.5 473 28.1 Hemiptera 274 1.5 202 12.0 Hymenoptera, Formicidae 10,501 56.0 1,228 73.1 Hymenoptera (other groups) 232 1.2 162 9.6 Lepidoptera (adults) 21 0.1 18 1.1 Lepidoptera (caterpillars) 85 0.4 65 3.9 Neuroptera 9 0.0 9 0.5 Myriapoda Diplopoda 25 0.1 19 1.3 Chilopoda 74 0.4 67 4.0 RESULTS A total of 20,284 specimens were retrieved from the traps and they were classified into 19 major groups representing 15 orders of Arachnida, Crustacea, Hexapoda and Myriapoda classes (Table 2). Among these groups, ants were the most abundant, consisting in 56% of all of the collected specimens (10,501), and the most frequent, found in 73% of the traps, and these differences were averagely maintained through the entire sampling period (Table 2, Figs. IV, V). Ants were represented by 22 species belonging to 16 genera and 3 subfamilies (Table 3). The most abundant species, F. cunicularia, L. niger and T. cf. immigrans represented alone over 85% of the collected specimens, and among them L. niger was the most abundant during all the sampling dates (Figs VI, VII). 99 ASSESSING ANT DIVERSITY IN AGROECOSYSTEMS: THE CASE OF ITALIAN VINEYARDS OF THE ADIGE… 101 Fig. IV - Frequency of the four main arthropod groups among the traps retrieved in the seven sampling dates. Fig. V - Arthropod specimens collected per trap during the seven sampling dates divided across the four main arthropod groups. 100 102 GIANNETTI ET AL. REDIA, Vol. 104, 2021 Table 3 - Ant species collected during the survey. Subfamily Species Occupied sites (N=10) Tot. Ind. % Ind. Branches traps (B) Formicinae Camponotus aethiops (Latreille, 1798) 1 4 0.0 Formica cinerea Mayr, 1853 4 19 0.2 X Formica cunicularia Latreille, 1798 10 1,169 11.1 X Lasius emarginatus (Olivier, 1792) 2 4 0.0 X Lasius fuliginosus Latreille, 1798 3 4 0.0 Lasius myops Forel, 1894 3 7 0.1 X Lasius niger Linnaeus, 1758 10 6,170 58.7 X Plagiolepis pygmaea (Latreille, 1798) 8 62 0.6 X Polyergus rufescens (Latreille, 1798) 3 64 0.6 Myrmicinae Aphaenogaster subterranea (Latreille, 1798) 5 9 0.1 Crematogaster scutellaris (Olivier, 1792) 4 4 0.0 X Messor ibericus Santschi, 1931 8 727 6.9 X Myrmica sabuleti Meinert, 1861 3 3 0.0 Myrmica specioides Bondroit, 1918 3 5 0.0 X Myrmecina graminicola (Latreille, 1802) 7 114 1.1 Pheidole pallidula (Nylander, 1849) 7 334 3.2 X Solenopsis fugax (Latreille, 1798) 9 132 1.2 X Strongylognathus testaceus (Schenck, 1852) 3 3 0.0 Temnothorax italicus (Consani, 1952) 5 9 0.0 Temnothorax unifasciatus (Latreille, 1798) 4 5 0.0 X Tetramorium cf. immigrans Santschi, 1927 10 1,643 15.6 X Ponerinae Hypoponera eduardi (Forel, 1894) 3 5 0.0 Fig. VI - Frequency on traps during the seven sampling dates of the three ant species detected in all orchards. 101 ASSESSING ANT DIVERSITY IN AGROECOSYSTEMS: THE CASE OF ITALIAN VINEYARDS OF THE ADIGE… 103 Fig. VII - Ant specimens collected per trap during the seven sampling dates for the three species detected in all vineyards. Fig. VIII - Species accumulation curves based on the number of ant species collected with traps placed in different positions. The vertical bars correspond to 30% of the standard error of the estimate. The S1 and S2 traps yielded a comparable performance in terms of number of captured ant species (Fig. VIII), and together granted the detection of all of the species encountered during this study, while only a subset of 13 species was collected with B traps. The number of species collected per vineyard varied from 7 to 16, while F. cunicularia, L. niger and T. cf. immigrans were found in every vineyard. Species accumulation curves showed that the sampling effort determined a clear plateau for most vineyards, with the exception of site I (Fig. IX; Table 4). 102 104 GIANNETTI ET AL. REDIA, Vol. 104, 2021 Fig. IX - Species accumulation curves based on the number of ant species collected at each site. The vertical bars correspond to 30% of the standard error of the estimate. Table 4 - Diversity indexes of each site based on collected species and % of traps occupied by the three species present in each vineyard. Site Species richness Shannon index (H) Equitability index (EH) % traps F. cunicularia % traps L. niger % traps T. cf. immigrans A 12 1.60 .31 68 69 41 B 8 1.28 .25 3 64 26 C 14 1.87 .37 34 59 32 D 7 1.00 .19 23 77 16 E 9 1.35 .26 36 71 14 F 14 2.39 .47 23 41 33 G 16 2.46 .48 27 71 14 H 12 1.62 .32 17 55 26 I 14 1.14 .22 7 67 14 J 10 1.64 .32 28 51 11 DISCUSSION As strongly documented from literature (e.g. AGOSTI et al., 2000; LACH et al., 2010), once again ants proved to be a convenient arthropod group for monitoring programs in agroecosystems, being consistently as the most abundant group in our survey. This study provides one of the few quantitative assessments conducted on the Italian ant fauna. We documented a moderately diverse fauna characterized by a high diversity of Formicinae and Myrmicinae (with a good diversity of Lasius genus, but also multiple species of Formica, Myrmica and Temnothorax genera) and by the notable absence of Dolichoderinae ants. We found an overwhelming prevalence of species characterized by very large distributions in Europe and beyond, only few Mediterranean taxa and no endemisms (JANICKI et al., 2016; GUÉNARD et al., 2017). This picture is not particularly different from that of recent sur - veys conducted on the Po Plain, but we detected no Eastern-Mediterranean species and even fewer Mediterranean or South European taxa (CASTRACANI et al., 2020). Moreover, it is worth noting that three notoriously disturbance-tolerant species, F. cunicularia, L. niger and the T. cf. immigrans were the most abundant species, as it was observed elsewhere in Northern Italy (CASTRACANI et al., 2020). While T. immigrans is probably an introduced species in Italy (CASTRACANI et al., 2020), the numerical dominance of these three species likely reflects their ability to fill empty niches created by human activities (see ARNAN et al., 2018; 2021). Only about half (54%) of the species we recorded was also detected on the vines themselves through the use of traps placed on their branches. Species like C. aethiops or T. italicus, which habitually visit plants (SEIFERT, 2018; GIANNETTI 103 ASSESSING ANT DIVERSITY IN AGROECOSYSTEMS: THE CASE OF ITALIAN VINEYARDS OF THE ADIGE… 105 et al., 2019), were probably only encountered on the soil just because of their low abundance. On the other hand, absence on the vines was expected for the social parasites such as the slave-maker P. rufescens or the inquiline S. testaceus, which are not active foragers. Other species such as A. subterranea, H. eduardi or M. graminicola were also not expected to climb into the vines because they usually forage on the soil surface or within the leaf litter (SEIFERT, 2018; GRASSO et al., 2020). While S. fugax belongs to the same category (SEIFERT, 2018), its finding on vines was rather unexpected. In comparison to the Australian and Brazilian vineyards’ ant faunas, we detected much fewer ant species. CHONG et al. (2011) sampled 50 vineyards from different Australian regions, achieving a vast geographic coverage, and detected 147 species, estimating each vineyard to be inhabited by 30-40 species (but recording only 5-24 species in one of the sampled regions). At the same time, ROSADO et al. (2012) recorded 72 species in total in Brazil, from 21 to 50 per vineyard. Our numbers are far lower and the most represented genera are different from those detected in these surveys, but this is unsurprising considering the ecological and biogeographical patterns of ant diversity worldwide: ant diversity is notoriously higher in the tropics, where genera that are dominant in the temperate ecozone have a modest presence and vice-versa (e.g. see JANICKI et al., 2016; GUÉNARD et al., 2017). On the other hand, possible comparisons with vineyards of the northern temperate ecosphere, which host more similar faunas, are not particularly numerous. For example, despite several papers dealt with peculiar ant species and their role in North American vineyards (e.g. KLOTZ et al., 2003; TOLLERUP et al., 2004; 2007; DAANE et al., 2006; 2007; NONDILLO et al., 2016; TOWNSEND et al., 2016; WESTERMANN et al., 2016; COOPER et al., 2019), no data on vineyard overall ant diversity are available. In some other cases, an ant species check-list is provided, but it included only species that were observed foraging on the vines (BELTRÀ et al., 2017). However, some interesting comparisons may be made with data published by GONÇALVES et al. (2017) from Portugal and MASONI et al. (2017) from Central Italy. The fauna from the Portuguese sites investigated by GONÇALVES et al. (2017) comprises 20 species in total, slightly less than ours, but the number of species per site is averagely much higher (15-20) than in our case, and so it is the number of species (9) common to all their 6 investigated vineyards. It is also a very different fauna in both ecological and biogeographic terms, consisting prevalently of species and genera associated with xero-Mediterranean climatic conditions and with a clear Western-Mediterranean characterization (e.g. Cataglyphis spp., West-Mediterranean Camponotus species such as C. cruentatus (Latreille, 1802) and C. sylvaticus (Olivier, 1792), the Iberian subendemic Iberoformica genus, Aphaenogaster iberica Emery, 1908 from the xerothermophilous testaceopilosa group, Crematogaster auberti Emery, 1869). On the other hand, MASONI et al. (2017) recorded a similar number of species (19) from 10 vineyards near Florence (Tuscany, Italy), and a slightly smaller number of species per vineyard than us (5-12). The ant assemblages documented by MASONI et al. (2017) present some relevant similarities such as a relatively high Myrmica diversity and the widespread presence of F. cunicularia and M. ibericus. Concerning the latter species, MASONI et al. (2017) refer to M. structor (Latreille, 1798), but Italy most likely only hosts its cryptic sister species M. ibericus (STEINER et al., 2018; SCHIFANI et al., 2021). However, there are also relevant differences, as the reduced diversity of Lasius, and, at the same time, the more widespread presence of thermophilous species such as P. pallidula and Mediterranean Tapinoma species from the Dolichoderinae subfamily. The ecological role of the overwhelming majority of ant species that inhabit vineyards across the globe, including of those we detected in our survey, is still virtually unknown. Only three ant species, the worldwide spread invasive Argentine ant Linepithema humile (Mayr, 1868), the South American L. micans (Forel, 1908) and the North American Formica perpilosa Wheeler, W.M., 1913 have been the subject of several studies considering them as significant pests requiring control strategies in vineyards of California and Brazil (KLOTZ et al., 2003; TOLLERUP et al., 2004; 2007; DAANE et al., 2006; 2007; SACCHETT et al., 2009; NONDILLO et al., 2016; WESTERMANN et al., 2016; COOPER et al., 2019). On the other hand, another invasive species, the red imported fire ant Solenopsis invicta Buren, 1972, was deemed a positive presence due to its significant predatory action on pest species in Texas’ vineyards (TOWNSEND et al., 2016). In conclusion, ant communities in agroecosystems are diverse and often species-rich, and documenting their identities is crucial to assess the possible services and disservices that different species assemblages may yield. While vineyards are worldwide spread, their ant faunas are rather different from place to place according to local climatic and biogeographic factors, so that the few available data do not show clear patterns, which could have originated from strong homogenizing ecological constraints derived from viticulture per se. Further investigation will be required to understand how the fauna of the vineyards from the Adige Valley compares with that of other agroecosystems or natural habitats from the same region and how different management practices may influence it. It will also be important to assess what is the role that different ant species may play in these environments to improve management practices accordingly. AUTHORS CONTRIBUTIONS Conceptualization: CC, DG, DAG, CI; Data collection: MD, MG, DG, FP; Specimen processing and identification: CC, MG, ES, FAS; Data curation and analysis: CC, ES; Visualization: CC, DG, ES; Writing—original draft preparation: DG, ES; Writing—review and editing: CC, MD, DAG, CI, AM, FP, FAS; Supervision: DAG, CI, AM; Funding acquisition: DAG, CI, AM. All authors have read and agreed to the published version of the manuscript. 104 164 SCHIFANI ET AL. REDIA, Vol. 105, 2022 MATERIALS AND METHODS Study areas We investigated 222 pear trees (Pyrus communis L.) from two organic orchards whose management was restricted to periodical lawn mowing (Fig. I). The surveys were conducted during the years 2018-2019 in the same period, to minimize potential differences between the years. Since we did not aim to evaluate the temporal variation of ant communities throughout the seasons, we chose to carry out our surveys in July which is considered a period of high ant activity for most species (Schifani et al., 2022a). Site 1, Northern Italy, Emilia-Romagna, continental climate zone: Pontescodogna (Parma province) (44.7378 N, 10.1954 E, 125 m a.s.l.). This orchard includes apple, cherry, fig, peach, pear and plum trees, arranged in 15 rows and partially surrounded by a deciduous oak forest, which is part of the Regional Natural Park “Boschi di Carrega”. We investigated 166 pear trees in July 2018. Site 2, Sicily, Mediterranean climatic zone: Polizzi Generosa (Palermo province), (37.8245 N, 14.0032 E, 750 m a.s.l.). This orchard includes apple, fig, hazel, peach and pear trees, only partly arranged in rows and partially surrounded by a deciduous oak forest, which is part of the Regional Natural Park “Parco delle Madonie”. We investigated 56 pear trees in July 2019. Data collected on each tree Data sampling was organized in daily sessions from 10:00 to 12:00 am until all trees were examined. The examination of each tree was conducted through a 5 minute continuous observation period, during which all ant specimens detected on each tree were collected. They were identified by a stereomicroscope following Seifert (2018, 2019; 2020) and Schifani et al. (2022). In addition, the circumference of each tree was measured 5 cm above the ground. Statistical analyses All statistical tests were performed using the software R 4.2.0 and RStudio-2022.02.2-485 (R Core Team, 2022; RStudio Team, 2022). The circumference of each tree was correlated with the number of arborealand ground-nesting ant species running Pearson’s correlation tests. The ant communities of the two study areas were compared using the Sørensen–Dice coefficient. To each species was assigned a chorotype following Vigna Taglianti et al. (1999), with the exception of the Maghrebian and SW-European distributions as suggested by Parenzan (1994) as NAW and ESW (see also Schifani et al., 2021). RESULTS We identified 20 ant species, belonging to 10 genera of the subfamilies Formicinae (65%), Myrmicinae (30%), and Dolichoderinae (5%) (Tab. 1). Among them 5 arboreal-nesting species were found. The similarity between ant communities identified in Emilia-Romagna (11 species) and Sicily (13 species) was 35%, according to the Sørensen–Dice coefficient, and 8 genera are shared between the two sites. In Emilia-Romagna, 73% of the identified species has a European or Eurasian distribution and 27% has a Mediterranean distribution. Conversely, in Sicily, 85% of the species identified has a Mediterranean distribution and 15% has a European or Eurasian distribution. Pearson’s correlation tests show significant positive relationships between tree circumference and the number of ant species per tree (R = 0.62, p < 0.001), the number of ground-nesting ants (R = 0.54, p < 0.001), and the number of arboreal-nesting ones (R = 0.33, p < 0.001) (Fig. II). DISCUSSION We are providing a first account of ant communities visiting pear trees in Italy. Our assessment highlighted significant diversity of species between sites at different latitudes, reflecting Italy’s remarkable biogeographical complexity. The ant fauna of Northern Italy was overwhelmingly characterized by European or Eurasian taxa, while these were a small minority in Sicily where Mediterranean species were very numerous (also see Castracani et al., 2020; Wang et al., 2022). At the same time, genera and species-groups are similar between the two investigated regions, suggesting an overall remarkable functional affinity: in both Emilia-Romagna and Fig. I - The two study areas investigated in Emilia-Romagna (Northern Italy) and Sicily. 111 TRUNK SIZE INFLUENCES SPECIES RICHNESS AND FUNCTIONAL COMPOSITION... 165 Fig. II - Results of the Pearson’s correlation tests between tree circumference and the number of ants found visiting the same tree on pear trees from Northern Italy and Sicily. Tab. 1 - Ant species visiting trees in the two study areas. Arboreal-nesting species are in bold. Subfamily Species Chorotype Visited trees Emilia Romagna Sicily Dolichoderinae Dolichoderus quadripunctatus (Linnaeus, 1771) TUE 8% - Formicinae Camponotus aethiops (Latreille, 1798) SEU - 27% Camponotus gestroi Emery, 1878 MED - 9% Camponotus lateralis (Olivier, 1792) MED - 3% Camponotus nylanderi Emery, 1921 ITAL - 18% Camponotus piceus (Leach, 1825) ESW 58% 30% Camponotus vagus (Scopoli, 1763) CEM 2% - Colobopsis truncata (Spinola, 1808) TEM 3% - Colobopsis imitans Schifani et al., 2021 NAW -3% Formica cunicularia Latreille, 1798 ASE 38% 6% Lasius casevitzi Seifert & Galkowski, 2016 THYRR - 12% Lasius niger (Linnaeus, 1758) ASE 34% - Lasius paralienus Seifert, 1992 EUR 51% - Plagiolepis pygmaea (Latreille, 1798) ASE 49% 18% Myrmicinae Crematogaster laestrygon Emery, 1869 NAW - 15% Crematogaster scutellaris (Olivier, 1792) WME 8% 18% Myrmica sabuleti Meinert, 1861 TUE 1% - Pheidole pallidula (Nylander, 1849) ESW -3% Temnothorax italicus (Consani, 1952) ITAL 2% - Temnothorax mediterraneus Ward et al., 2014 WME - 4% 112 166 SCHIFANI ET AL. REDIA, Vol. 105, 2022 Sicily most species belong to the subfamily Formicinae. Although counting only about half of the Italian Myrmicinae species (Schifani, 2022), this group plays a particularly important role in ant-plant interactions in temperate areas (Wilson & Hölldobler, 2009). Out of 10 occurring genera, only Dolichoderus, Pheidole, and Myrmica are not shared between the two investigated areas. Sicily has a larger amount of Camponotus and Crematogaster species, while the more cold tolerant genera Lasius and Formica play a more important role in Emilia-Romagna. Besides that, the two communities often host either of two closely related, often vicariant species, such as Colobopsis truncata and Co. imitans, Lasius paralienus and L. casevitzi, or at least species of the same genus with similar ecology, such as Temnothorax italicus and T. mediterraneus (Schär et al., 2020; Schifani, et al., 2022). In addition, Camponotus piceus was the sole common ant in both sites. The number of species we found in each community reflects that of comparable studies in other temperate regions (Tschinkel & Hess, 1999), and it is obviously much lower than in tropical forests (e.g. Klimes et al., 2015). We found few more species in Sicily, but on average each tree hosted less ants, maybe because July is a less favorable month for ant activity under the hot and arid Mediterranean climate (Retana & Cerdá, 2000; Schifani et al., 2020b). Our results show a significant increase of both arboreal and ground nesting species when larger trees are present. This contrasts with the outcome of other studies, where the occurrence of dominant tree inhabiting Crematogaster spp. on larger trees reduces ant species richness (Tschinkel & Hess, 1999). The dominant arboreal species Cr. scutellaris seemingly does not affect the presence of other ants on the trees we investigated. Also, it is worth mentioning that at least two species (Camponotus lateralis and Co. imitans) can take advantage of the presence of Cr. scutellaris (Schifani et al., 2022). Arboreal nesting species represent a small portion of the observed ant diversity and are almost exclusively limited to medium and large trees. The lack of sufficient deadwood for nesting in younger trees may be the main factor affecting their presence (Tschinkel & Hess, 1999; Schlaghamerský & Omelková, 2007), although the relationships between ants and deadwood availability is still little explored if compared to the studies of other insects (e.g. Campanaro et al., 2011; Thomaes et al., 2018). Artificial nests may be adopted to overcome this issue whenever the presence of tree inhabiting ants, such as Cr. scutellaris, which may play a role in pest control (e.g. Castracani et al., 2017; Giannetti et al., 2019), is needed (Philpott & Foster, 2005). ACKNOWLEDGEMENTS We thank Emanuele Genduso and Roberto Reggiani for granting us access to the pear orchards examined in this study. We also thank Tommaso Vighi for assisting us during data collection, and two anonymous referees for their comments on the manuscript. This study benefited from the equipment and framework of the COMP-HUB Initiative, funded by the “Departments of Excellence” program of the Italian Ministry for Education, University and Research (MIUR, 2018–2022). REFERENCES Baraibar B., Carrión E., Recasens J., Westerman P.R., 2011 – Unravelling the process of weed seed predation: developing options for better weed control. - Biological Control, 56: 85-90. Boomsma J. J., Brady S.G., Dunn R.R., Gadau J., Heinze J., Keller L., Moreau C.S., Sanders N.J., Schrader L., Schultz T.R., Sundström L., Ward P.S., Wcislo WT., Zhang G., Ehab A., Adams R.M.M., Aron S., Bacci M., Bengston S.E., Bernadou A., Blaimer B.B., Boulay R., Bornberg-Bauer E., Branstetter M.G., Economo E.P., Feinerman O., Feldhaar H., Feldmeyer B., Fisher B., Foitzik S., Frank E.T., Grasso D.A., Helanterä H., Holman L., Hughes W., Jongepier E., Kronauer D., Kulmuni J., Linksvayer T.A., Longino J.T., McKenzie S.K., Orivel J., Rabeling C., Ran H., Rubin B.E.R., Seal J.N., Seifert B., Shik J.Z., Tinaut A., Tsutsui N.D., Tysklind N., Vargo E., Vilijakainen L., Yannick W., Xu Y., Yek S.H., 2017 – The global ant genomics Alliance (GAGA). - Myrmecological News, 25: 61-66. Bulgarini, G., Castracani, C., Mori, A., Grasso, D.A., Maistrello, L., 2021 – Searching for new predators of the invasive Halyomorpha halys: the role of the black garden ant Lasius niger. – Entomologia Experimentalis et Applicata, 169: 799-806. Campanaro A., Toni I., Hadersen S., Grasso D.A., 2011 – Monitoring of Lucanus cervus by means of remains of predation (Coleoptera: Lucanidae). - Entomologia Generalis, 33: 79-89. Campolo O., Palmeri V., Malacrinò A., Laudani F., Castracani C., Mori A., Grasso D.A., 2015 – Interaction between ants and the Mediterranean fruit fly: new insights for biological control. - Biological Control, 90: 120-127. Castracani C., Mori A. 2006 – The role of permanent grasslands on ant community structure: ants (Hymenoptera: Formicidae) as ecological indicators in the agro-ecosystems of the Taro River Regional Park (Italy). - Myrmecologische Nachrichten, 9: 47-54. Castracani C., Maienza A., Grasso D.A., Genesio L., Malcevschi A., Miglietta F., Vaccari F.P., Mori A., 2015 – Biochar–macrofauna interplay: Searching for new bioindicators. - Science of the Total Environment, 536: 449-456. Castracani C., Bulgarini G., Giannetti D., Spotti F.A., Maistrello L., Mori A., Grasso D.A., 2017 – Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys. - Journal of Pest Science, 90: 1181-1190. Castracani C., Spotti F.A., Schifani E., Giannetti D., Ghizzoni M., Grasso D.A., Mori A., 2020 – Public 113 TRUNK SIZE INFLUENCES SPECIES RICHNESS AND FUNCTIONAL COMPOSITION... 167 engagement provides first insights on Po Plain ant communities and reveals the ubiquity of the cryptic species Tetramorium immigrans (Hymenoptera, Formicidae). - Insects, 11: 678. de Bruyn L.A.L., 1999 – Ants as bioindicators of soil function in rural environments. - Agriculture, Ecosystems & Environment, 74: 425-441. Frizzi F., Masoni A., Ottonetti L., Tucci L., Santini G., 2020 – Resource-dependent mutual association with sap-feeders and a high predation rate in the ant Crematogaster scutellaris: help or harm in olive pest control?. - Biocontrol, 65: 601-611. Giannetti D., Castracani C., Spotti F.A., Mori A., Grasso D.A., 2019 – Gall-colonizing ants and their role as plant defenders: from’bad job’ to ’useful service’. - Insects, 10: 392. Giannetti D., Schifani E., Castracani C., Ghizzoni M., Delaiti M., Pfenner F., Spotti F.A., Mori A., Ioriatti C., Grasso D.A., 2021 – Assessing ant diversity in agroecosystems: the case of Italian vineyards of the Adige Valley. - Redia, 104: 97-109. Giannetti, D., Schifani, E., Gugliuzzo, A., Zappalà, L., Biondi, A., Grasso, D.A. (2022). – Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus. - Biological Control, 174: 105032. Grasso D.A., Mori A., Le Moli F., 1998 – Chemical communication during foraging in the harvesting ant Messor capitatus (Hymenoptera, Formicidae). Insectes Sociaux, 45: 85-96. Grasso D.A., Mori A., Le Moli F., 1999 – Recruitment and trail communication in two species of Messor ants (Hymenoptera, Formicidae). Italian Journal of Zoology, 66: 373-378. Hölldobler B., Wilson E.O. 1990 – The ants. - Harvard University Press. Klimes P., Idigel C., Rimandai M., Fayle T.M., Janda M., Weiblen G.D., Novotny V., 2012 – Why are there more arboreal ant species in primary than in secondary tropical forests?. Journal of Animal Ecology, 81: 1103-1112. Klimes P., Fibich P., Idigel C., Rimandai M., 2015 – Disentangling the diversity of arboreal ant communities in tropical forest trees. - PloS One, 10: e0117853. Lach L., Lori L., Parr L., Catherine L., Abbot K., 2010 – Ant Ecology. - Oxford University Press. La Pergola A., Alicata A., Longo S., 2008 – Survey of the ants (Hymenoptera Formicidae) in citrus orchards with different types of crop management in Sicily. - IOBC/wprs Bulletin, 38: 233-237. Masoni A., Frizzi F., Brühl C., Zocchi N., Palchetti E., Chelazzi G., Santini G., 2017 – Management matters: A comparison of ant assemblages in organic and conventional vineyards. - Agriculture, Ecosystems & Environment, 246: 175-183. Offenberg J., 2015 – Ants as tools in sustainable agriculture. - Journal of Applied Ecology, 52: 1197-1205. Offenberg J., Damgaard C., 2019 – Ants suppressing plant pathogens: a review. - Oikos, 128: 1691-1703. Ottonetti L., Tucci L., Chelazzi G., Santini G., 2008 – Stable isotopes analysis to assess the trophic role of ants in a Mediterranean agroecosystem. - Agricultural and Forest Entomology, 10: 29-36. Parezan P., 1994 – Proposta di codificazione per una gestione informatica dei corotipi W-paleartici, con particolare riferimento alla fauna italiana. - Entomologica, 28: 93-98. Parker J., Kronauer D.J., 2021 – How ants shape biodiversity. - Current Biology, 31: R1208-R1214. Paulson G.S., Akre D.K., 1992 – Introducing ants (Hymenoptera: Formicidae) into pear orchards for the control of pear psylla, Cacopsylla pyricola (Foerster) (Homoptera: Psyllidae). Journal of Agricultural Entomology, 9: 37-39. Philpott S.M., Foster P.F., 2005 – Nest‐site limitation in coffee agroecosystems: artificial nests maintain diversity of arboreal ants. - Ecological applications, 15: 1478-1485. Philpott S.M., Armbrecht I., 2006 – Biodiversity in tropical agroforests and the ecological role of ants and ant diversity in predatory function. - Ecological Entomology 31: 369-77. R Core Team, 2022 – R: A language and environment for statistical computing. - R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/. Retana J., Cerdá X., 2000 – Patterns of diversity and composition of Mediterranean ground ant communities tracking spatial and temporal variability in the thermal environment. - Oecologia, 123: 436-444. RStudio Team, 2022 – RStudio: Integrated Development for R. - RStudio, PBC, Boston, MA URL http:// www.rstudio.com/. Sanchez J.A., Carrasco‐Ortiz A., López‐Gallego E., La‐Spina M., 2020 – Ants (Hymenoptera: Formicidae) reduce the density of Cacopsylla pyri (Linnaeus, 1761) in Mediterranean pear orchards. - Myrmecological News, 30: 93-102. Schär S., Menchetti M., Schifani E., Hinojosa J.C., Platania L., Dapporto L., Vila R., 2020 – Integrative biodiversity inventory of ants from a Sicilian archipelago reveals high diversity on young volcanic islands (Hymenoptera: Formicidae). - Organisms Diversity & Evolution, 20: 405-416. Schifani E., Castracani C., Giannetti D., Spotti F.A., Reggiani R., Leonardi S., Mori A., Grasso D.A., 2020a – New Tools for Conservation Biological Control: Testing Ant-Attracting Artificial Nectaries to Employ Ants as Plant Defenders. - Insects, 11: 129. Schifani E., Scupola A., Alicata A., 2020b – Morphology, ecology and biogeography of Myrmecina sicula André, 1882, rediscovered after 140 years (Hymenoptera, Formicidae). – Biogeographia 35: 105-116. Schifani E., Nalini E., Gentile V., Alamanni F., Ancona C., Caria M., Cillo D., Bazzato E., 2021 – Ants of Sardinia: an updated checklist based on new 114 168 SCHIFANI ET AL. REDIA, Vol. 105, 2022 faunistic, morphological and biogeographical notes. - Redia, 104: 21-35. Schifani E., Giannetti D., Csősz S., Castellucci F., Luchetti A., Castracani C., Spotti F.A., Mori A., Grasso D.A., 2022 – Is mimicry a diversification-driver in ants? Biogeography, ecology, ethology, genetics and morphology define a second West-Palaearctic Colobopsis species (Hymenoptera: Formicidae). - Zoological Journal of the Linnean Society, 194: 1424-1450. Schifani E., 2022 – The new Checklist of the Italian Fauna: Formicidae. - Biogeographia, 37: ucl006. Schlaghamersky J., Omelkova M., 2007 – The present distribution and nest tree characteristics of Liometopum microcephalum (Panzer, 1798) (Hymenoptera: Formicidae) in South Moravia. - Myrmecological News, 10: 85-90. Seifert B., 2018 – Ants of Northern and Central Europe. - Lutra Verlagsund Vertriebsgesellschaft, Tauer, Germany. Seifert B., 2019 – A taxonomic revision of the members of the Camponotus lateralis species group (Hymenoptera: Formicidae) from Europe, Asia Minor and Caucasia. - Soil Organisms, 91: 7-32. Seifert B., 2020 – A taxonomic revision of the Palaearctic members of the ant subgenus Lasius s. str. (Hymenoptera, Formicidae). - Soil Organisms, 92: 15-86. Solida L., Grasso D.A., Testi A., Fanelli G., Scalisi M., Bartolino V., Mori A., Fanfani A., 2011 – Differences in the nesting sites microhabitat characteristics of two syntopic species of Messor harvester ants in a phytosociological homogeneous grassland area. - Ethology Ecology & Evolution, 23: 229-239. Thomaes A., Dhont P., Dekeukeleire D., Vandekerkhove K., 2018 – Dispersal behaviour of female stag beetles (Lucanus cervus) in a mosaic landscape: when should I stay and where should I go. - Insect Conservation and Diversity, 11: 523-533. Tschinkel W.R., Hess C.A., 1999 – Arboreal ant community of a pine forest in northern Florida. - Annals of the Entomological Society of America, 92: 63-70. Vigna Taglianti, A., Audisio, P. A., Biondi, M., Bologna, M. A., Carpaneto, G. M., De Biase, A., Fattorini S., Piattella E., Sindaco R., Venchi A., Zapparoli M., 1999 – A proposal for a chorotype classification of the Near East fauna, in the framework of the Western Palearctic region. - Biogeographia, 20: 31-59. Wang, R., Kass, J.M., Galkowski, C., Garcia, F., Hamer, M.T., Radchenko, A., Salata, S., Schifani, E., Yusupov, Z.M., Economo, E.P., & Guénard, B. (2022) – Geographic and climatic constraints on bioregionalization of European ants. - Journal of Biogeography (in press). Way M.J., Khoo K.C., 1992 – Role of ants in pest management. - Annual review of Entomology, 37: 479503. Wilson E.O., Hölldobler B., 2009 – The superorganism: The beauty, elegance, and strangeness of insect societies. - W. W Norton & Company, New York. 115 Section III - First data on ant diversity and distribution in Italian agroecosystems Chapter 11 Developing the DNA barcoding library of European ants: the species of the Italian agroecosystems _______________________________________________________________________________ 116 Developing the DNA barcoding library of European ants: the species of the Italian agroecosystems Mattia Menchetti1, Enrico Schifani2, Donato A. Grasso2, Roger Vila1 1 Institut de Biologia Evolutiva (CSIC-Univ. Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain 2 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy Introduction DNA barcoding is a widespread specimen identification method that is based on sequencing a short DNA section of a specimen and comparing it to a library of sequences of already identified specimens. The ability of DNA barcoding to successfully provide species-level identification can greatly vary across taxonomic groups, since different species may sometimes share the same haplotype (barcode sharing) because of different possible mechanisms (e.g., introgression, incomplete lineage sorting). However, DNA barcoding remains a cost-effective tool across many different taxonomic groups and can also be used for the simultaneous identification of multiple taxa from the same sample (metabarcoding). The most basic limitation to the use of DNA barcoding and metabarcoding is related to the availability of a reliable reference library. Sequences in the libraries must be correctly linked to species-level identifications, as many organisms as possible must sequenced, and for each taxon, as many representative of different populations should be included in order to cover spatial variation and assess intraspecific variation. The construction of a DNA barcoding library for European ants is a key part of the Genetic Map of European ants project (AntGem - https://www.mattiamenchetti.com/antgem). 117 To obtain a library containing at least about 80% of the over six hundred European ant species, the project produced thousands of new barcode sequences and re-analyzed those that were already available. Here, we present an overview of the data collected for the 34 ant species that were encountered and studied through the chapters of this thesis for their presence and role in the Italian agroecosystems. Materials and methods DNA was extracted from one or a few legs of each ant specimen. DNA-barcoding (mitochondrial gene cytochrome c oxidase I, COI, 658 bp) data was generated at two institutes: the Centre for Biodiversity Genomics, University of Guelph, Canada, using the primers LepF1 and LepR1 (deWaard et al. 2008); the Butterfly Diversity and Evolution Lab (BDEL), following the protocol by Schr et al. (2020) and using the primers LCO1490/HC02198 (Folmer et al. 1994). In the latter case, PCR products were visualized by gel electrophoresis and sent to Macrogen Europe for Sanger sequencing. Raw sequences were edited and aligned in Geneious Prime 2020.2.4 (Kearse et al. 2012). Chromatograms and sequences have been inspected for the presence of, respectively, double peaks and stop codons. Results A total of 836 new sequences were produced for the 34 ant species listed in the previous chapters, which summed with the available ones that were analyzed are 1,192 sequences (from 4 to 80 per species – see Table 1). For each species, we assembled a haplotype network and a Principal Coordinate Analysis (PCoA) to illustrate intraspecific diversity patterns (Fig. 1). Furthermore, to illustrate phylogeographic patterns, we built a geographic map of each species using the colors derived from the PCoA to plot the spatial distribution of the haplotypes found across different populations (Fig. 2). 118 Discussion Our results suggest that DNA barcoding may be used effectively to identify species belonging to cryptic species complexes, such as Tapinoma subboreale or Tetramorium immigrans whose morphological identification can be very demanding (requiring the use of male genitalia in the first case, and of male genitalia or several time-consuming morphometric measurements in the second – see Seifert 2012; Wagner et al. 2017). At the same time, about one-fourth of the ant species examined here showed barcode sharing and were therefore not unequivocally distinguishable by DNA barcoding only. This group included species of presumably recent divergence for which incomplete lineage sorting may be the underlying mechanism (e.g., Colobopsis spp., see Schifani et al. 2022) or species with complex introgression history (e.g., Myrmica sabuleti, see Blatrix et al. 2020). The DNA fragment used for barcoding in ants also proved to be a valid tool to explore biogeographic patterns in many species, as illustrated in the example of Crematogaster scutellaris. DNA barcoding appears as a promising tool to facilitate ant identification, but more caution is needed in comparison to other groups of organisms in which barcode sharing is rarer. Acknowledgments The Genetic Map of European Ants project is made possible by the collaboration of several ant enthusiasts, while numerous experts played a crucial role in it: Bonnie Blaimer (Museum für Naturkunde – Berlin, Germany), Lech Borowiec (University of Wroclaw – Wroclaw, Poland), Leonardo Dapporto (University of Florence – Florence, Italy), Fede García (Barcelona, Spain), Kiko Gómez (Barcelona, Spain), José María Gómez Durán (Madrid, Spain), Paul D.N. Hebert (University of Guelph – Guelph, Canada), Albena Lapeva-Gjonova (Sofia University “Sv. Kliment Ohridski” – Sofia, Bulgaria), Francisca Ruano (University of Granada – Granada, Spain), Sebastian Salata (University of Wroclaw – Wroclaw, Poland), Jose Alberto Tinaut Ranera (University of Granada – Granada, Spain), Smi Schr (Switzerland), and Tomasz Suchan (Polish Academy of Sciences – Kraków, Poland). 119 Table 1. DNA barcode data collected for the 34 ant species mentioned in the previous chapters of this thesis. Species New barcode sequences Total number of barcode sequences Barcode sharing with other European ants Max intraspecific p-dist Median intraspecific p-dist Camponotus aethiops 55 67 0.096 0.021 Camponotus vagus 46 50 0.070 0.005 Camponotus piceus 41 48 0.065 0.009 Camponotus lateralis 46 50 0.026 0.011 Camponotus nylanderi 11 15 0.017 0.003 Camponotus gestroi 8 8 0.115 0.107 Colobopsis truncata 38 64 Yes 0.023 0.004 Colobopsis imitans 4 14 Yes 0.025 0.002 Crematogaster laestrygon 6 6 0.055 0.051 Crematogaster scutellaris 56 61 0.032 0.021 Crematogaster sordidula 36 39 0.104 0.021 Dolichoderus quadripunctatus 30 45 0.045 0.003 Formica cinerea 16 21 Yes 0.040 0.008 Formica cunicularia 46 52 Yes 0.070 0.035 Lasius casevitzi 11 21 0.009 0.002 Lasius emarginatus 43 54 Yes 0.029 0.005 Lasius myops 29 35 0.046 0.006 Lasius niger 39 62 0.008 0.000 Lasius paralienus 14 20 0.018 0.012 Messor ibericus 23 51 0.003 0.000 Myrmica sabuleti 20 27 Yes 0.035 0.006 Myrmica specioides 15 15 0.025 0.017 Nylanderia jaegerskioeldi 11 12 0.003 0.000 Pheidole pallidula 47 52 0.135 0.103 Plagiolepis pygmaea 47 66 0.110 0.003 Solenopsis fugax 36 38 0.063 0.046 Tapinoma magnum 24 80 Yes 0.046 0.014 Tapinoma subboreale 16 18 0.023 0.014 Temnothorax affinis 15 21 0.039 0.020 Temnothorax italicus 4 4 Yes 0.006 0.003 Temnothorax mediterraneus 5 11 0.034 0.029 Temnothorax unifasciatus 11 18 Yes 0.121 0.086 Tetramorium immigrans 13 69 0.024 0.006 Tetramorium semilaeve 32 53 0.037 0.018 120 the ant species and communities that inhabit them, with integrative approaches offering the most promising perspectives to unravel complex diversity patterns (Chapters 9-11). This can also lead to learning how different management approaches can influence these ant communities, thus influencing the different services and disservices they may provide, as already observed in other regions (Perfecto & Vandermeer 1996). It also contributes to evaluating the effects of management practices on biodiversity in agricultural systems, which is crucial to insect conservation across Europe (Wagner et al. 2021). The study of ants' role in Mediterranean agroecosystems has just begun during the last few decades, and the diversity of these environments will make their exploration a long but fascinating journey. Many directions still must be investigated, and while complexity may, unfortunately, delay the discovery of practical answers to some agronomical problems, it will certainly reward those who seek with several cues on wider ecological and evolutionary aspects on the evolutionary history of ants and the arthropod communities they are part of. 127 6. Acknowledgements My journey would not have been possible without the support and great trust of my supervisor, Donato A. Grasso, who first introduced me to the topic of ant role in agroecosystems in 2017, wonderfully communicating the beauty of a research line that naturally leads to answers that are relevant to both basic and applied research. A special thanks to Daniele Giannetti for his staunch support throughout all the experiments that formed this thesis, as well as to the rest of the members of the Insect Ethology, Ecology and Sociobiology Lab of the University of Parma - Cristina Castracani, Fiorenza A. Spotti, and Alessandra Mori – a research group that welcomed me and supported me like a family throughout these years and introduced me to many aspects of the amazing world of myrmecological research. I wholeheartedly express my gratitude to Roger Vila and Mattia Menchetti, who warmly hosted me at the Institute of Evolutionary Biology in Barcelona for some extraordinarily intense and enthusiastic months of work, together with the other colleagues at the Butterfly Biodiversity and Evolution Lab and the IBE staff. I am also grateful to my former Entomology professors, Stefano Colazza and Ezio Peri (University of Palermo) whose guidance encouraged me to take this step in my career and who kept supporting me throughout. The research presented in this thesis would not have been possible without the involvement of many researchers and students I had the privilege to work with, coauthoring, or acknowledged in the Chapters of this thesis. I also wish to thank the PhD course coordinator, Pierluigi Viaroli, and the staff of the University of Parma for their support and patience, as well as Dean Adams and Stefano Leonardi for their classes and teachings. Exploring the world of scientific research during these years would not have been the same without the amazing experience of meeting and collaborating with a community of dozens of other myrmecologists, entomologists, and biodiversity enthusiasts in Italy and abroad, many of whom I was lucky to have as coauthors. I thank all the friends in Palermo, Parma, Barcelona, and elsewhere, whose affection and joy surrounded me, and, finally my parents, for their unwavering and unparalleled support and love. 128 7. References Armbrecht, I., Perfecto, I., & Silverman, E. (2006). Limitation of nesting resources for ants in Colombian forests and coffee plantations. Ecological Entomology, 31(5), 403-410. https://doi.org/10.1111/j.1365-2311.2006.00802.x Anjos, D. V., Tena, A., Viana-Junior, A. B., Carvalho, R. L., Torezan-Silingardi, H., Del-Claro, K., & Perfecto, I. (2022). The effects of ants on pest control: a metaanalysis. Proceedings of the Royal Society B, 289(1981), 20221316. https://doi.org/10.1098/rspb.2022.1316 Bale, J. S., Van Lenteren, J. C., & Bigler, F. (2008). Biological control and sustainable food production. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1492), 761-776. https://doi.org/10.1098/rstb.2007.2182 Banks, C. J., & Macaulay, E. D. M. (1967). Effects of Aphis fabae Scop, and of its attendant ants and insect predators on yields of field beans (Vicia faba L.). Annals of Applied Biology, 60(3), 445-453. https://doi.org/10.1111/j.1744-7348.1967.tb04499.x Baraibar, B., Ledesma, R., Royo-Esnal, A., & Westerman, P. R. (2011a). Assessing yield losses caused by the harvester ant Messor barbarus (L.) in winter cereals. Crop Protection, 30(9), 1144-1148. https://doi.org/10.1016/j.cropro.2011.05.010 Baraibar, B., Carrión, E., Recasens, J., & Westerman, P. R. (2011b). Unravelling the process of weed seed predation: Developing options for better weed control. Biological Control, 56(1), 85-90. https://doi.org/10.1016/j.biocontrol.2010.09.010 Bolton, B. 2023. An online catalog of the ants of the world. Available from https://antcat.org. Borbély, C., & Nagy, C. (2022). Providing sugar sources for ants improves the biological control of Aphis spp. in apple orchards. Biological Control, 175, 105056. https://doi.org/10.1016/j.biocontrol.2022.105056 129 Branstetter, M. G., Danforth, B. N., Pitts, J. P., Faircloth, B. C., Ward, P. S., Buffington, M. L., Gates, M. W., Kula, R. R., & Brady, S. G. (2017). Phylogenomic insights into the evolution of stinging wasps and the origins of ants and bees. Current Biology, 27(7), 1019-1025. http://dx.doi.org/10.1016/j.cub.2017.03.027 Campolo, O., Palmeri, V., Malacrinò, A., Laudani, F., Castracani, C., Mori, A., & Grasso, D. A. (2015). Interaction between ants and the Mediterranean fruit fly: New insights for biological control. Biological Control, 90, 120-127. https://doi.org/10.1016/j.biocontrol.2015.06.004 Castracani, C., Bulgarini, G., Giannetti, D., Spotti, F. A., Maistrello, L., Mori, A., & Grasso, D. A. (2017). Predatory ability of the ant Crematogaster scutellaris on the brown marmorated stink bug Halyomorpha halys. Journal of Pest Science, 90, 11811190. https://doi.org/10.1007/s10340-017-0889-1 Cheng, S., Zeng, L., & Xu, Y. (2015). Mutualism between fire ants and mealybugs reduces lady beetle predation. Journal of Economic Entomology, 108(4), 1560-1569. https://doi.org/10.1093/jee/tov117 Cocco, A., Pacheco da Silva, V. C., Benelli, G., Botton, M., Lucchi, A., & Lentini, A. (2021). Sustainable management of the vine mealybug in organic vineyards. Journal of Pest Science, 94, 153-185. https://doi.org/10.1007/s10340-020-01305-8 Daane, K. M., Sime, K. R., Fallon, J., & Cooper, M. L. (2007). Impacts of Argentine ants on mealybugs and their natural enemies in California’s coastal vineyards. Ecological Entomology, 32(6), 583-596. https://doi.org/10.1111/j.13652311.2007.00910.x De Bruyn, L. L., & Conacher, A. J. (1990). The role of termites and ants in soil modification-a review. Soil research, 28(1), 55-93. https://doi.org/10.1071/SR9900055 130 De Bruyn, L. L. (1999). Ants as bioindicators of soil function in rural environments. In: Invertebrate Biodiversity as Bioindicators of Sustainable Landscapes (pp. 425441). Elsevier. https://doi.org/10.1016/S0167-8809(99)00047-X Delabie, J. H. C., Da Encarnacao, A. M. V., Cazorla, I. M. (2021). Relations between the little fire ant, Wasmannia auropunctata, and its associated mealybug, Planococcus citri, in Brazilian cocoa farms. In: Exotic Ants (pp. 91-103). CRC Press. Della Lucia, T. M., Gandra, L. C., & Guedes, R. N. (2014). Managing leaf‐cutting ants: peculiarities, trends and challenges. Pest Management Science, 70(1), 14-23. https://doi.org/10.1002/ps.3660 Giannetti, D., Mandrioli, M., Schifani, E., Castracani, C., Spotti, F. A., Mori, A., & Grasso, D. A. (2021). First report on the acrobat ant Crematogaster scutellaris storing live aphids in its oak-gall nests. Insects, 12(2), 108. https://doi.org/10.3390/insects12020108 Guénard, B., Weiser, M. D., Gomez, K., Narula, N., & Economo, E. P. (2017). The Global Ant Biodiversity Informatics (GABI) database: synthesizing data on the geographic distribution of ant species (Hymenoptera: Formicidae). Myrmecological News, 24, 83-89. Hölldobler, B., & Wilson, E. O. (1990). The ants. Harvard University Press, Cambridge, USA. Hölldobler, B., & Wilson, E. O. (2008). The Superorganism: The Beauty, Elegance and Strangeness of Insect Societies. WW Norton & Co, New York, USA. Huang, H. T., & Yang, P. (1987). The ancient cultured citrus ant. BioScience, 37(9), 665-671. https://doi.org/10.2307/1310713 Kaplan, I., & Eubanks, M. D. (2002). Disruption of cotton aphid (Homoptera: Aphididae)—natural enemy dynamics by red imported fire ants (Hymenoptera: 131 Formicidae). Environmental Entomology, 31(6), 1175-1183. https://doi.org/10.1603/0046-225X-31.6.1175 Kass, J. M., Guénard, B., Dudley, K. L., Jenkins, C. N., Azuma, F., Fisher, B. L., Parr, C. L., Gibb, H., Longino, J. T., Ward, P. S., Chao, A., Lubertazzi, D., Weiser, M., Jetz, W., Guralnick, R., Blatrix, R., Des Lauriers, J., Donoso, D. A., Georgiadis, C., Gomez, K., Hawkes, P. G., Johnson, R. A., Lattke, J. E., MacGown, J. A., Mackay, W., Robson, S., Sanders, N. J., Dunn, R. R., & Economo, E. P. (2022). The global distribution of known and undiscovered ant biodiversity. Science Advances, 8(31), eabp9908. https://doi.org/10.1126/sciadv.abp9908 Lach, L., Parr, C., & Abbott, K. (Eds.). (2010). Ant ecology. Oxford University Press, Oxford, UK. Mgocheki, N., & Addison, P. (2009). Interference of ants (Hymenoptera: Formicidae) with biological control of the vine mealybug Planococcus ficus (Signoret)(Hemiptera: Pseudococcidae). Biological Control, 49(2), 180-185. https://doi.org/10.1016/j.biocontrol.2009.02.001 Offenberg, J. (2015). Ants as tools in sustainable agriculture. Journal of Applied Ecology, 52(5), 1197-1205. https://doi.org/10.1111/1365-2664.12496 Offenberg, J., & Damgaard, C. (2019). Ants suppressing plant pathogens: a review. Oikos, 128(12), 1691-1703. https://doi.org/10.1111/oik.06744 Offenberg, J., Jensen, I. C., & Hansen, R. R. (2022). Combatting plant diseases with ant chemicals: A review and meta‐analysis. Journal of Applied Ecology, 59(1), 25-38. https://doi.org/10.1111/1365-2664.14017 Parker, J., & Kronauer, D. J. (2021). How ants shape biodiversity. Current Biology, 31(19), R1208-R1214. https://doi.org/10.1016/j.cub.2021.08.015 132 Perfecto, I., & Vandermeer, J. (1996). Microclimatic changes and the indirect loss of ant diversity in a tropical agroecosystem. Oecologia, 108, 577-582. https://doi.org/10.1007/BF00333736 Plata, Á., Gómez-Martínez, M. A., Beitia, F. J., & Tena, A. (2023). Native ants facilitate the invasion by Delottococcus aberiae in Mediterranean citrus. Journal of Pest Science, 1-13. https://doi.org/10.1007/s10340-023-01615-7 Queiroz, J. M., & Oliveira, P. S. (2001). Tending ants protect honeydew-producing whiteflies (Homoptera: Aleyrodidae). Environmental Entomology, 30(2), 295-297. https://doi.org/10.1603/0046-225X-30.2.295 Rico-Gray, V., & Oliveira, P. S. (2008). The ecology and evolution of ant-plant interactions. University of Chicago Press, Chicago, USA. Risch, S. J., & Carroll, C. R. (1982). The ecological role of ants in two Mexican agroecosystems. Oecologia, 55, 114-119. https://doi.org/10.1007/BF00386726 Romiguier, J., Borowiec, M. L., Weyna, A., Helleu, Q., Loire, E., La Mendola, C., Rabeling, C., Fisher, B.L., Ward, P.S., & Keller, L. (2022). Ant phylogenomics reveals a natural selection hotspot preceding the origin of complex eusociality. Current Biology, 32(13), 2942-2947. https://doi.org/10.1016/j.cub.2022.05.001 Schultheiss, P., Nooten, S. S., Wang, R., Wong, M. K., Brassard, F., & Guénard, B. (2022). The abundance, biomass, and distribution of ants on Earth. Proceedings of the National Academy of Sciences, 119(40), e2201550119. https://doi.org/10.1073/pnas.2201550119 Styrsky, J. D., & Eubanks, M. D. (2007). Ecological consequences of interactions between ants and honeydew-producing insects. Proceedings of the Royal Society B: Biological Sciences, 274(1607), 151-164. https://doi.org/10.1098/rspb.2006.3701 133 Van Mele, P. (2008). A historical review of research on the weaver ant Oecophylla in biological control. Agricultural and Forest Entomology, 10(1), 13-22. https://doi.org/10.1111/j.1461-9563.2007.00350.x Wagner, D. L., Grames, E. M., Forister, M. L., Berenbaum, M. R., & Stopak, D. (2021). Insect decline in the Anthropocene: Death by a thousand cuts. Proceedings of the National Academy of Sciences, 118(2), e2023989118. https://doi.org/10.1073/pnas.2023989118 Wang, B., Lu, M., Peng, Y. Q., & Segar, S. T. (2021). Direct and indirect effects of invasive vs. native ant-hemipteran mutualism: a meta-analysis that supports the mutualism intensity hypothesis. Agronomy, 11(11), 2323. https://doi.org/10.3390/agronomy11112323 Wezel, A., Bellon, S., Doré, T., Francis, C., Vallod, D., & David, C. (2009). Agroecology as a science, a movement and a practice. A review. Agronomy for Sustainable Development, 29, 503-515. https://doi.org/10.1051/agro/2009004 134 Index of other articles produced during the years 2020-2023 Ant taxonomy, systematics, and evolution 1. Akbar, S.A., Bharti, H., Schifani, E., & Wachkoo, A.A. (2023). Taxonomic study of the ant genus Vollenhovia (Hymenoptera, Formicidae) in India, with two new species from the Western Ghats biodiversity hotspot. European Journal of Taxonomy, 908(1), 77-107. https://doi.org/10.5852/ejt.2023.908.2339 2. Akbar, S.A., Schifani, E., Bharti, H., & Wachkoo, A.A. (2023). New species of the Tetramorium tortuosum group (Hymenoptera: Formicidae) from the Western Ghats hotspot, with a key to the Indian and Sri Lankan taxa. Annales Zoologici Fennici, 60(1), 109-126. https://doi.org/10.5735/086.060.0112 3. Schifani, E., & Alicata, A. (2023). Nomenclatural changes on some Mediterranean Aphaenogaster Mayr, 1853 taxa (Hymenoptera, Formicidae). Zootaxa, 5277(1), 59-70. https://doi.org/10.11646/zootaxa.5277.1.2 4. Schifani, E., Alicata, A., Borowiec, L., García, F., Gentile, V., Gómez, K., Nalini, E., Rigato, F., Schär, S., Scupola, A., Vila, R., & Menchetti, M. (2023). Unrecognized for centuries: distribution and sexual caste descriptions of the West European Aphaenogaster species of the subterranea group (Hymenoptera, Formicidae). Zookeys, 1153, 141-156. https://doi.org/10.3897/zookeys.1153.98297 5. Schifani, E., Alicata, A., Menchetti, M., Borowiec, L., Fisher, B.L., Karaman, C., Kiran, K., Oueslati, W., Salata, S., & Blatrix, R. (2022). Revisiting the morphological species groups of West-Palearctic Aphaenogaster ants (Hymenoptera: Formicidae) under a phylogenetic perspective: toward an evolutionary classification. Arthropod Systematics & Phylogeny, 80, 627-648. http://dx.doi.org/10.3897/asp.80.e84428 6. Schifani, E., Prebus, M.M., & Alicata, A. (2022). Integrating morphology with phylogenomics to describe four island endemic species of Temnothorax from Sicily and Malta (Hymenoptera, Formicidae). European Journal of Taxonomy, 833, 143-179. https://doi.org/10.5852/ejt.2022.833.1891 7. Schifani, E., Giannetti, D., Csősz, S., Castellucci, F., Luchetti, A., Castracani, C., Spotti, F.A., Mori, A., & Grasso, D.A. (2022). Is mimicry a diversification-driver in ants? Biogeography, ecology, ethology, genetics and morphology define a second West-Palaearctic Colobopsis species (Hymenoptera: Formicidae). Zoological Journal of the Linnean Society, 194, 1424-1450. http://doi.org/10.1093/zoolinnean/zlab035 8. Schifani, E., Scupola, A., Menchetti, M., Bazzato, E., & Espadaler, X. (2021). Morphology and Phenology of Sexuals and New Distribution Data on the Blind Mediterranean Ant Hypoponera abeillei (Hymenoptera, Formicidae). Sociobiology, 68(4), e7261. https://doi.org/10.13102/sociobiology.v68i4.7261 9. Degueldre, F., Mardulyn, P., Kuhn, A., Pinel, A., Karaman, C., Lebas, C., Schifani, E., Bračko, G., Wagner, H.C., Kiran, K., Borowiec, L., Passera, L., Abril, S., Espadaler, X., & Aron, S. (2021). Evolutionary history of inquiline social parasitism in Plagiolepis ants. Molecular Phylogenetics and Evolution, 155, 107016. https://doi.org/10.1016/j.ympev.2020.107016 135 Ant ecology and behavior 1. Schifani, E., Grasso, D.A., Gobbi, M., Spotti, F.A., Pedrotti, L., Vettorazzo, E., Mori, A., & Castracani, C. (2024). Ant diversity along altitudinal gradients in the European Alps: insights for conservation under a changing climate. Journal of Insect Conservation, in press. 2. Bazzato, E., Lallai, E., Caria, M., Schifani, E., Cillo, D., Ancona, C., Pantini, P., Maccherini, S., Bacaro, G., & Marignani, M. (2023). Focusing on the role of abiotic and biotic drivers on cross-taxon congruence. Ecological Indicators, 151, 110323. https://doi.org/10.1016/j.ecolind.2023.110323 3. Castracani, C., Giannetti, D., Spotti, F.A., Schifani, E., Ghizzoni, M., Delaiti, M., Penner, F., Leonardi, S., Mori, A., Ioriatti, C., & Grasso, D.A. (2023). Ants as mealybug detectors: a novel tool for monitoring Planococcus ficus infestation based on ant behaviour. Agricultural and Forest Entomology, 25(2), 237-250. https://doi.org/10.1111/afe.12547 4. Giannetti, D., Schifani, E., Castracani, C., Spotti, F.A., Mori, A., & Grasso, D.A. (2022). Different nest entrance architecture by Colobopsis and Temnothorax ants colonizing oak galls. Insectes Sociaux, 69, 383-388. https://doi.org/10.1007/s00040-022-00884-2 5. Bazzato, E., Lallai, E., Caria, M., Schifani, E., Cillo, D., Ancona, C., Alamanni, F., Pantini, P., Maccherini, S., Bacaro, S., & Marignani, M. (2022). Land-use intensification reduces multi-taxa diversity patterns of Small Woodlots Outside Forests in a Mediterranean area. Agriculture, Ecosystems and Environment, 340, 108149. https://doi.org/10.1016/j.agee.2022.108149 6. Schifani, E., Castracani, C., Spotti, F.A., Giannetti, D., Mori, A., & Grasso, D.A. (2022). Tool use in pavement battles between ants: first report of Tetramorium immigrans (Hymenoptera, Formicidae) using soil-dropping as an interference strategy. Insectes Sociaux, 69, 355-359. https://doi.org/10.1007/s00040-022-00876-2 7. Giannetti, D., Schifani, E., Castracani, C., Mori, A., & Grasso, D.A. (2022). The introduced oak Quercus rubra and acorn-associated arthropods in Europe: an opportunity for both carpophagous insects and their ant predators. Ecological Entomology, 47(4), 515-526. https://doi.org/10.1111/een.13136 8. Giannetti, D., Schifani, E., Castracani, C., Spotti, F.A., Mori, A., & Grasso, D.A. (2022). Unlike rolling stones: not every Myrmecina species actively rolls away from danger (Hymenoptera, Formicidae). European Zoological Journal, 89(1), 15-21. https://doi.org/10.1080/24750263.2021.2011967 9. Giannetti, D., Mandrioli, M., Schifani, E., Castracani, C., Spotti, F.A., Mori, A., & Grasso, D.A. (2021). First report on the acrobat ant Crematogaster scutellaris storing live aphids in its oak-gall nests. Insects, 12(2), 108. https://doi.org/10.3390/insects12020108 10. Castracani, C., Spotti, F. A., Schifani, E., Giannetti, D., Ghizzoni, M., Grasso, D. A., & Mori, A. (2020). Public engagement provides first insights on po plain ant communities and reveals the ubiquity of the cryptic species Tetramorium immigrans (Hymenoptera, Formicidae). Insects, 11(10), 678. https://doi.org/10.3390/insects11100678 136 [Document text truncated for crawler view.]