Importance of invasive species and ectoparasites in Corixidae communities in south-west Spain

Recommended citation: Céspedes V (2019) Importance of invasive species and ectoparasites in Corixidae communities in south-west Spain. PhD Thesis. Universidad de Sevilla, Spain. Cover image is an original creation of Mamen RS and illustrations of chapter I and chapter V are original creations of Ruben Izquierdo (@mibuho.es) and Paula Martin Art (@paulamartinart) respectively (intellectual property ownership). Any form of reproduction, distribution, public communication or transformation of this property without prior authorization of the authors is forbidden. “Importancia de la especie invasora y los ectoparásitos en la comunidad Corixidae en el suroeste de España” Memoria presentada por la Licenciada en Ciencias Ambientales, Vanessa Céspedes Castejón, para optar al título de Doctora por la Universidad de Sevilla PhD Thesis – Sevilla 2019 Fdo. Vanessa Céspedes Castejón UNIVERSITY OF SEVILLE DOÑANA BIOLOGICAL STATION Wetland Ecology Department Doctoral programme “Integrate Biology” “Importance of invasive species and ectoparasites in Corixidae communities in south-west Spain” Dissertation submitted by Vanessa Céspedes Castejón to obtain the PhD degree with the International Mention by the University of Seville Seville, 2019 Supervisors/Directors: Andy J. Green and Marta I. Sánchez Tutor: Carlos Granado Florencio Dr. Andy J. Green Profesor de Investigación en la Estación Biológica de Doñana (EBD-CSIC); Dra. Marta I. Sánchez, Profesora Contratada Doctora en el INMAR (Instituto de Investigación Marina) en la Universidad de Cádiz como director y codirectora, y Dr. Carlos Granado Florencio, Catedrático del departamento de Biología Vegetal y Ecología de la Universidad de Sevilla, como tutor CERTIFICAN: Que los trabajos de investigación desarrollados en la Memoria de Tesis Doctoral “Importance of invasive species and ectoparasites in Corixidae communities in south-west Spain” son aptos para ser presentados por la Licenciada Vanessa Céspedes Castejón ante el Tribunal que en su día se designe, para aspirar al grado de Doctora por la Universidad de Sevilla. Y para que así conste, y en cumplimiento de las disposiciones legales vigentes, firman el presente documento en Sevilla, a 13 de Marzo de 2019. Directores: Fdo. Andy J. Green Fdo. Marta I. Sánchez Fdo. Tutor. Carlos Granado Florencio The work developed by Vanessa Céspedes in this thesis, including field sampling, as well as her visits and stays to other research centres, have been funded by the following institutions: Ministry of Economy and Competitiveness and Spanish Ministry of Science and Innovation (MICINN), Andalusian Regional Government (ARG) and Doñana Biological Station (EBD) (see details below).  Severo Ochoa doctoral contract “SVP-2013-067595” from MICINN  Mobility grant “EEBB-I-15-10016” from MICINN –Belgium  Evolutionary Stress Ecology and Ecotoxicology, University of LeuvenBelgium  Mobility grant EEBB-I16-10819 from the MICINN – England  Mobility grant EEBB-I-17-12675 from the MICIN–Spain  Innovation, science and industry Council from the ARG: research project no. P10-RNM6262.  Wetland Ecology Department of EBD. “Vivir es como avanzar por un museo: es luego cuando empiezas a entender lo que has visto” Audrey Hepburn 14 ESUMEN La presente tesis aborda las interacciones entre las especies nativas y la especie invasora en los ecosistemas acuáticos y el papel de los factores bióticos y abióticos involucrados en la invasión. Nos centramos en los insectos acuáticos de la familia Corixidae en el suroeste de España incluyendo a la especie invasora Trichocorixa verticalis. Esta especie, procedente de Norte América, es el único insecto estrictamente acuático categorizado como especie invasora de Europa, ya que el mosquito tigre sólo presenta la larva en fase acuática. Trichocorixa verticalis fue detectada por primera vez en 1997 en la Península Ibérica y desde entonces se está expandiendo por el oeste de la Península y el norte de Marruecos. Es abundante en aguas salinas permanentes, como Marismas del Odiel y en los estanques de peces del Parque Natural de Doñana (Veta la Palma), pero también ha sido localizada en otros sitios a menor salinidad, como las lagunas del Parque Nacional de Doñana, además de lagunas poco profundas y protegidas en otras partes de Andalucía. Trichocorixa verticalis es aún una especie poco conocida en su rango introducido, por lo tanto, nuestro primer estudio fue sobre su ciclo de vida (Capítulo 1), primordial para comprender sus rasgos biológicos y si les proporciona una ventaja respecto a las especies nativas. Igualmente importante es, conocer sus interacciones (incluyendo predador-presa, parásito-hospedador) con el resto de la comunidad. En su zona de distribución nativa (Norte América), se ha observado un efecto “top-down” de T. verticalis regulando las poblaciones del crustáceo (Artemia franciscana) en medios hipersalinos. En este contexto, en el Capítulo 2 se analiza la interacción presadepredador (Artemia parthenogenetica–T. verticalis) con el fin de determinar los posibles impactos de la especie invasora en el área nativa. Es además objeto de estudio de esta tesis las interacciones entre los parásitos y los coríxidos acuáticos. Los parásitos son fundamentales en el funcionamiento de los ecosistemas y tienen un papel clave en el éxito o fracaso de una invasión. El Capítulo 3 proporciona la primera descripción detallada de las interacciones entre ácaros acuáticos y sus huéspedes en Doñana, así como las relaciones entre los ácaros adultos de vida libre y las características de su entorno acuático. En el Capítulo 4 se estudió las asociaciones entre el ectoparasitismo y la supervivencia y fecundidad de especies de coríxidos nativos, así como las interacciones entre el ectoparasitismo y la salinidad, factor abiótico clave afectado por el cambio global. Finalmente, en el Capítulo 5 se analizó la respuesta inmune al parasitismo por ácaros en las especies nativas e invasora como una aproximación para determinar la implicación de los parásitos en el proceso de invasión. R Resumen 15 Capítulo 1 Estudiamos el ciclo de vida de Trichocorixa verticalis en dos áreas del oeste de Andalucía donde la especie invasora habita de forma permanente durante todo el año: Veta la Palma, perteneciente al Espacio Natural de Doñana (provincia de Sevilla) y las salinas del Paraje Natural Marismas del Odiel (provincia de Huelva). Los resultados muestran que la especie invasora puede reproducirse en invierno, ventaja que no tienen las especies nativas, y que son capaces de reproducirse hasta una salinidad de 70g/l. Observamos que existe una adaptación del tamaño corporal a la temperatura, disminuyendo durante los meses de verano, y que presenta una mayor fecundidad que las especies nativas. Utilizando microcosmos y aproximaciones experimentales determinamos una duración del ciclo de 54 días, menos que la media existente en las especies nativas. “Las ventajas de la especie invasora respecto al ciclo biológico, podrían explicar su éxito de invasión en humedales salinos - permanentes, ocupando diferentes hábitats y superando a los coríxidos nativos” Capítulo 2 Investigamos el papel de la especie invasora como depredador clave en la red trófica. Analizamos experimentalmente la tasa de depredación sobre el crustáceo Artemia parthenogenetica, en relación a su abundancia, tamaño y estado de infección (presencia de parásitos céstodos). Los resultados obtenidos demuestran el importante papel de este invasor como depredador en los ecosistemas hipersalinos. Encontramos que las hembras de T. verticalis, de mayor tamaño que los machos, tienen una tasa de depredación mayor y depredan más cuando el número de presas es mayor. Observando preferencia por las artemias de menor estadío y por las artemias infectadas. Por tanto, el tamaño de las poblaciones de Artemia puede disminuir por la depredación de T. verticalis, con efectos cascada a través de la red alimentaria. Por ejemplo, la depredación podría reducir los cambios de los parásitos céstodos que completan su ciclo a través de la ingestión de los hospedadores aviares finales. Por otro lado, la Artemia es un filtrador crucial en el ecosistema hipersalino, una disminución de su abundancia incrementaría la densidad de fitoplancton y la turbidez, con posibles efectos cascada. En conclusión, T. verticalis podría alterar Capítulos 16 las relaciones tróficas en los ecosistemas hipersalinos al controlar las poblaciones de Artemia. La presencia de Trichocorixa podría explicar en parte la ausencia del crustáceo a salinidades por debajo de los 100 g/l donde la especie invasora es abundante. “Trichocorixa verticalis podría causar efectos top-down produciendo cascadas tróficas” Capítulo 3 Describimos las interacciones entre ácaros ectoparásitos y sus huéspedes en Doñana, en relación a las condiciones ambientales e incluyendo no sólo coríxidos sino otros insectos como coleópteros. También incluimos las fases adultas (de vida libre) de los ácaros. La abundancia y la riqueza de ácaros adultos fue mayor en el mes de junio, debido probablemente al ciclo de los ácaros. Ambos factores se relacionaron negativamente con la salinidad y la turbidez. La prevalencia y la intensidad de infección fueron mayor en el mes de mayo y en lagunas con el hidroperíodo más largo. Adultos y larvas de los ácaros fueron más abundantes y con mayor riqueza de especies en la zona de dunas de la Reserva Biológica de Doñana que en las marismas (marismas de agua dulce). Describimos nuevas relaciones (no identificadas previamente) entre ácaros y larvas de los insectos de las familias Dysticidae, Notonectidae y Naucoridae (Coleoptera y Hemiptera). “Doñana es un lugar inexplorado con respecto a sus ectoparásitos y sus interacciones con los hospedadores. La variedad de hábitats acuáticos lo convierten en un lugar perfecto para estudiar las interacciones huésped–parásito” Capítulo 4 Estudiamos las implicaciones del ectoparasitismo y la salinidad en el fitness en dos especies nativas de coríxidos que ocupan hábitats de baja salinidad. Demostramos el impacto negativo de los ectoparásitos en la supervivencia, la fecundidad y la viabilidad de huevos de los coríxidos, afectando al proceso de muda de las ninfas, esencial para completar el ciclo de vida. Las salinidades más altas también tuvieron un impacto negativo en el tiempo de supervivencia y la fecundidad. También encontramos evidencias de interacción entre Resumen 17 parasitismo y salinidad. La exposición a alta salinidad junto con el parasitismo reveló tasas muy altas de mortalidad, y efectos en la fecundidad de las hembras y al éxito de eclosión de los huevos. “Los aumentos generalizados de salinidad debido al cambio climático y a la acción antrópica local en las lagunas mediterráneas podrían tener implicaciones importantes para las comunidades de insectos y ácaros acuáticos, aumentando los efectos del parasitismo” Capítulo 5 Analizamos la respuesta inmune de los coríxidos al ectoparasitismo con el objetivo de determinar sus implicaciones en la competencia interespecífica y en la invasión de T. verticalis. Para responder a esta cuestión comparamos la actividad fenoloxidasa entre la especie invasora y varias especies nativas, utilizando datos de campo e infecciones controladas en el laboratorio. La fenoloxidasa es una enzima importante en la respuesta del sistema inmunológico de los insectos a daño mecánico, parásitos, o patógenos bacterianos, fúngicos y virales. Trichocorixa verticalis mostró los valores basales más bajos de fenoloxidasa, lo que sugiere una baja inversión en inmunidad a costa de una buena adaptación a los entornos de alta salinidad. Además, nuestros resultados proporcionan uno de los pocos ejemplos en los que los parásitos inmuno-suprimen al huésped invasor en mayor grado que a las especies nativas, lo que sugiere una desventaja competitiva para el invasor. “La especie invasora presenta una respuesta inmunitaria más débil que las especies nativas, y los efectos de los ectoparásitos le perjudican más. Este es un ejemplo raro en el que la especie invasora presenta una desventaja al parasitismo en comparación con las especies nativas” UMMARY The present thesis tackles the interactions between native and alien species in aquatic ecosystems and the role of biotic and abiotic factors involved in an invasion. The main focused is on aquatic insects of the family Corixidae in south-west Spain, including the alien species Trichocorixa verticalis. This species, native to North America, is the only strictly aquatic insect categorised as an invader in Europe, considering that the tiger mosquito is only aquatic in its larval phase. Trichocorixa verticalis was detected for the first time in the Iberian Peninsula in 1997, and since then it is spreading through the south and west of the Peninsula as well as northern Morocco. It is abundant in permanent saline waters, such as the salt ponds of Marismas del Odiel Natural Park and the fish ponds of Doñana Natural Park (Veta la Palma), but is also recorded in less saline waterbodies within Doñana National Park as well as protected shallow lakes elsewhere in Andalusia. Trichocorixa verticalis is a poorly known species in the introduced range, therefore our first study was of its life cycle (Chapter 1), essential to understand its biological traits and how it gains a notable advantage over native species. Moreover, it is also very important to understand its interactions with the rest of the aquatic community (including predator-prey and host-parasite interactions). In its native distribution Trichocorixa verticalis has been observed to exert a “topdown” effect whereby it controls crustacean populations (Artemia franciscana) in hypersaline environments. In this context, in Chapter 2 predator-prey (Artemia parthenogenetica–T. verticalis) interactions are analysed to determine the possible impacts of the invasive species on native prey. Furthermore, another objective of this thesis is to study the interactions between ectoparasites and corixids. Parasites are fundamental in the functioning of aquatic ecosystems and potentially have a key role in the success or failure of invasions. Chapter 3 provides the first detailed description of the interactions between aquatic mites and their insect hosts in Doñana, as well as the relations between free-living adult mites and the characteristics of their aquatic environment. In Chapter 4 we study the associations between ectoparasitism and the survival and fecundity of native corixid species, as well as the interactions between ectoparasitism and salinity as a key abiotic factor affected by global change. Finally, in Chapter 5 we analyse the immune response of native and invasive corixid species to mite parasitism and consider the implications for the invasion process. S Summary 21 Chapter 1 We studied the Trichocorixa verticalis life cycle in two areas of western Andalusia with permanent wetlands where the species can be observed all year round: Veta la Palma, inside to Doñana Natural Park (Sevilla province) and the salt ponds within Marismas de Odiel Natural Park (Huelva province). We show that the alien species is able to reproduce in winter, unlike native Corixidae species, and reproduce in salinities up to 70g/l. We observe a body size change in response temperature, with a decrease in summer months, and a higher fecundity than recorded for native corixid species. Using microcosms and experimental approaches, we estimate a duration of 54 days for the life cycle, which is less than reported for native species. “The biological cycle of the alien species provides advantages that may explain its invasion success in permanent, saline wetlands, occupying new habitats and outcompeting native corixids” Chapter 2 We investigated the role of the invasive species as a key predator in a trophic web. We compared experimentally the predation rate on the crustacean Artemia parthenogenetica in relation to changes in prey abundance, size and parasitic status (presence of cestode larvae). The results obtained demonstrate the important role in hypersaline ecosystems of the invader as a predator. We found that T. verticalis females, which are bigger than males, prey on more Artemia when prey abundance is high. Predation rates are also greater for smaller Artemia life stages, and also for infected prey. Therefore, the size of Artemia populations may be decreased by T. verticalis predation, with effects cascading through the food-web. For example, such predation may reduce the changes of cestode parasites completing their cycle through ingestion by avian final hosts. Furthermore, Artemia is a crucial filter-feeder in hypersaline ecosystems, and a decrease in their abundance will increase phytoplankton density and turbidity. In conclusion, T. verticalis may alter trophic relations in hypersaline ecosystems through the control of Artemia populations. The presence of T. verticalis may explain the absence of the crustacean in the Odiel salt ponds at salinities below 100 g/l, where the invader is abundant. Chapters 22 “Trichocorixa verticalis can have top-down predatory effects, creating trophic cascades” Chapter 3 We describe the interactions between ectoparasitic mites and their hosts in Doñana, considering the environmental conditions and including Hemiptera and other insects such as Coleoptera. We also study the (free-living) adult mite phase. The abundance and species richness of mite adults was highest in June, probably due to their life cycle. Both factors were negatively related to salinity and turbidity. Prevalence and infection intensity of mite larvae were higher in May, and in sites with a long hydroperiod. Adult and larval mites were more abundant and species-rich in dune ponds within Doñana Biological Reserve than in the marshland (freshwater marshes) of the National Park. We describe new (previously unrecorded) relationships between particular mite species and larval stages of the Dysticidae, Notonectidae and Naucoridae insect families (Coleoptera and Hemiptera). “Doñana is an unexplored place regarding its ectoparasites and their interactions with hosts. The variety of aquatic habitats make it a perfect place for studying host–parasite interactions” Chapter 4 We studied the implications of ectoparasitism and salinity for the fitness of two native Corixidae characteristic of low salinity habitats. We found evidence that ectoparasitic mites have a strong negative impact on survival, lifespan, egg laying rate and egg fertility of corixids, as well as the moulting process of nymphs essential to complete the lifecycle. Higher salinities also had a negative impact on survival time and fecundity. We also found evidence of an interaction between salinity and parasitism effects. Exposure to high salinities as well as parasitism revealed extremely high death rates, and effects on fecundity and egg viability. Summary 23 “Expected salinity increases due to local anthropic actions and climate change in Mediterranean ponds could have major implications for aquatic insect communities, enhancing the effects of parasitism” Chapter 5 We analysed the immune response of corixids to ectoparasitism with the aim of determining its implications in interspecific competition and the invasion of T. verticalis. To do this, we compared the phenoloxidase activity between the alien species and several native species, using both field data and controlled infections in the laboratory. Phenoloxidase is an important enzyme in immune system responses of insects, associated with mechanical damage, eukaryotic parasites or bacterial, fungal or viral pathogens. Trichocorixa verticalis showed the lowest baseline phenoloxidase values suggesting low investment in immunity as an adaptation to high salinity environments. Furthermore, our results provide one of few examples where parasites immunosuppress an invasive host to a greater extent than native hosts, which represents a competitive disadvantage to the invader. “The invasive species shows a weaker immune response than native species, and the effects of ectoparasites are stronger. This is a rare example where, under parasitism, an invader is at a disadvantage compared with native species” General Introduction 30 interactions it stablishes in the invaded under different environmental conditions area are very important to understand the invasion success and potential impact. Life history traits are of key importance in determining the invasiveness of a species (Grabowski et al. 2007). For example, generation time, sexual maturity, fecundity, body size and saline tolerance can allow one species to outcompete another and control the success or failure of an invasion. The life cycle of T. verticalis and its population ecology is poorly known. Laboratory and field studies in key habitats for this species are necessaries to describe the cycle and understand the spatial and seasonal variation in population density in relation with environmental factors. At the same time, ecological interactions such as predator-prey and host-parasites relationships are of particular importance to understand the invasion process. Moreover, the role of environmental factors in shaping these interactions is important to understand current and future distribution. Water salinity is of particular relevance, with a major influence on biotic communities and the potential to interact with other stressors. Trichocorixa verticalis occurs in coastal and inland saline habitats up to 70–80 g l−1 (e.g. Jansson 2002). In its native distribution T. verticalis is considered omnivorous (Kelts 1979, Simonis 2013) being considered a voracious predator of anostraceans and cladocerans (Wurtsbaugh 1992, Simonis 2012) with potential of strongly influencing ecosystem functioning. For example, Wurtsbaugh (1992) observed in Great Salt Lake (USA) that T. verticalis showed a strong predation rate of Artemia franciscana nauplii and Simonis (2013) revealed a high consumption rate of adult cladocerans (Simonis 2013). Both studies demonstrate how T. verticalis can cause a strong top-down effect and trophic cascades. However, there is no information about the potential impact of T. verticalis in invaded ecosystems. On the other hand, there is little information on how T. verticalis interact with parasites in the invaded area. There are different mechanisms through which parasites can operate during the invasion process and identifying them is important to understand the potential role of parasites in an invasion. Invasive species may lose their own parasites during the invasion process and it would confer a competitive advantage over native species (“Enemy release Hypothesis”, Fig. 2A). Invasive species may also acquire native parasites in new ecosystem (“Parasite acquisition”, Fig. 2B). When the parasite cannot reproduce in the exotic species it could led to a net decrease of parasite abundance in native populations (“Dilution effect”) (Ostfeld and Keesing 2000). However, when the acquired parasite can reproduce in the exotic species it could increase the impact of parasites in the native communities (“Parasite spillback”) (Kelly et al. 2009). The thirst case is when the invader introduce parasite from its native distribution to the invaded community (“Parasite introduction or spillover”, Fig. 2C). PhD Thesis –Vanessa Céspedes 31 31 31 Figure 2: Possible outcomes for parasitism following the invasion of a new habitat. (A) Enemy release; an invader may benefit from a reduction in parasite diversity and/or prevalence as a result of invasion. (B) Parasite acquisition; an invader may acquire parasites in the new habitat. It may cause parasite dilution or parasite spillback (C) Parasite introduction; parasites introduced with the invader may infect novel host species in the new habitat (parasite spillover). Adaptation of Dunn 2009. Water mites are common ecotoparasites of corixids, which can strongly impact hosts populations and influence biological interactions between corixid species (Smith 1977, Sánchez et al. 2015). However, there is little information about the potential role of these parasites in the invasion dynamics of the American corixid. The only study in the invaded area showed that T. verticalis was more susceptible to the infection that native species (Sánchez et al. 2015). However, these authors did not explore the impact of infection in both native and invasive species, which is critical information to evaluate the role of parasites in the invasion process. For example, parasites may infect hosts but fail to evade its immune system (Rigaud and Moret 2003). On the other hand, because immune defences are costly (Bonneaud et al. 2003, Moret 2003), in the absence of parasites, invaders may reallocate energy resources from the immune system to growth, dispersal or reproduction, leading to increased competitive ability (Blossey and Nötzold 1995). Consequently, invasive species with reduced immune response may be at risk from infection by new parasites and pathogens, with collateral physiological costs (Cox 2001). 34 BJECTIVES AND THESIS OUTLINE This thesis explores several key questions about invasive species focusing on the aquatic insect Trichocorixa verticalis. This invasive species originating from North America is the dominant species in many saline wetlands of south Spain, but it remains a poorly known species. In this thesis we describe its life cycle, its potential impact in the native ecosystems and role of parasites (water mites) in its invasion success. The thesis is integrated by five chapters (See Fig. 3) that correspond to the following specific objectives: Chapter 1 Describing the life history traits to elucidate the life cycle of Trichocorixa verticalis. We used laboratory experiments, mesoscosms and field studies to assess the fecundity, duration of each developmental stage, and spatial and seasonal variation in population density and reproductive activity. We also related this variation with adult sex ratio and body size. Chapter 2 Studying the potential impact of T. verticalis through predation of native preys. We focused in hypersaline ecosystems where this invasive species is present all year round. Previous studies in the native area show that T. vertically can predate on Artemia in hypersaline ecosystems and cause important cascading effects. In laboratory experiments we determined the effect of Artemia developmental stage, body size, its parasitic status and Artemia density on prey selection by T. verticalis. Chapter 3 Describing the interactions between water mites and different insect host in temporary water bodies of Doñana National Park. We assessed the prevalence and intensity of infection, O PhD Thesis –Vanessa Céspedes 35 35 35 and related them with environmental variables such as hidroperiod, salinity, depth, temperature between other physicochemical variables. Chapter 4 To study the individual al combined effect of water mite parasites and salinity on native corixids. We specifically examined the effects of Hydrachna Skorikowi, one of the most prevalent species in Doñana, and different levels of salinity on Sigara lateralis and Corixa affinis adult’s fecundity and survival. We also evaluated the effect of salinity on Sigara lateralis nymph’s survival. Chapter 5 To evaluate the effect of water mite parasites on two related key physiological variables in native and invasive corixids: the phenoloxidase activity (as measure of immune function) and fat levels (as measure of body condition). We rated it with the role of parasites in the success of T. verticalis. The previous chapters correspond to five scientific articles (see below). Chapters 1-2-4 have already been published in international peer reviewed journals indexed in SCI. The Chapter 3 is in preparation for its publication and chapter 5 is under second review. Chp.1 Céspedes V, Coccia C, Carbonell JA, Sánchez MI & Green AJ (2019) The life cycle of the alien boatman Trichocorixa verticalis (Hemiptera Corixidae) in saline and hypersaline wetlands of south-west Spain. Hydrobiologia (IF 2.16) 827: 309 Chp.2 Céspedes V, Sánchez MI & Green AJ (2017) Predator–prey interactions between native brine shrimp Artemia parthenogenetica and the alien boatman Trichocorixa verticalis: influence of salinity predator sex and size abundance and parasitic status of prey. PeerJ (IF 2.12) 5 e3554. Chp.3 Céspedes V, Valdecasas AG, García-Jiménez R, Sánchez MI & Green AJ. Water mites and their interactions with aquatic insect hosts in dune ponds and temporary marshes in south-west Spain. In preparation Objetives and thesis outline 36 Chp.4 Céspedes V, Valdecasas AG, Green AJ & Sánchez MI (2019) Water boatman survival and fecundity are related to ectoparasitism and salinity stress. PLOS ONE (IF 2.76). DOI: 10.1371/journal.pone.0209828 Chp.5 Céspedes V, Stock R, Green AJ & Sánchez MI. Eco-immunology of native and invasive water bugs in response to water mite parasites: insights from phenoloxidase activity. Second review in Biological Invasions (IF 3.05). The 1-5 chapters (articles) on which this thesis is based are: Figure 3: Scheme of the thesis structure. The main studied aspects are show for each Chapter (Chp.) with the invader as focus on this thesis. 38 eneral study area in South-west Spain Mediterranean temporary ponds, such as those of Doñana National Park (South-West Spain), allow the study of interactions between water mites and native and invasive (T. verticalis) corixids in conjunction with environmental stressors. Temporary ponds are among the most threatened habitats in the Mediterranean region (Grillas et al. 2004, Zacharias et al. 2007). They are considered “hotspots” of biodiversity and endemism (Boix et al. 2016), and are a priority habitat under the European Union Habitats Directive. There are at least 394 genera of 149 families of aquatic invertebrates in temporary ponds of the Mediterranean basin (Boix et al. 2016). Doñana National Park has been recognized as a critical area for Biodiversity since the early 1950s (Mountford 1958). The area, dominated by natural temporary wetlands but also permanent ponds, fish ponds, salt ponds and ricefields, is protected as a Ramsar Site, Biosphere Reserve, Special Protection Area for birds and Natural World Heritage. Doñana National Park also includes a rich network of more than 3,000 temporary dune ponds that vary greatly in size, hydroperiod and salinity (Green et al. 2017). Despite the great ecological value of temporary ponds of Doñana and the increasing interest they have attracted during the last two decades (e.g. DiazPaniagua et al. 2010, Florencio et al. 2009, 2013, Serrano et al. 2005, 2006) they are still poorly studied. While research on vertebrates and the largest invertebrates are abundant (GómezRodríguez et al. 2009), microinvertebrates and parasites have been poorly studied (Céréghino et al. 2011). Permanent wetlands such as fish ponds or salt ponds are widely recognized as of high value for waterbird conservation (Rendón et al. 2008), however these environments act as reservoirs during the summer for invertebrates which are able to disperse, including invasive species. For example, in Veta la Palma fish ponds (east of the National Park) have been found several exotic species such as the gastropod Potamopyrgus antipodarum (Frisch et al. 2006, Rodríguez-Pérez 2006) or T. verticalis (Van de Meutter et al. 2010). Despite these permanent ponds facilitate the expansion and colonization of exotic species, play an important role in the biodiversity conservation in Doñana (Rodríguez-Pérez et al. 2006, 2009). In the case of T. verticalis could work how a refuge for parasites during desiccation in summer. During the summer, temperature and salinity increase, with the concomitant reduction of their depth and surface area in Doñana ponds, which completely dry out by August (Serrano et al. 2006). In these conditions, temporal pond become disappear and corixid densities become high and allow for increasing contact between ectoparasites and their hosts, although salinity exert strong controls on aquatic G PhD Thesis –Vanessa Céspedes 39 39 39 communities, eliminating sensitive species (Frisch et al. 2006) and impacting on host-parasite interactions (Muhling et al. 2018). Trichocorixa verticalis is found at salinities ranging from 0.6 to 75 g.l-1 (Rodríguez-Pérez et al. 2009, Coccia et al. 2016a, Carbonell et al. 2017), and has become invasive in saline and hypersaline permanent water bodies, where it outcompetes native halotolerant species such as Sigara selecta or S. stagnalis. In contrast, in temporary ponds of lower salinity T. verticalis is less abundant, and co-occurs with native Sigara (mainly S. scripta and S. lateralis) along different parts of the salinity gradient (Rodríguez-Pérez et al. 2009, Carbonell et al. 2016b, 2017), with overlap in their dietary niches (Coccia et al. 2016b). To date, there is no evidence that this invader is displacing native species or has been observed an aggressive behavior over native communities (Carbonell et al. 2017). tudy community Corixidae family The Corixidae is the largest family of aquatic Hemiptera with approximately 500 species and 33 genera know in the world (Savage 1989). Some species occupy different habitats, such as lotic or lentic, continental or coastal, in different salinities, from freshwater to hypersaline waters. The most important habitat requirements are availability of oviposition sites, water depth and salinity (Díaz-Paniagua et al. 2019). The body is usually dark above, often mottled or cross-banded with yellow, brown or black on the pronotum and leathery hemelytra. The rostrum is short, triangular and broadly attached to head. Forelegs are short, scoop-like pala, middle and hind legs are long (Fig. 1 A/B). Corixids are also key in the food web of ecosystems where they occur, being one of the most abundant group (Nieser et al. 1994) and contributing with a high biomass (Barahona et al. 2005). Although Corixidae are aquatic in all their life stages, adults have a marked tendency to leave water and disperse flying. In relation to the life cycle, this family have five larval instars each one lasting from 4 to 10 days (e.g. Scudder 1976). Species can have one single generation or several ones (polivoltinism, Savage 1989, Barahona et al. 2005). In most genera of temperate areas, the species overwinter as adults and mating takes place in spring. However, some species e.g. the invasive Trichocorixa verticalis (Fieber 1851) may pass the winter as an egg or larval stage (Poisson 1935, Rodríguez-Pérez et al. 2009) in its native distribution (specifically the subspecies T. verticalis interiores). S References 46 Mountford G (1958) Portrait of A Wilderness: The story of the Coto Donana expeditions. Hutchinson, Londres. Muhling BA, Gaitán CF, Stock CA, Saba VS, Tommasi D, Dixon KW (2018) Potential salinity and temperature futures for the Chesapeake Bay using a statistical downscaling spatial disaggregation framework. Estuaries and Coasts, 41(2) 349–372 Nielsen GJ, Davids C (1976) Contributions to the knowledge of the morphology and biology of the larvae of four European Eylais species (Acari, Hydrachnellae). 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Rodríguez‐Pérez H, Green AJ (2006) Waterbird impacts on widgeongrass Ruppia maritima in a Mediterranean wetland: comparing bird groups and seasonal effects. Oikos, 112(3), 525-534 Sailer RI (1948) The genus Trichocorixa (Corixidae: Hemiptera). In H. B. Hungerford (Ed.), The corixidae of western hemisphere (Hemiptera) (Vol. 32, pp. 289–407). The university of Kansas science bulletin. Sala J, Boix D (2005) Presence of the nearctic water boatman Trichocorixa verticalis verticalis (Fieber, 1851) (Heteroptera, Corixidae) in the Algarve region (S Portugal). Graellsia, 61(1), 31-36 Sánchez MI, Coccia C, Valdecasas AG, Boyero L, Green AJ (2015) Parasitism by water mites in native and exotic Corixidae: Are mites limiting the invasion of the water boatman Trichocorixa verticalis (Fieber, 1851)? Journal of insect conservation, 19(3), 433-447. Savage AA (1989) Adults of the British aquatic Hemiptera Heteroptera: a key with ecological notes. Scientific Publication, No. 50. 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Diversity and Distributions, 12, 443–455. Williamson H, Fitter A (1996) The characters of successful invaders. Biological Conservation, 78, 163–170. Wurtsbaugh WA (1992) Oecologia Food-web modification by an invertebrate predator in the Great Salt Lake (USA). Oecologia, 89, 168–175. Zacharias I, Dimitrou E, Dekker A, Dorsman E (2007) Overview of temporary ponds in the Mediterranean region: Threats, management and conservation issues. Journal of Environmental Biology, 28: 1-9. Chapter 1 “The life cycle of the alien boatman Trichocorixa verticalis (Hemiptera, Corixidae) in saline and hypersaline wetlands of south-west Spain” 52 Chapter 1: Life cycle of Trichocorixa verticalis 53 NTRODUCTION A major question in invasion biology is what factors determine whether a species becomes invasive or not (Drake et al. 1989). Those factors include both the species traits and aspects of the habitat to which the species is introduced (Richardson and Pyšek 2006). Thus, postulated mechanisms of invasions have focused either on a) traits of the invaders themselves (e.g. the “tens rule”) and the concept of species invasiveness (Williamson and Fitter 1996); or b) the ecological interactions between the invader and the recipient community (e.g. “invasional meltdown”, “novel weapons”, “enemy release” hypotheses, see Jeschke et al. 2012 for review) and the invasibility of an ecosystem. Life history traits are of key importance in determining the invasiveness of a species (Rajagopal et al. 1999, Grabowski et al. 2007). Short generation time, early sexual maturity, high fecundity, large body size and euryhalinity, among others, can enable the establishment and population increase of an alien species in new aquatic environments, allowing one species to outcompete another (Bij de Vaate et al. 2002, Grabowski et al. 2007). The Corixidae is the largest family of aquatic Hemiptera, with species inhabiting different habitats, such as lotic or lentic, continental or coastal, at different salinities, from freshwater to hypersaline waters. They constitute an important functional element in such ecosystems, contributing significantly to energy flow through the ecosystem. Corixids are detritivores responsible for the cycling of the organic matter (Kumari and Kumer 2003), and are also key predators of zooplankton at intermediate levels in the food web with the potential for cascading effects (Henrikson and Oscarson 1981, Simonis 2013); in turn, they are an important part of the diet of other invertebrates, fish and waterbirds (Henrikson and Oscarson 1978, Giles et al. 1990, Euliss and Jarvis 1991). Trichocorixa verticalis (Fieber 1851) is a small euryhaline corixid (Hemiptera) (length 3.55mm) originally distributed across a wide latitudinal range in North America and the Caribbean, but the subspecies T. verticalis verticalis is an alien in South Africa, New Caledonia and the Western Mediterranean (Morocco, Spain and Portugal, Guareschi et al. 2013). In its native range, Trichocorixa verticalis occurs in coastal and inland saline habitats up to 70-80 g.l-1 (Wurtsbaugh 1992, Hutchinson 1959, Jansson 2002). The life history of this species (e.g. number of generations per year, overwintering strategy) can be expected to vary considerably over such a broad natural range (covering c.25 degrees of latitude, Guareschi et al. 2013). Some studies have reported two or three generations per year (Tones 1977, Kelts 1979), and in Canadian lakes that freeze in winter, T. verticalis interiores produces eggs that go through diapause in winter and early spring (Tones 1977). Within the Trichocorixa genus, T. kanza and T. calva overwinter as adults (Sailer 1948). I Hydrobiologia (2019) 827:309–324 54 In Europe, T. verticalis verticalis is now well established as an alien in the southwestern part of the Iberian Peninsula, but is projected to expand widely in coastal Europe over time (Guareschi et al. 2013). In south-west Spain, it is found at salinities ranging from 0.6 to 75 g.l-1 (Rodríguez-Pérez et al. 2009, Coccia et al. 2016b, Carbonell et al. 2017), and has become invasive in saline and hypersaline permanent water bodies, where it outcompetes native halotolerant species such as Sigara selecta or S. stagnalis. In salt ponds, T. verticalis is an effective predator of brine shrimp Artemia (Céspedes et al. 2017). In contrast, in temporary ponds of lower salinity T. verticalis is less abundant, and co-occurs with native Sigara (mainly S. scripta and S. lateralis) along different parts of the salinity gradient (Rodríguez-Pérez et al. 2009, Carbonell et al. 2016 2017), with overlap in their dietary niches (Coccia et al. 2016a). There remains a lack of basic studies on the ecology of T. verticalis. As a result, the mechanisms underlying the success and invasiveness of this alien insect, and the reasons why permanent, saline habitats are more invasible, are currently unclear. T. verticalis is smaller than Sigara spp. and unable to displace them through aggressive behavior, and is more susceptible to size-limited predators such as Odonata larvae (Coccia et al. 2014, Carbonell et al. 2017). Although it coincides with other invasive species in the introduced range (Rodríguez-Pérez and Green 2012, Walton et al. 2015), there is no evidence that these species facilitate invasion by T. verticalis as would be suggested by the invasional meltdown hypothesis (Simberloff and Von Holle 1999). For example, alien fish predators showed no preference between T. verticalis and native S. lateralis (Coccia et al. 2014). Similarly, there is no evidence to support the “Enemy release hypothesis” (see Jeschke et al. 2012), since T. verticalis is more susceptible to ectoparasitic water mites than are native Corixidae, although these parasites are absent in saline and hyper-saline habitats (Sánchez et al. 2015). T. verticalis shows high plasticity in its thermal ecology and is better able to acclimate to high salinities and high temperatures than S. lateralis (Coccia et al. 2013). Its eggs are more tolerant to high salinities than co-occurring Sigara species, and its wing morphology suggests it has a stronger dispersal ability (Carbonell et al. 2016). Based on preliminary data from different seasons (Rodríguez-Pérez and Green 2012) and on laboratory studies by Carbonell et al. 2016, we hypothesized that the invasiveness of T. verticalis in permanent, saline wetlands could be related to higher rates of population growth than native Sigara species, because it is able to reproduce throughout the annual cycle and/or has a higher fecundity. In order to test this hypothesis, herein we investigate the life cycle of T. verticalis, and its population ecology during one annual cycle in permanent hypersaline salt ponds and saline fish ponds in South–West Spain. Our specific objectives were: (a) to quantify its life cycle (fecundity and duration of each developmental instar) in the laboratory, (b) to assess spatial and seasonal variation in population density and reproductive activity in a selection of ponds Chapter 1: Life cycle of Trichocorixa verticalis 55 representative of habitats where the species is invasive, and relate this variation with environmental variables, (c) to quantify seasonal variation in adult sex ratio and body size. Study areas in South-West Spain Our study was conducted in two separate wetland complexes in Andalusia (Fig. 1). Veta la Palma (VLP; 6° 14 ´W, 36°57´N) in Seville province is an area of former marshland of 3,125 ha in the delta of the River Guadalquivir that was transformed into a network of over 30 extensively farmed fishponds between 1990 and 1993 (Rodríguez-Pérez and Green 2012, Walton et al. 2015). All the ponds are supplied with water from the Guadalquivir estuary and are shallow (average depth 30 cm, maximum 50 cm) with a broad salinity range of 3 – 55 g.l-1 and flat-bottomed except for a deeper (1 m) perimetral canal for fish extraction (Rodríguez-Pérez and Green 2012). VLP is highly important for waterbirds (Kloskowski et al. 2009, Walton et al. 2015) and is included within the Doñana Natural Park which itself is listed within the Natura 2000 Network, as a wetland of international importance (Ramsar site), and within a Biosphere Reserve (Green et al. 2018). The shoreline vegetation of VLP ponds is dominated by Phragmites australis and the alien Spartina densiflora, whereas submerged vegetation is dominated by Ruppia maritima. The invertebrate community and food webs in these ponds were studied by Rodríguez-Pérez and Green (2012) and Walton et al. (2015). Previous studies at VLP have shown that T. verticalis is highly dominant, with much lower densities of native corixids Sigara spp. (Rodríguez-Pérez et al. 2009, Van de Meutter et al. 2010, Coccia et al. 2016a). Several other non-native invertebrates and fish are present (Rodríguez-Pérez and Green 2012, Walton et al. 2015). Trichocorixa verticalis was first recorded in these ponds in 2001 (Rodríguez-Pérez et al. 2009) but is likely to have arrived earlier, and may have invaded them as soon as they were created. The diet of T. verticalis in VLP was studied by Coccia et al. (2016a). We studied three VLP ponds of similar depth but varying in salinity range (G3 = 3-29 g.l-1; A3 = 4-18 g.l-1 and A7 = 9 – 53 g.l-1; see Fig. 1 and Table S1 for more details). The Odiel Marshes (37°17′N 06°55′W, Fig. 1) are located in Huelva province at the mouths of the rivers Odiel and Tinto. These marshes are protected as a Biosphere Reserve, Ramsar site, Natura 2000 site and Natural Park owing to their importance for migratory waterbirds (Sánchez et al. 2006a 2006b). They contain 7,185 ha of intertidal mudflats, of which 1,120 ha have been transformed into industrial salt ponds. Seawater is pumped through a series of ponds and salinity increases via evaporation until crystallisation. Water first circulates through a series of primary evaporation ponds (salinity 25-70 g.l-1) where corixids are abundant, followed by secondary evaporation ponds (salinity 40-125 g.l-1) where brine shrimp Artemia are abundant but corixids are limited to the ponds of lower salinity (Sánchez et al. 2006a). Finally, there is a series of crystallisation ponds where salt precipitates and corixids are absent. T. verticalis is the only corixid Hydrobiologia (2019) 827:309–324 62 had a strong negative correlation with air temperature, a result also recorded at E4 and for adults at E3 (Fig. S1). Within a given pond, correlations between corixid abundance and parameters indicating resource availability such as chlorophyll a, Total P and Total N were inconsistent and often negative (Fig. S1), suggesting that such correlations were largely driven by confounding effects of temperature and evaporation. Chlorophyll a was significantly correlated with adult abundance in three ponds (two positive, one negative) and with nymph abundance in three ponds (one positive, two negative). Figure 3: Mean density ±SE of T. verticalis adults and nymphs against salinity (monthly measures through a complete annual cycle at three ponds in VLP in 2012, and four ponds in Odiel in 2015. Note the log scale for density. Adult sex ratio Overall, adult sex ratios did not differ from parity in VLP but were slightly female-biased in Odiel. Of 792 individuals sexed in VLP, 50.19% were females (95% confidence intervals = 46.7 - 53.7). Of 4293 individuals sexed in Odiel, 55.85% (54.4 - 57.3) were females. Furthermore, sex ratios differed markedly between VLP and Odiel for a given month (Fig. 4). At VLP, sex ratios were significantly male-biased during four of 12 months (i.e. the ratio 0.5 lies outside the range between the 95% confidence intervals, Fig. 4) and were significantly female-biased during April. In contrast, in Odiel, adult samples were never significantly male-biased, but were female-biased during five months (Fig. 4). Chapter 1: Life cycle of Trichocorixa verticalis 63 Figure 4: Adult sex ratio for adult T. verticalis throughout the annual cycle. Total proportions of females (± 95% confidence interval) are provided for four ponds combined at Odiel in 2015, and two ponds (A7 and G3) combined at VLP in 2012. In September in Odiel, no confidence intervals are shown because of the small sample size (3 females, 1 male). Figure 5: Length of adult T. verticalis (mean ± S.E.) for each month from A7 pond at VLP (see Table S4 for analysis). Significant differences determined by posthoc analysis with Bonferroni correction are indicated. Different letters above bars indicate differences between months (P<0.05). Asterisks above bars indicate differences between sexes for a given month (* (P<0.01), ** (P<0.001)). Hydrobiologia (2019) 827:309–324 64 Seasonal variation in adult length There were highly significant effects of month (F 11,339 = 31.8, P < 0.001) and sex (F1,339 = 141.4, P < 0.001) and their interaction (F11,339 = 2.3, P = 0.009) on corixid body length (Table S2). Females were consistently longer than males. Generally, T. verticalis were longer from December to April, and shorter between May and September (Fig. 5). Mean body length for a given month was significantly negatively correlated with temperature and salinity for both males and females (Fig. S2). Seasonal changes in abundance of life history stages In both VLP and Odiel, at least one nymph instar was present during each month, except that nymphs were entirely absent from both sites in August (Fig. 6). In a given month, there were often nymphs from several instars or all five, indicating overlapped cohorts (Fig. 6). In both VLP and Odiel, all instars were recorded during the mid-winter months of December and January. Duration of life history stages in laboratory conditions Results from microcosm experiments indicated that the egg stage had a mean duration of 10 days and nymphs took a mean of 30 days to reach adulthood (Fig. 7, Tables S3-S4). Adults laid eggs during a mean period of 14 days after they reach adulthood (Fig. 7, Table S4), such that the estimated total generation time was of 54 days (Fig. 7, Tables S3-S4). Each female laid a mean of 31 eggs, split between 1-4 days of egg-laying with a mean of 11.5 eggs laid in a single day (Table S4). Chapter 1: Life cycle of Trichocorixa verticalis 65 Figure 6: Monthly life history stages (instars I-V and adults “A”) - frequency histograms for Trichocorixa verticalis in Veta La Palma in 2012 (A7 pond) and in Odiel in 2015 (four ponds combined). The total numbers of individuals (adults + nymphs) for each month are provided above the figures. Hydrobiologia (2019) 827:309–324 66 Figure 7: Approximate timeframe for each instar of the T. verticalis life cycle, based on microcosm experiments. An indoor experiment was performed at a stable temperature of 22 ºC and a salinity of 10 g.l-1, and an outdoor one at a salinity of 20 g.l-1 and at a mean temperature 23.2 ºC (see methods). Instars from hatching to adult were quantified indoors (Table S3), and from adult to reproduction outdoors (Table S4). The entire cycle lasted 54 days. Drawings to scale and the main structures are represented, but details are insufficient to allow for taxonomic identification of T. verticalis. Credit: Vanessa Céspedes and Ruben Izquierdo @mibuho.es. Chapter 1: Life cycle of Trichocorixa verticalis 67 ISCUSSION We have shown T. verticalis to be abundant throughout the annual cycle in permanent, saline and hypersaline wetlands in south-west Spain, and found evidence that its success as an invader is explained by an ability to reproduce throughout the year, and a high fecundity. Our study sites typify the habitats in which T. verticalis is invasive in the Western Mediterranean region. We were not able to compare alien and native corixid species in the same wetlands because native Sigara species were almost absent from our study area. Continuous reproduction, voltinism and generation time To date, T. verticalis is the only corixid species in the Western Mediterranean observed to have nymphs in winter months. The abundance of both nymphs and adults almost all year long suggests that near-continuous reproduction allows T. verticalis to outcompete native Sigara species (S. lateralis, S. selecta, S. scripta and S. stagnalis) in permanent habitats by providing a high population growth rate. In contrast, these Sigara species do not breed through the winter, and they overwinter as adults (Perán 1997, Barahona et al. 2005). Egg deposition by corixids in temperate zones usually occurs in the spring (univoltine species) and summer (bivoltine species) (Griffith 1945, Fernando 1959). Even when polivoltinism is present, corixids typically overwinter as adults. In contrast, the abundance of T. verticalis nymphs of all instars throughout the winter in our study sites (Fig. 6) can only be explained by winter reproduction. The breeding phenology and life-cycles of corixids can be expected to vary considerably between different populations of species such as T. verticalis that have an extensive native range. Intraspecific flexibility in the number of generations per year is likely in response to thermal differences between habitats at different latitudes or altitudes (Ward and Stanford 1982). The ability of T. verticalis to breed through winter can largely explain its invasiveness in permanent, saline wetlands in coastal Iberia where it is not interrupted by desiccation and where populations can accumulate through successive generations. This is likely to be related to the mild winter temperatures in this part of Spain where water temperatures remain well above freezing point and winter temperatures have increased in recent decades (Espinar et al. 2015). T. verticalis is exposed to much lower winter temperatures in its native range, and hence may be preadapted to breeding during the Iberian winter. Indeed, we have found evidence that reproduction may be halted in high summer, or at least be inhibited by high temperatures and not low ones (Fig. S2, S1). However, D Hydrobiologia (2019) 827:309–324 68 reproduction throughout the winter is unlikely if the species expands to northern Europe (as predicted by current models, Guareschi et al. 2013) where winters are much colder. Our laboratory experiments show that a generation takes about 54 days (under conditions of 10-20 g.l-1 salinity; and mean water temperature of 21-23 ºC), suggesting that there could be time to complete at least six generations per year in the field under continuous reproduction and with overlapping cohorts. The available literature suggests that its competitors S. scripta and S. selecta have 4 – 5 generations per year in the Western Mediterranean (Perán 1997, Barahona et al. 2005). In hyposaline and mesosaline Mediterranean streams in eastern Spain, S. scripta and S. selecta had multivoltine cycles and four overlapping cohorts, each with a generation time of 2 to 3 months (Perán 1997, Barahona et al. 2005). Nymphs of S. selecta occurred in almost all instars for most of the year, except during winter, when only adults were recorded. The mean temperature threshold for mating and oviposition in S. selecta was about 13oC. We measured the duration of each developmental instar of T. verticalis under a single temperature regime in microcosms, to provide baseline information on the development time for this alien species. Development times are likely to be highly dependent on food supply and temperature, thus we must be cautious when extrapolating our experimental results from microcosms to field conditions. It is likely that generation time in our study wetlands is longer in winter when temperatures are relatively low, and this would be consistent with seasonal changes recorded in body length (see below). Sexual development, egg laying and metabolic rates were highly dependent on temperature in Sigara alternata (Sweeney and Schnack 1977). When keeping several T. verticalis individuals together in the same laboratory mesocosm during our indoor experiment, we only recorded the first observation for each development instar, because it was not possible to distinguish between individuals. Hence, in some cases our data may underestimate the development times of average individuals. Oviposition rates The high oviposition rate we recorded for T. verticalis (11.5 eggs/day per female) supports earlier findings from the laboratory experiment of Carbonell et al. (2016) who found that T. verticalis showed at least twice the laying rate recorded for S. lateralis, S. scripta and S. selecta. This marked difference in fecundity between the alien and Sigara spp. was retained when correcting for hatching success (Carbonell et al. 2016) which we were unable to quantify in our own study. When considering days from the laying of the first to the last egg for each individual (≥ 1 egg laid), we found that female T. verticalis laid 7 - 24.8 eggs per day (mean 11.52 + 0.8 S.E.) at Chapter 1: Life cycle of Trichocorixa verticalis 69 20 g.l-1, and comparable data from Carbonell et al. 2016 reveal that female T. verticalis laid 1.5-53 (mean 13.35 + 5.64 S.E.) eggs per day at 25 g.l-1. Furthermore, in an unpublished experiment conducted more recently in our lab at a salinity of 15 g.l-1 and a temperature of 20oC, female S. lateralis laid only 0.25 - 4 (mean 1.7 + 0.81 S.E.) eggs per day (data from Céspedes et al. submitted) showing that T. verticalis are consistently more fecund than their competitors, at least at high salinities. The exceptional fecundity and high aerial dispersal ability (Carbonell et al. 2016) of T. verticalis is also likely to promote its rapid expansion along coastal areas of the Iberian Peninsula and beyond (Guareschi et al. 2013). Our observations suggest that T. verticalis females only have one bout of egg-laying which lasts no more than four days. However, under some field conditions, or at some times of the year, females may live longer and lay eggs over an extended period, especially as our experiment was done outdoors when microcosms were exposed to hot maximum temperatures. In a related laboratory study on S. selecta at 18-22 oC and 37 g.l-1, the mean oviposition period was about 3 weeks, although some females extended this to more than 1 month. During this period, days with intense oviposition alternated with periods of little or no oviposition. Mean total fecundity was 31 eggs and the mean rate of oviposition was 1.4 eggs per day (Barahona et al. 2005). Adult sex ratio and body length Adult T. verticalis sex ratio was often female biased in Odiel, but often male biased in VLP. Seasonal patterns were inconsistent, and could have multiple causes related to the relative survival rates of males and females at different instars, sex ratio at hatching, or different dispersal rates between sexes (Boda and Csabai 2009, Carbonell et al. 2016). Since they are smaller, males may be subject to different predation pressure, although there is no evidence for this from a laboratory experiment with fish predation (Coccia et al. 2014). In a previous study of T. verticalis in native Canada in lakes that freeze in winter (i.e. very different temperatures to our study area), biased sex ratios were recorded from May to July because females reached sexual maturity a week ahead of males (Aiken and Malatestinic 1995). Barahona et al. (2005) found the sex ratio in S. selecta to vary, being female biased for most of the year but male-biased or balanced in spring, and suggested this was due to the effect of higher female longevity following the emergence of the first spring generation. We found female T. verticalis to be consistently larger than males, as expected (Coccia et al. 2013). However, we also found that adult body size varied between months, and was lower during summer. Development is likely to be more rapid during warmer months, leading to maturity Hydrobiologia (2019) 827:309–324 70 at a smaller size when the benefits of early maturity may be greater e.g. to enable rapid dispersal to colonize other waterbodies. Environmental conditions that favor or limit the reproduction of T. verticalis Generally, the densities of T. verticalis recorded in saline VLP and hypersaline Odiel were similar (Fig. 3), showing T. verticalis thrives over an extensive salinity range of 8-74 g.l-1. There is very limited reproduction from August to October when temperatures and salinities are both high (Fig. 2, S1). In VLP, reproduction is most pronounced in spring when temperatures and salinities begin to increase. In hypersaline Odiel, reproduction peaks in November when winter rainfall reduces salinity. Differences between our seven study ponds in overall density of adults or nymphs along the annual cycle were not explained by salinity (Fig. 3). Nevertheless, changes in corixid abundance between months within an individual pond were often negatively correlated with salinity, especially in hypersaline ponds, suggesting that extreme salinities can limit abundance and reproduction, although there was also evidence that high temperatures can be more limiting (Fig. S1). There was no consistent evidence from nutrient or chlorophyll concentrations to suggest that T. verticalis abundance was determined by pond productivity. In the native range, T. verticalis is reportedly unable to reproduce in abundance at salinities of > 60 g.l-1 (Wurtsbaugh 1992, Simonis 2013). This is roughly consistent with our results, including the finding that the ratio of nymphs to adults dropped in our most saline pond E4, above 70 g.l-1 (Fig. 3). T. verticalis is rarely recorded in salt ponds of salinities >80 g.l-1 at Odiel (Sánchez et al. 2006a). Depth is also important, and in Mediterreanean wetlands corixid densities are generally higher in shallow areas of below 30 cm (Fuentes 2013). T. verticalis density is low at depths of over 50 cm, and this may explain why Odiel pond E1 had a low density, since it was unusually deep (see methods). Preference for shallow areas may help to explain why T. verticalis densities drop off at the hottest times of the year, since water temperatures in the shallows then often exceed air temperatures. Habitat temporality, ectoparasites and relationship with Artemia Temporary habitats are abundant within and around Doñana in south-western Spain (Green et al. 2018). In these habitats, T. verticalis is rarely dominant and coexists with abundant S. lateralis and S. scripta (Coccia et al. 2016, Carbonell et al. 2017). The lower success of T. verticalis in temporary ponds is likely to be related to the rebooting of competition each year after ponds reflood, since all corixid species then need to recolonize the sites and T. verticalis retains no numerical advantage from higher fecundity in previous years. Furthermore, these habitats have Chapter 1: Life cycle of Trichocorixa verticalis 71 a lower salinity, and are frequented by ectoparasitic water mites that are particularly likely to infest the alien species (Sánchez et al. 2015). In salt ponds, T. verticalis preys on Artemia (Céspedes et al. 2017), and in Iberian salt ponds there is an ongoing replacement of native Artemia by the highly invasive American A. franciscana (Horvath et al. 2018) which is syntopic with T. verticalis in their native range. There is a strong parallel between the invasions by American corixids and brine shrimps, since A. franciscana continues to reproduce throughout the winter in Iberian salt ponds unlike the native Artemia, and is also more fecund than them (Redon et al. 2015). The invasion of A. franciscana might potentially benefit T. verticalis by boosting their food supply during winter, but there is no evidence that they facilitated the invasion by the corixid (e.g. Artemia are absent from VLP). Conclusions Our study has improved our understanding of the life cycle of T. verticalis within the introduced range. The invasion success of T. verticalis in permanent, saline wetlands is due to a particularly high population growth rate that allows the alien to dominate the corixid community. There was no strong trend in abundance and reproduction across the salinity range from 8-74 g.l1, except that little or no reproduction was recorded at over 70 g.l-1. Reproduction was confirmed throughout winter months, when average daily mean air temperatures were around 9.4 ºC and average daily maxima were around 16.2 ºC, and no such winter reproduction is reported for native Corixidae. Months of lowest abundance are those of highest salinities and/or of highest temperatures. Laboratory experiments and field sampling suggest T. verticalis could complete one or two more generations per year than competing Sigara species. T. verticalis also has higher fecundity than native Sigara spp. Future studies should compare the life cycle of T. verticalis and native Sigara spp in temporary habitats of lower salinity. Acknowledgements E. Martinez (Director of Marismas del Odiel Natural Park), and Doñana Natural Space provided permission for fieldwork. J. Miguel Medialdea and Pesquerías Isla Mayor, S.A. provided facilities in VLP. Miguel Lozano Terol, Raquel López Luque, Natalia Ospina-Alvarez and Simona Kacmarcikova helped with laboratory and fieldwork. Andres Millán and Josefa Velasco provided helpful advice. Ruben Izquierdo and the “MiBuho” company provided the graphic design for Fig. 7. The staff of the Aquatic Ecology (LEA-EBD) and GIS and Remote Sensing (LAST-EBD) laboratories of EBD-CSIC provided essential support. This research was funded by the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía project (P10-RNM-6262) to AJG, a Severo Ochoa predoctoral contract (SVP-2013067595) from the Spanish Ministry of Science and Innovation (MICINN) to VC, a JAE predoctoral grant from CSIC and a post doc project 3160330 financed by FONDECYT to CC, a predoctoral FPU grant to JAC and a Ramón y Cajal postdoctoral contract from MICINN to MIS. Two anonymous referees greatly improved an earlier version of the manuscript. Hydrobiologia (2019) 827:309–324 78 Table S5: Matrices of Spearman non-parametric correlations using environmental variables and abundances of Trichocorixa verticalis adults and nymphs (see details in Table S1) for three ponds (G3, A3, A7) in VLP and four ponds (E1, E2, E3, E4) in Odiel. Significant P values: * 0.05, ** 0.01, *** 0.001. The intensity of the grey colour in the cells increases with more significant p values. Chapter 1: Life cycle of Trichocorixa verticalis 79 Hydrobiologia (2019) 827:309–324 80 Chapter 1: Life cycle of Trichocorixa verticalis 81 Table S6: Matrix of Spearman non-parametric correlations between environmental variables and the body length of adults (females and males) (see Fig. 5) for A7 pond in VLP. Significant P values: * 0.05, ** 0.01, *** 0.001. The intensity of the grey colour in the cells increases with more significant p values. Chapter 2 “Predator–prey interactions between native brine shrimp Artemia parthenogenetica and the alien boatman Trichocorixa verticalis: influence of salinity, predator sex, and size, abundance and parasitic status of prey” 87 Chapter 2: Predation of Artemia by T. verticalis 88 NTRODUCTION Biological invasions are one of the most important drivers of global change and biodiversity loss (Vilà et al. 2011, Simberloff et al. 2013), and are of particular concern in aquatic ecosystems (Bunn and Arthington 2002, Dudgeon et al. 2006) with impacts at multiple levels of organization (Simon and Townsend 2003). Trichocorixa verticalis (Fieber 1851) (hereafter T. verticalis) is one of the few strictly aquatic insects that can be considered as an ‘‘alien’’ species (Guareschi et al. 2013), and is the only alien aquatic Hemipteran in Europe. This corixid (length <5.5 mm) is native to North America and the Caribbean islands (Tones and Hammer 1975, Tones 1977, Kelts 1979, Wurtsbaugh and Berry 1990), where it can be the dominant Corixidae species in saline wetlands (Wurtsbaugh 1992, Aiken and Malatestinic 1995) or even survive in the open sea (Hutchinson 1931, Gunter and Christmas 1959) owing to its high osmoregulatory ability (Scudder 1976). The subspecies T. verticalis verticalis is now introduced to north-west Africa and the southwestern Iberian Peninsula, where it is often the dominant corixid in saline and hyper-saline wetlands (Carbonell et al. 2017, Guareschi et al. 2013, Rodríguez-Pérez et al. 2009). Its success at high salinities is related to its osmoregulatory ability at the egg, nymph and adult stages, its high fecundity (Carbonell et al. 2016), and the release from parasitism by water mites that infect T. verticalis at lower salinities (Sánchez et al. 2015). In the native range, T. verticalis has been found to be an important predator in saline ecosystems, with the potential to cause major changes via trophic cascades, being one of few predators able to survive in highly mineralized aquatic ecosystems (Wurtsbaugh 1992, Simonis 2013). In the hypersaline Great Salt Lake (USA), the densities of T. verticalis and Artemia franciscana (Branchiopoda, Anostraca) were negatively correlated, and microcosm experiments indicated that predation by T. verticalis reduces the abundance of Artemia nauplii resulting in an increase in phytoplankton abundance (Wurtsbaugh 1992, Wurtsbaugh and Berry 1990). A similar trophic cascade effect of T. verticalis was observed more recently in a mesocosm experiment in which T. verticalis was predating on cladocerans (Moina macrocopa and Daphnia pulex), releasing phytoplankton from grazing (Simonis 2013). The strength of the cascade caused by T. verticalis adults was stronger than that produced by nymphs, which are smaller and less effective as predators (Simonis 2013). The sex of adult T. verticalis might also be expected to influence predation rates, since females are larger with higher energetic expenditure (females are 8% longer, see results). Salinity variation may also influence predation efficiency, especially as T. verticalis and Artemia overlap in the field at the extreme upper end of the salinity range tolerated by T. verticalis (Wurtsbaugh 1992). I Peer J (2017) 5: e3554 95 Figure 2: Predation of adult Artemia according to availability. Number of A. parthenogenetica prey consumed by T. verticalis in 24 h as a function of number of prey available, T. verticalis sex, and salinity (Experiment 2). (A) Salinity 25 g.l-1(B) Salinity 55 g.l-1; female. (C) Salinity 25 g.l-1(D) Salinity 55 g.l-1; male. Shown are range, quartiles, median, and arithmetic mean. Figure 3: Predation of adult Artemia according to parasitic status. Number of Artemia adult prey consumed (infected or uninfected by cestode parasites) by T. verticalis in 24 h (Experiment 3). Results pooling data from different T. verticalis sexes and salinities. Shown are range, quartiles, median, and arithmetic mean. Chapter 2: Predation of Artemia by T. verticalis 96 ISCUSSION Our results confirm our expectations that T. verticalis in the introduced range (subspecies T. v. verticalis) predate brine shrimps, as previously reported for another subspecies (T. v. interiores) in the native range (Wurtsbaugh 1992). We found evidence that predation rates are higher for the larger female T. verticalis, and that T. verticalis are sensitive to the number and size of their prey, to their parasitic status, and to the environmental salinity. Our results suggest T. verticalis readily predate Artemia parthenogenetica, as previously shown for A. franciscana, with which they coincide in their native range (Wurtsbaugh 1992). In his experiments, Wurtsbaugh (1992) found a significant effect of T. verticalis on the density of nauplii larvae (P< 0.01) but not on adult density (P=0.06). In our experiment, we found T. verticalis had a significant preference for the smallest life stage offered (metanauplii), but they also consumed a high proportion of the adult Artemia parthenogenetica. In addition, we have confirmed in the laboratory that T. verticalis readily predate A. franciscana adults (unpublished observations). Invasive alien species sometimes have an advantage because native prey species may not recognize them as predators (Sih et al. 2010). The fact that T. verticalis would not encounter A. parthenogenetica in their native American range seems to make no difference to their ability to recognize them as suitable prey. Even T. verticalis from other habitats in the introduced range which had no prior experience of Artemia seem to instantly recognize native Artemia as prey, and feed readily on them in microcosms (personal observation). Similarly, in a previous experimental study, fish and Odonata larvae predated T. verticalis and the native corixid Sigara lateralis at a similar rate, although Odonata larvae showed a slight preference for T. verticalis as expected from their smaller size (Coccia Boyero and Green 2014). T. verticalis has little difficulty grasping adult Artemia which they are able to roll into a ball before commencing feeding (personal observation, see videos in additional information). Although we used small experimental containers, increasing the chances of encounters between T. verticalis and Artemia, T. verticalis also actively hunt Artemia adults in larger 3.75 l microcosms (personal observation). T. verticalis also capture and feed on live benthic chironomid larvae in the laboratory, but with more difficulty as these larvae often wriggle free. In salt ponds, chironomid larvae are an alternative prey item to Artemia, and are more abundant in ponds of lower salinity where Artemia are rarer (Sánchez et al. 2006a). Copepods are also potential prey items, although it is unclear if they are important in T. verticalis diet or not (Wurtsbaugh 1992). T. verticalis and the native Sigara corixids have similar piercing and sucking mouthparts that allow feeding on softbodied invertebrate prey, although they are omnivorous and feed on algae, detritus and periphyton D Peer J (2017) 5: e3554 97 as well as zooplankton and dipteran larvae (Kelts 1979, Murillo and Recasens 1986, Simonis 2013, Coccia et al. 2016) Brine shrimps and other Anostracans have poor defense mechanisms against predators, and are easy prey for birds, fishes and aquatic insects. Anostracans therefore rely on occupying habitats that are relatively free of predators. Brine shrimps do this by occupying habitats that are too saline for insect predators, whereas fairy shrimps typically do this by occupying temporary aquatic habitats soon after they are flooded and before they are colonized by predators. Hence the addition of a new predator such as T. verticalis, which is able to tolerate hypersaline environments, may have a considerable impact on the distribution of Artemia in the introduced range. Our knowledge of the influence of T. verticalis on food webs and the abundance of other aquatic organisms in the introduced range is currently very limited, and restricted to a stable isotope study in lower salinity fish ponds and temporary ponds (Coccia et al. 2016), and studies of the niche space occupied by T. verticalis and native Sigara corixids (Van De Meutter et al. 2010, Carbonell et al. 2016 2017). This is the first study to consider the influence of T. verticalis when it invades salt pond systems occupied by Artemia. Artemia are keystone species and are the most important filter feeders in salt pond ecosystems (Sánchez et al. 2013, 2016a). In the Odiel salt ponds, field studies have shown that Artemia are low in abundance or absent in ponds of salinities below 100gl-1 where corixids are present (Sánchez et al. 2006b). Predation by T. verticalis and other predators such as the alien fish Fundulus heteroclitus are likely to restrict Artemia to ponds of higher salinities. As we predicted, we found that the larger female T. verticalis had higher predation rates than males. The evidence we found for prey size selection has implications for the cascading effects predation has on phytoplankton densities and dynamics (Simonis 2013) since the filter feeding rates of Artemia increase strongly with body length (Sánchez et al. 2016a). We found significant differences in predation rates between salinities of 55 gl-1 and 25 gl-1, but the results were not very consistent between experiments. In experiment 2, more Artemia adults were predated at the higher salinity, this increase being more marked for male T. verticalis. In experiment 3, more uninfected Artemia adults were predated at the lower salinity, with no salinity effect for infected prey. The lower salinity is closer to the physiological optimum for T. verticalis (Coccia et al. 2013) which is a highly abundant species in fish ponds in SW Spain of a similar salinity (Van De Meutter et al. 2010, Walton et al. 2015). It is possible that T. verticalis increased Artemia predation rates at a higher salinity as a means of compensating for the higher physiological costs of osmoregulation. The change in experimental salinity may also potentially have influenced predation rates via changes in the behaviour of the Artemia prey (Sánchez et al. Chapter 2: Predation of Artemia by T. verticalis 98 2009b) e.g. if uninfected prey became less active with a weaker escape response at the lower salinity. We found that T. verticalis were more likely to predate adult Artemia parthenogenetica when they are parasitized by cestodes, as previously recorded for avian predators (Sánchez et al. 2009a). This may be connected with the bright, red coloration of infected native Artemia (Redón et al. 2015a) owing to an increased carotenoid content (Sánchez et al. 2016b). However, it is unclear whether T. verticalis would use colour as a cue, as occurs with other Heteroptera (Notonectidae, Immonen et al. 2014) and our results may perhaps have been the product of reduced mobility or escape response of infected Artemia. Preference for infected Artemia may also be associated with their higher lipid content (Sánchez et al. 2016b). Predation of infected individuals is of less significance for the reproductive rate of the Artemia population, because cestodes severely reduce the fecundity of infected individuals (Redón et al. 2015a, Sánchez et al. 2016b). This predation by T. verticalis represents a major cost to the cestode parasites since there is no chance of them completing their life cycle. In conclusion, T. verticalis is likely to be important in invaded saline ecosystems owing to its ability to exert top down control on Artemia and other prey, causing trophic cascades. In hypersaline systems, the invasion is particularly important since native corixids were absent or rare prior to the arrival of T. verticalis, adding an important predator that is likely to restrict the abundance and distribution of Artemia. Future work should compare the functional responses of T. verticalis and competing native corixids when feeding on zooplankton and other prey (Dick et al. 2014) to clarify the consequences of the invasion on prey communities at lower salinities tolerated by both native and alien corixids. Given the projected expansion of T. verticalis over large areas of Europe and the Palaearctic (Guareschi et al. 2013) this alien species may have widespread impacts. Acknowledgments We are grateful to Raquel López and Ana Badosa for field assistance and to C. Coccia, J.A. Carbonell, A. Millan and J. Velasco for their help and suggestions. The Aquatic Ecology Laboratory (LEA) at EBD-CSIC assisted with the experiments. Research permits were provided by the Marismas de Odiel Natural Park, of the Regional Andalusian Government (Consejería de Medio Ambiente). 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Canadian Journal of Fisheries and Aquatic Sciences 47:100–109. “Water mites and their interactions with aquatic insect hosts in dune ponds and temporary marshes in south-west Spain” Chapter 3 Chapter 3 111 the 40 ponds sampled in May, we compared the abundance and species richness of adult mites between marsh sites and dune ponds with Mann Whitney U tests. We did similar analyses for the 17 ponds sampled in June (when most ponds were dry already). We compared the habitat use of adults for each species with the characteristics of the available habitats, combining repeated samples in May, April and June for all 40 ponds (Table 1, Fig. 2). Prior to realised niche analysis we performed a Spearman correlation between environmental variables and removed one of each pair of variables that had a strong correlation (rs ≥ 0.7, see Table S2 for remaining variables, which included e.g. phosphate, ammonia, water temperature (ºC) and salinity (g.l-1)). Realised niche for adults and larvae along the salinity gradient Of the 10 mite species recorded as adults, we recorded larvae of five (Eylais infundibulifera, Arrenurus cuspidifer, Hydrachna skorikowi, H. conjecta and H. globosa) which are ectoparasites on Heteroptera, Coleoptera and Odonata (see Table 2 for details). For these five species, we combined data on the presence of adult and larval ectoparasitic mites for ponds sampled in the month of May, and related the data to the salinity range in these ponds. The realised niches of these five species for salinity were assessed by estimating Outlying Mean Index (OMI) and niche breadth (Dolédec et al. 2000), which allowed a characterisation and comparison of species niches based on observational data. Species OMI or species marginality index is a simple measure that integrates the niche specialisation of species according to their selected habitats. It represents the deviation of a species’ distribution from the overall mean conditions of sampling localities. Its significance is tested by random permutations that assess whether the observed species’ position in the gradient differs significantly from a randomised distribution. Niche breadth is a measure of the amplitude in the distribution of each species along the sampled gradient (Dolédec et al. 2000). The salinity variable and species abundance were logarithmically transformed prior to the OMI analyses, which were performed using the ADE-4 package (Thioulouse et al. 1997) in R-studio (ver. 1.1.453 – © 2009-2018 330 RStudio, Inc). Prevalence of all water mite larvae Total prevalence of larvae (of whichever mite species) was calculated for each host taxon focussing on the three groups: Hemiptera, Coleoptera and Odonata, In the 8 dune ponds (Sopetón, Dulce, Hermanillos, Pajas I and II, Taraje and Santa Olalla) sampled each month (May, June and July) (see Table 2), prevalence was compared between months with a Fisher’s Exact Test at https://www.danielsoper.com/statcalc. The confidence interval for prevalence in a given month was calculated at: http://www.sample-size.net/confidence-interval-proportion/ Host-parasite interactions 112 For those 28 ponds sampled in May that had a minimum abundance of corixids that allowed their total prevalence to be calculated, we related prevalence to habitat features. First, individuals of Corixa, Sigara and Trichocorixa were combined in each pond, and ponds with less than N=10 were excluded from the analysis. Then prevalence was related to hydroperiod and compared between dune ponds and marsh sites. We also conducted Spearman correlations between the prevalence of larval water mites and key environmental variables, both for all 28 ponds with a prevalence measure in May, and separately for the 19 dune ponds and the 9 marsh sites (Table S2). Host - parasite interactions between insects and water mites The network of interactions between mite species and their hosts was analysed using the package igraph and network in R studio (ver. 1.1.453 – © 2009-2018 330 RStudio, Inc) and command “cluster_fast_greedy”. Intensity of larval infection The total intensity (number of parasites per individual host) was calculated for each host taxon both for the 8 ponds and for all ponds (Table 3), and also for each month in the 8 ponds (April, May and June) (Table 4). We used Kruskal Wallis test or Mann Whitney tests to compare intensity between host taxa or between months. RESULTS Water mite adults Ten different water mite species were recorded at the adult stage (Table 1). Seasonal differences in eight dune ponds For eight dune ponds sampled intensively (Table S1) we compared the abundance and species richness of adult mites in April, May and June (median abundance = 4, 8, 19; median richness = 1, 2, 2 respectively). Both the abundance and species richness of adults was significantly higher in June than in either April or May (P ≤ 0.015). There were no significant differences between April and May (P > 0.300). Chapter 3 113 40 ponds sampled in May Both abundance and species richness of adult mites was significantly lower (P= 0.001) in 20 marsh sites (median abundance = 5, median richness = 1) than in 20 dune ponds (median abundance = 21, median richness = 2). Adult abundance and richness were also significantly higher in ponds with a longer hydroperiod (P ≤ 0.006), comparing ponds which retained water in July (median abundance = 21, median richness = 2) with those that were dry (median abundance = 7, median richness = 1). 17 ponds sampled in June The abundance and species richness of adult mites was significantly lower (P ≤ 0.029) in six marsh sites (median abundance = 0, median richness = 0) than in 11 dune ponds (median abundance = 6, median richness = 1). Again, there were significant differences between sites with a long (median abundance = 7, median richness =1) and short (median abundance = 0, median richness = 0) hydroperiod (P ≤ 0.009). Habitat use by adults of different species Hydrachna skorikowi occupies both marsh and dune ponds (Table 1) and occupies intermediate conditions along the environmental gradient of available habitat (Fig. 2). The three Eylais species had a similar realised niche (see Fig. 2), and two of them were the only mite species to be recorded more frequently in marsh sites than in dune ponds (Table 1). Eylais infundibulifera showed a preference for relatively high salinities (Fig. 2) and was the only mite species that showed a significant difference in the physicochemical variables between sites that were sampled, and those that were occupied (P <0.0001, comparing the observed species’ position in the habitat with a randomised distribution). Arrenurus cuspidifer, Eylais degenerata and Hydrachna conjecta had the narrowest niches, but this is inevitable since they were recorded in few ponds (Table 1). Realised niche for mite adults and larvae along the salinity gradient Larvae were recorded for five mite species (see Table 2 for details). Larvae and adult of Arrenurus cuspidifer (combined total for adults and larvae =4); Hydrachna globosa (n=21) and Hydrachna conjecta (n=178) were only recorded in dune ponds (Table 1, Fig. 2). Hydrachna skorikowi had 722 individuals in dune ponds and 37 in marsh sites, whereas Eylais infundibulifera had 11 individuals in dune and 72 in marsh sites. Host-parasite interactions 114 Table 1. Summary of frequency of occurrence of 10 different mite species in Doñana ponds in 2014, combining data from April, May and June for all ponds sampled at least once (N = 40 ponds). Mite species N ponds with only adults N ponds with only larvae N ponds with both adults and larvae Total N ponds with presence of the species Habitat presence* Eylais extendens 3 0 0 3 Marsh >Dune Eylais degenerata 1 0 0 1 Dune Eylais infundibulifera 9 1 3 13 Marsh >Dune Hydrachna globosa 2 3 1 6 Dune Hydrachna conjecta 0 3 0 3 Dune Hydrachna skorikowi 2 8 13 23 Marsh <Dune Hydrodroma pilosa 1 0 0 1 Dune Hydryphantes flexuosus 3 0 0 3 Dune Piona nodata 10 0 0 10 Dune Arrenurus cuspidifer 3 1 0 4 Dune *Marsh > dune: proportion of ponds with presence of the species was higher in marsh; Dune > Marsh: proportion of ponds with presence of the species was higher in Dune Salinity niches of each species overlapped considerably, but a degree of segregation along the salinity gradient was observed for the five species (Fig. 3). Only E. infundibulifera used a salinity range that differed significantly from the mean conditions of the sampling sites, being associated with relatively higher salinities (Fig. 3). Chapter 3 115 Figure 2: Realised niche (ellipses) of adults of different mite species across the physical chemistry gradients in the study area obtained from outlying mean index analyses combining data from April, May and June for all ponds sampled at least once (N = 40 ponds). Vectors represent the explicative variables in OMI ordination. Dots are sites of species occurrences. Hydrodroma pilosa and Eylais degenerata are not represented because they were only recorded in a single pond. E. infundibulifera was the only species with a significant difference between habitats occupied and those available (P <0.0001, comparing the observed species’ position in the habitat with a randomised distribution). Host-parasite interactions 116 Table 2: Details of host abundances (N tot.), larval prevalence (Prev.) and confidence interval (CI, 95%) for each month and for each host species (focussing on Hemiptera, Coleoptera and Odonata). Data are from 8 dune ponds sampled monthly (Table S2). L=larvae; A=adults. In the lower section of the Table, details are given of the abundance of adult mites in the same ponds, and of the codes to identify the species in column P (Parasites). Chapter 3 117 Figure 3: Outlying Mean Index (dot) and realised niche breadth (horizontal bars) for five mite species that are ectoparasites of Heteroptera, Coleoptera or Odonata along the salinity gradients in the study area obtained from outlying mean index analyses in the month of May. Salinity was log-transformed. Mite data include both adults and larvae. Small vertical bars at the bottom correspond to the position of sites along the salinity gradient. * mean salinity value of the sampling sites. Only Eylais infundibulifera showed a significant difference along the sampled salinity gradient with a randomised distribution (P <0.001). Prevalence of water mite larvae In ponds sampled in May, overall there were significant positive correlations between prevalence and maximum depth, sampling date and the abundance and species richness of adults, as well as negative correlations with salinity and nutrient concentrations (Table S2). In the marsh sites (which were generally more saline and eutrophic, Fig. 2), prevalence was negatively correlated with ammonia concentration but positively correlated with adult species richness. Within the dune ponds, prevalence was positively related with sampling date, but negatively correlated with temperature which itself may be an indicator of depth (Table S2). Host-parasite interactions 118 Figure 4: Prevalence (%, mean ± 95% confidence interval) for May and June in eight dune ponds (see Table S2) with mite parasites Hydrachna skorikowi (HS). Results are shown above bars for significant differences (p < 0.05) between May and June for each species. Fisher´s exact tests were used to compare the prevalence between all the individuals of a given host species for each month. When there were more than 200 individuals in the samples, a X2 test was used instead. Prevalence in April was zero for these host taxa (Table 2). Figure 5: Prevalence (%, mean ± 95% confidence interval) for May and June of eight dune ponds (see Table S2) with several mite parasites: Eylais infundibulifera (EI), Hydrachna conjecta (HC), Arrenurus cuspidifer (AC) and Hydrachna globosa (HG). Results are shown above bars for significant differences (P < 0.05) between May and June for each species (Fisher´s exact tests, or X2 test when there were > 200 individuals in the samples). Prevalence in April was zero for these host taxa (Table 2). Chapter 3 119 Table 3: Summary of potential insect hosts recorded (Hemiptera, Coleoptera and Odonata), and the abundance of infected and uninfected individuals for each insect taxon (S= Stage, with two options: L=larvae, A=adults), as well as the prevalence and intensity of infection by different mite species (P). Data are presented for number of individuals (N) and number parasitized (Np) both for all study ponds (all ponds) and for the 8 ponds sampled monthly (8 ponds). Prevalence is presented with confidence intervals (CI, 95%). Mean, median and range for intensity of infection is also given, indicating the mite species involved. Arrenus cuspidifer (AC), Hydrachna skorikowi (HS), Hydrachna conjecta (HC), Hydrachna globosa (HG), Eylais infundibulifera (EI). Data combined for April, May and June. Host-parasite interactions 120 Figure 6: Larva Cybister tripunctatus with external parasite Hydrachna globosa. Credits Antonio G. Valdecasas & Vanessa Céspedes Host - parasite interactions between insects and water mites Figure 7 summarises the host-parasite interactions detected in our study. Besides the host-parasite interactions previously described in the literature, we recorded many new interspecific interactions, listed in Table 5. Hydrachna globosa shows a previously unreported interaction with Dytiscidae larvae (Table 4). When comparing the seven Cybister individuals with other host species in the dune ponds, their prevalence of infection was particularly high (Table 3, Fig. 4 and 5) as was the intensity of infection (Table 4, Fig. 6) with a median of 5 H. globosa larvae for infected Cybister hosts. As well as the novel interaction between H. globosa and Coleoptera, we recorded the first interactions between H. skorikowi and the Hemipteran families Notonectidae and Naucoridae (Table 4 and 5). On the other hand, we failed to detect the hosts of the mites Eylais degenerate, E. extendens, Hydrodroma pilosa, Hydryphantes flexuosus. In the case of the latter two species this was because we did not include the diptera and trichoptera in our host study (Table 5). Piona nodata is not thought to be parasitic (Table 5). Chapter 3 127 the generally higher salinity and nutrient status of marsh sites, although potentially also with the absence of permanent waters in the marsh area that facilitate rapid recolonisation of the marsh during the wet cycle. We found good evidence of specialization and niche differentiation, with Eylais infundibulifera being the one species strongly associated with marsh habitats. Doñana dune ponds generally exhibited moderate nutrient concentrations in comparison to other Mediterranean temporary ponds (Della Bella et al. 2008, Waterkeyn et al. 2008), whereas the marsh system has a catchment area affected by nutrient pollution from intensive agriculture and poorly treated urban wastewaters (Paredes et al. 2018). High levels of turbidity have been shown to be an important determinant of macroinvertebrate density and diversity (Stewart and Downing 2008, Sharma and Rawat 2009), and in Doñana dune ponds is often due to the treading effect of cattle which generally has a strong negative effect (Declerck et al. 2006). We found evidence of strong seasonality. In ponds with a long hydroperiod, water mite adults were most abundant and diverse in June when most other ponds were already dry or coming towards the end of the hydroperiod month. This is consistent with our finding that the prevalence of mite larvae was highest in May, with a positive correlation with date within that month (i.e. a peak in late May). Prevalence in April was nearly zero, suggesting that mites mainly lay their eggs in March or April, and that many of the adults recorded in June were larvae the month before. As ponds dry out, it is likely that many of the Hemiptera and other insects that are forced to disperse by flight disperse mite larvae into new habitats. Acknowledgements Doñana Natural Space provided permission for fieldwork. Miguel Lozano Terol and Raquel López Luque helped with laboratory and fieldwork. The staff of the Aquatic Ecology (LEA-EBD) and GIS and Remote Sensing (LAST-EBD) laboratories of EBD-CSIC provided essential support. This research was funded by Severo Ochoa predoctoral contract (SVP-2013-067595) from the Spanish Ministry of Science and Innovation (MICINN) to VC, Ramón y Cajal postdoctoral contract from MICINN to MIS. 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Host-parasite interactions 132 SUPPLEMENTARY MATERIAL Table S1: Adult water mite abundance in 8 ponds on different sampling date (see Supplementary Appendix I for details about species identification). In some samples the identification to species level was not possible. For genus Hydrodroma see Appendix II. No adults= absence of water mite adults in the sample. Pond Location April May June Verdes 36°59'4.34"N 6°27'48.58"O No adults 2 Hydrachna skorikowi 6 Piona nodata No adults Sopetón 36°57'32,32"N 6°26'59.41" O 4 Hydrachna skorikowi 1 Piona nodata 18 Hydrachna skorikowi 1 Eylais infundibulifera 11 Hydrachna sps. 23 Eylais infundibulifera Hermanillos 36°58'54.22"N 6°27'12.27" O 2 Hydrachna skorikowi 3 Hydrachna sps. 9 Piona nodata 1 Eylais infundibulifera 3 Piona nodata 7 Eylais infundibulifera Pajas 36°58'47.99"N 6°27'54.64" O 23 Hydrachna skorikowi 88 Hydrachna skorikowi 2 Eylais infundibulifera 1 Piona nodata 10 Hydrachna globosa 1 Hydryphantes flexuosus 6 Eylais infundibulifera 2 Hydrachna sps. Dulce 36°58'44.51"N 6°29'05.29" O 26 Hydrachna sps. 53 Piona nodata 9 Hydrachna sps. 29 Piona nodata 1 Arrenurus cuspidifer 2 Eylais infundibulifera 2 Hydryphantes flexuosus 21 Hydrachna skorikowi 2 Eylais infundibulifera Taraje 36°59'20.50"N 6°29'36.48"O 11 Hydrachna skorikowi 21Hydrachna skorikowi 1 Piona nodata 1 Arrenurus cuspidifer 1 Hydrachna globosa 6 Hydrachna skorikowi 1 Hydrodroma cf. pilosa 1 Eylais infundibulifera Pajas II 36°58'50.99"N 6°28'1.34"O 1 Hydryphantes flexuosus 3 Hydrachna skorikowi 25 Hydrachna sps. 1 Hydryphantes flexuosus 3 Hydrachna skorikowi 1 Hydrachna skorikowi Santa Olalla 36°58'41.64"N 6°28'43.33"O No adults 1 Hydrachna skorikowi 1 Arrenurus cuspidifer 6 Hydrachna skorikowi Chapter 3 133 Table S2: Correlation matrix for prevalence of mite larvae, abundance of adult mites, and species richness of adult mites (ASR) in each pond in May together with physico-chemical variables. Data for prevalence correspond to combined total prevalence in the sum of all individuals from the genera Corixa, Sigara and Trichocorixa, with a minimum combined abundance of 10 individual corixids for a pond to be included in the analysis. Data for ponds sampled during May (*P-values for Spearman correlations: ***=<0.001, **=0.01, *p=0.05). Data for adult mite abundance and species richness are from 20 dune ponds and 20 marsh sites (N=40). In blue: data for prevalence are combined from 19 dune ponds and 9 marsh sites. In yellow: dune ponds. In green: marsh sites. Prevalence Total Dune pond Marsh site Abundance Abundance 0.72** 0.18 0.44 Total Dune pond Marsh site ASR ASR 0.66** 0.2 0.51* 0.90*** 0.82*** 0.94*** Total Dune pond Marsh site Salinity -0.83*** -0.23 -0.03 -0.78*** 0.35 -0.75*** -0.78*** 0.5* -0.67** Turb -0.34 0.03 0.29 -0.74** -0.70** -0.08 -0.56* -0.93*** 0.12 DepthMax 0.53* -0.13 -0.23 0.79*** 0.56* 0.13 0.7** 0.57* -0.05 Date 0.87*** 0.57* 0.48 0.66* -0.17 0.73** 0.66** -0.36 0.81*** TempWater -0.58* -0.53* -0.15 -0.55* -0.33 -0.54* -0.4 -0.05 -0.38 Ammonia -0.51* 0.32 -0.63** -0.35 -0.39 -0.42 -0.51* -0.64* -0.51* Nitrites -0.5 0.38 -0.35 -0.16 -0.59* -0.13 -0.39 -0.75*** -0.23 Nitrates -0.49 0.29 -0.35 -0.22 -0.70** -0.13 -0.45 -0.82*** -0.24 TN -0.81*** 0.13 -0.54 -0.75*** -0.03 -0.79*** -0.82*** -0.33 -0.84*** Phosphate -0.56* 0.33 -0.35 -0.29 -0.62* -0.14 -0.53* -0.77*** -0.24 TP -0.44 0.25 -0.36 -0.22 -0.66** -0.12 -0.48 -0.84*** -0.21 pH 0.72** -0.16 0.16 0.68** 0.3 -0.3 0.79*** 0.43 -0.13 Host-parasite interactions 134 Supplementary Appendix I. Chemical measurements for 40 ponds in each sampled month. The 8 dune ponds sampled monthly are shown in bold. April May June Habitat Name pond pH TempWater (ºC) Cond. (mS/cm) pH TempWater (ºC) Cond. (mS/cm) pH Temp Cond. (mS/cm) Marsh GUADIAMAR 8.31 19.00 5.05 6.67 24.50 7.33 6.00 30.00 9.26 Marsh FAO 7.79 18.80 1.34 6.69 26.00 1.04 6.46 29.00 1.43 Marsh ENTREMUROS 7.94 18.00 2.80 6.64 21.00 8.17 6.64 31.00 8.17 Marsh LOBO 9.10 19.20 17.50 7.10 27.50 28.01 Marsh ROSALIMAN 8.59 18.50 4.61 6.67 28.00 6.76 Marsh CANAL 8.44 30.30 15.72 6.67 28.00 31.80 Marsh 3N3MP 8.27 18.80 37.60 6.64 34.00 47.40 Marsh 6N2MP 7.88 22.10 12.40 6.68 26.00 49.40 Marsh 7N2PP 9.00 21.80 25.80 6.76 38.00 33.00 Marsh 8N4PP 8.62 24.80 15.01 6.79 32.00 35.70 Marsh 9N3PP 7.22 22.20 14.02 6.63 32.10 38.30 Marsh 9N2PP 7.70 22.70 24.60 6.73 33.00 45.30 Marsh 0N2GP 9.42 26.10 25.00 6.69 36.00 31.10 Marsh 4N1MP 9.67 25.90 27.70 6.69 27.20 44.50 Marsh 8N1MS 9.22 29.30 21.20 6.65 24.30 48.50 Marsh 0N1GP 8.28 29.30 26.00 6.50 28.00 35.60 Marsh 3N1PP 8.94 27.50 29.10 6.70 22.30 46.50 Marsh MARTINAZO 6.51 17.50 0.33 6.58 26.60 5.62 6.64 28.00 15.12 Marsh M-DOMINGUEZ 6.65 25.00 0.34 6.65 23.90 0.41 6.29 29.60 0.80 Marsh MANECORRO 6.61 31.90 0.90 6.33 21.30 1.71 6.43 26.30 2.60 Dune Pond VERDES 6.40 25.00 1.12 6.64 18.50 3.50 6.33 27.00 4.47 Dune Pond PAJAS II 6.59 25.00 8.91 6.65 25.90 13.10 6.37 32.90 24.63 Dune Pond PAJAS I 6.84 25.00 1.13 6.66 23.90 2.80 6.29 29.30 5.10 Dune Pond HERMANILLOS 6.62 26.20 4.48 6.63 29.10 7.64 6.30 23.00 13.73 Dune Pond DULCE 6.55 15.30 1.31 6.75 28.20 1.63 6.30 29.10 1.92 Dune Pond TARAJE 6.63 27.50 5.29 6.75 28.20 8.91 6.43 33.00 17.39 Dune Pond SOPETON 6.91 27.00 1.93 6.73 19.70 2.25 6.33 30.10 2.71 Dune Pond SANTA OLALLA 6.78 23.60 6.73 7.01 26.70 8.80 6.32 29.00 9.81 Dune Pond ZAC. ORILLA 6.59 19.10 0.02 6.59 18.80 0.17 6.32 21.50 0.19 Dune Pond ZAC. NSE 6.58 24.70 0.68 7.01 23.70 5.22 6.50 23.10 8.66 Dune Pond BAMBINO 6.59 25.40 0.98 6.68 20.90 1.35 6.32 21.00 2.48 Dune Pond BOLIN 6.00 25.00 1.87 7.12 29.25 2.04 Dune Pond 68 6.53 16.40 1.84 6.70 21.20 3.17 Dune Pond 70 6.75 23.60 0.65 6.60 25.50 2.02 Dune Pond ZAHILLO 7.22 30.10 2.77 6.59 22.20 11.36 Dune Pond 67 5.90 19.30 2.26 6.70 21.20 3.17 Dune Pond NAVAZO ARAGON 6.63 23.30 0.44 6.66 22.60 0.59 Dune Pond GLORIETA 6.42 30.80 0.35 6.68 25.10 29.60 Dune Pond TIKAPE 6.54 24.60 2.02 7.01 21.00 3.04 Dune Pond PICCOLA 6.61 25.50 5.76 7.20 23.70 8.75 Chapter 3 135 Supplementary Appendix II. Larvae and adults imaging were carried out with a Leica TCS SPE Laser Confocal Scanning Microscope. Each serial optical stack was processed with Fiji/ImageJ, Amira (version 5.5.0) and Photoshop CS5 extended. For each species some of its diagnostic characteristics is illustrated. (A) Adult Eylais extended. Fourth segment of palps. Arrows pointing to pectinate setae. Amira Voltex volume rendering; (B) Adult Eylais infundibulifera. Ocular plate. Fiji Maximum Intensity Projection; (C) Adult. Eylais cf. degenerata. Gnathosoma plate. Fiji Maximum Intensity Projection; (D) Larvae Hydrachna conjecta. Coxae. Amira Voltex volume rendering; (E) Larvae Hydrachna globosa. Coxae. Amira Voltex volume rendering; (F) Larvae Hydrachna skorikowi. Coxae. The larvae of Hydrachna skorikowi are identifiable by the similar length of lateral border of coxa I and II, and the presence of a ‘thick cone’ of setae on coxa III. Amira Voltex volume rendering; (G) Adult Hydrodroma cf. pilosa. Cuticle sculpture. Amira Voltex volume rendering. See Annexe II “Taxonomic Key to Genera Hydrodroma in Europe” for more details; (H) Adult Hydryphantes flexuosus. Ventral side. Fiji Maximum Intensity Projection; (I) Adult Piona nodata. Ventral side. Fiji Maximum Intensity Projection; (J) Adult Arrenurus cuspidifer. Ventral side. Fiji Maximum Intensity Projection. Host-parasite interactions 136 143 Chapter 4: Water mites, salinity and water bugs 144 NTRODUCTION The ecological consequences of being parasitized and the net fitness cost for the host critically depend on environmental conditions. Parasites often interact in complex ways with other stressors (reviewed in Sures 2008), and the outcome of the interaction can be positive or negative for the host. The parasite and the stressor may often have additive or synergistic effects. For example, Kelly et al. 2010 showed that the herbicide glyphosate and the trematode parasite Telogaster opisthorchis Macfarlane 1945, act synergistically on survival and the development of spinal malformations in juvenile Galaxias anomalus Stokell 1958. Less often, parasites can ameliorate the effect of the stressor. For example, Sánchez et al. 2016 showed that cestode parasites increased brine shrimp resistance to metalloid arsenic, by enhancing the host antioxidant defences. The outcome of the interaction between different stressors also depends on the particular life-history trait considered. For example, Coors and De Meester 2008 experimentally evaluated joint effects of parasites, predation and contaminants on Daphnia magna (Strauss 1820), and found all possible of joint effects – additive, synergistic and antagonistic – depending on the trait considered, and demonstrated that multiple stressors reduced population growth by up to 28%. Parasitism should therefore be studied in the context of key environmental stressors. Particularly important is interaction with climate change, which is predicted to have important effects on parasitism, disease transmission and possibly virulence, with a major impact in aquatic ecosystems (Marcogliese 2001). On the other hand, we need to integrate studies across multiple species and developmental stages. For example, infection data from adult stages may underestimate the impact of parasites in natural populations, because early developmental stages are generally more sensitive (Kefford et al. 2004). Many studies have focused on vectorand foodborne diseases affecting humans, livestock, or domestic animals (Harvell et al. 1999, Rose et al. 2001, Marcogliese 2008, Sternberg 2014). However, less attention has been devoted to hostparasite interactions that do not involve humans (Marcogliese 2001). One interesting model system for studying interactions between environmental stress and parasitism is water mites infecting aquatic insects. Water mites or Hydrachnidia are the most diversified group of the Acari in freshwater ecosystems, with more than 300 genera and 6000 species (Gledhill 1985, Zhang et al. 2011). They occur in almost all fresh and brackish aquatic environments around the world where they can reach densities exceeding 2000 specimens per square meter (Smith 2009). Water mites have a complex life cycle involving ecto-parasitic and free living stages (Proctor et al. 2015). While the deutonymph (second larval stage) and adult instars are typically free-living predators feeding mainly on insect eggs, insect larvae and microcrustaceans, the larval stage is morphologically distinct and ecto-parasitic (Böttger 1976). I Plos One (2019) 14(1): e0209828 145 Larvae mainly parasitize aquatic and semiaquatic insects, with relatively weak host-specificity (Di Sabatino et al. 2000). They feed on the host haemolymph and after a period of engorgement they detach themselves. One of the most common genera is Hydrachna, which often parasitize waterboatmen (Heteroptera: Corixidae). Corixids are key links in aquatic food webs being both primary and secondary consumers, and serving as food for predatory vertebrates such as fish and birds (Kortegaard 1974, Applegate et al. 1977). Water mites can strongly impact their host populations and influence biological interactions between corixid species (Smith 1977, Sánchez et al. 2015). Mediterranean temporary ponds, such as those of Doñana National Park (south-west Spain), allow the study of interactions between water mites and corixids in conjunction with environmental stressors. In Doñana, Hydrachna skorikowi Piersig, 1900, is one of the most abundant and widespread watermites (Sánchez et al. 2015). Water salinity is a key natural stressor in arid and semiarid zones of the Mediterranean basin. It has a major influence on biotic communities and is affected by a range of natural and anthropogenic processes. During the summer, temperature and salinity increase, with the concomitant reduction of their depth and surface area in Doñana ponds, which completely dry out by August (Serrano et al. 2016). In these conditions, corixid densities become high and allow for increasing contact between ectoparasites and their hosts, although salinity exert strong controls on aquatic communities, eliminating sensitive species (Frisch et al. 2006). Human water use and climate change in Doñana and in the Mediterranean region in general tend to increase salinities (Jeppensen et al. 2015, Green et al. 2017). In the present work, we used laboratory experiments to study the interactions between corixids and water mites and how they were affected by environmental stressors. We examined the effect of salinity on adult corixids (Sigara lateralis (Leach 1817) and Corixa affinis (Leach 1817)) and how the results depend on the presence of ectoparasites (H. skorikowi). The salinity gradient is known to structure corixid communities in Mediterranean wetlands (Carbonell et al. 2017), but the role of parasitism has not previously been explored. We compared both adult corixid mortality and female fecundity (number of eggs and hatching success) between unparasitized individuals and those infected by mites. We also examined the relationship between the presence of H. skorokowi and the mortality rates of S. lateralis larvae. METHODS Study area Doñana is a Mediterranean wetland complex, situated on the Atlantic coast of south-west Spain (36°58'41''N, 6°20'40''W). It has the highest degree of environmental protection in Spain Chapter 4: Water mites, salinity and water bugs 146 (National Park) and is one of the most emblematic protected areas in Europe. It was designated a UNESCO Man and Biosphere Reserve in 1980, a Ramsar Site (Wetland of international importance) in 1982, and a UNESCO World Heritage Site in 1994 (Green et al. 2017). The climate is sub-humid, with a well-defined seasonality. The area has very dry and hot summers and wet and cool winters (with rainfall mostly occurring between October and March). The region includes a rich network of more than 3000 temporary dune ponds that vary greatly in size, hydroperiod and salinity (Green et al. 2017). It also includes a few permanent and semipermanent lagoons formed in an area where discharges of an adjacent dune and regional aquifers coincide (Díaz-Paniagua et al. 2015). The ionic composition of Doñana pond waters is dominated by Chloride (Cl-) and Sodium (Na+) as a result of the solubilisation of salts from the sediment and airborne marine salt deposition (Serrano and Aragónes 1995, López and Toja 1995). Calcium, magnesium, sulphate and silica are usually present at much lower concentrations. The study was conducted on corixids and mites collected in one of the larger semi-permanent oligohaline lagoons of Doñana National Park, Laguna Dulce (see López et al. 1991 for a limnological description). Study model Twelve species of water boatmen Corixidae can be found in Laguna Dulce, which is representative of larger dune ponds in Doñana (see Florencio et al. 2009, plus S1 and Table S2s for more details), but only a fraction of them are regularly encountered. Sigara lateralis and Corixa affinis are the most common species of their respective genera (see Table S2). Corixids used in our study are mainly omnivorous (Murillo 1986, Simonis 2013). Their piercing-sucking mouthparts allow them to feed on both plants and animals. They are known to be predators on other invertebrates such as cladocera or Artemia (Cambpbell 1979, Wurtsbaught 1992, Céspedes et al. 2017). However, larvae generally feed at lower trophic levels, consuming much periphyton and phytoplankton (Kelts 1979, Coccia et al. 2016). Depending on the species and the availability of different foods, they can be more or less herbivorous (see Coccia et al. 2016 for niche differences in different ponds). Corixids from Doñana are commonly parasitized by two water mite species: Hydrachna skorikowi (Family Hydrachnidae) and Eylais infundibulifera (Koenike, 1897; Family Eylaidae) (see Table S3 and Sánchez et al. 2015). Larvae of Hydrachnidae are strictly aquatic and can use dissolved oxygen from the water; besides Nepomorpha (Heteroptera), they also parasitize other aquatic insects such as Dytiscidae and Hydrophilidae (Coleoptera) (Davids et al. 2006, Aykut et al. 2018). Eylais larvae are aerial (Lanciani 1969), requiring an air-oxygen supply to survive and are restricted to areas such as under the wings, tergites, the underside of the elytra and hemelytra of the host (Nielsen 1976, Lorenzo-Carballa et al. 2011). Plos One (2019) 14(1): e0209828 147 Experiments Samples of corixids of different stages (larvae and adults, and both infected by H. skorikowi and uninfected) were collected with a hand net of 250 μm mesh in June 2014. All specimens were transported to the laboratory in containers filled with water from the lagoon for subsequent experiments. Experiment with corixid larvae The objective of this experiment was to explore the relationship between water mite infection and larvae mortality. We were unable to include a salinity gradient due to the shortage of infected larvae required for testing multiple experimental factors. On 9 June, we collected larvae of S. lateralis parasitized with a single H. skorikowi (mite identification was confirmed later, see below) and unparasitized individuals from Laguna Dulce. We collected a total of 30 instar II, 30 instar III, 35 instar IV and 35 instar V of unparasitized S. lateralis, and 13 instar II, 11 instar III, 15 instar IV and 21 instar V of parasitized S. lateralis. All the larvae were individually placed in plastic containers with aquarium stones and sterilized water from the collection area (350 ml). To minimize mortality of this particularly vulnerable stage, individuals were placed in a climatic chamber under conditions simulating natural environment (25 ºC and 12:12 photoperiod). On alternate days, we added 1 ml of algae (Tetraselmis chuii – Easy Algae®) as food. The water level of each container was checked and adjusted every day. Oxygen level was measured regularly, always being between 93-98 % (saturation). During 15 days we daily checked for mortality of corixid individuals. From day 15 we checked for moulting to adult stage until the last individual moulted (30 days). We removed those individuals for which mites moulted and became detached from the host (5 in total). Experiment with adult corixids The objective of this experiment was to explore the relationship between mite infection and adult mortality and female fecundity (number of eggs and hatching success) under different salinity conditions. We selected adult Sigara lateralis and C. affinis parasitized with a single Hydrachna skorikowi, or unparasitized adults, from Laguna Dulce. We rejected individuals infected with more than a single mite so as to simplify the experimental design. Corixids were acclimatized for 48 hours in a climate chamber at 20 ºC and 12h/12h photoperiod. These conditions were selected to make results comparable with other studies (for example Kefford et al. 2004). Each individual was placed in a 250 ml container with a mesh (10 mm2 size and 1 mm2 core) serving as substrate for eggs, and randomly allocated to one of 4-5 salinity treatments (depending on the corixid species). According to the natural conductivity range Chapter 4: Water mites, salinity and water bugs 148 of the habitat commonly occupied by selected species (including lower and upper extreme values (Kelly et al. 2010, Sánchez et al. 2015), salinity treatments were: 0.5, 5, 10 and 15 g.l-1 for S. lateralis and 0.5, 5, 10, 15 and 20 g.l-1 for C. affinis. We used 10 S. lateralis and 5 C. affinis for each combination of salinity treatment, sex and parasitic status (a total of 160 S. lateralis and 100 C. affinis). The different saline solutions were prepared by dissolving marine salt (Ocean Fish, Prodac@, Citadella, Italy) in distilled water. We also used a control group with water from the lagoon where corixids were collected (0.8 g.l-1). It included 40 females and 40 males (20 parasitized and 20 unparasitized of each sex) of S. lateralis and the same for C. affinis. Corixids were fed every day with 2 frozen chironomids each. The water level of each container was checked and adjusted every day. Mortality was checked every day, and the number of eggs produced by S. lateralis within two weeks counted with the aid of a stereomicroscope. C. affinis did not reproduce in the laboratory. Hatched larvae were also counted during a month, to estimate hatching success. Experiments were run for 15 days for S. lateralis, and 35 days for C. affinis. This difference is due to the greater longevity of the larger C. affinis in the laboratory. We removed those individuals (three S. lateralis) for which mites moulted and became detached from the host. Water mite identification Prior to identification, water mite larvae were detached from the host under a Bausch and Lomb stereo microscope. Subsequently, larvae were mounted and studied with a Leica TCS SPE Laser Confocal Scanning Microscope (see Lorenzo-Carballa et al. 2011 for detailed procedure). Optical serial sections were acquired and processed with Fiji/ImageJ (version 1.48d; downloaded from http://fiji.sc/Fiji), Amira (version 5.5.0) and Photoshop CS5 extended. The larvae of Hydrachna skorikowi are identifiable by the similar length of lateral border of coxa I and II, and the presence of a ‘thick cone’ of setae on coxa III (Davids 1973). Statistical analysis Cox regression models were used to examine the survival of corixids (time to death from the start of the experiment) in relation with water mite infection and larval stage. The interaction stage x infection status was also included in the model. We also used cox regression models to analyse the survival time of Sigara lateralis adults in relation to salinity (0.5, 5, 10 and 15 g.l-1 together with water from the collection site as a control (0.8 g.l-1)), infection status (infected by H. skorokowi or uninfected), corixid sex and their interactions. A similar cox regression analysis was conducted for C. affinis adults, incorporating the additional salinity treatment of 20 g.l-1 (Supplementary Material). Plos One (2019) 14(1): e0209828 149 GLMs were used to analyze the number and hatchability of S. lateralis eggs in relation with salinity (0.5, 5, 10 and 15 g.l-1, plus control water from the collection site (0.8 g.l-1)) and parasitic status (infected by H. skorikowi or uninfected) within two weeks. A Poisson error distribution with a log link function was applied for the number of eggs, and a binomial error distribution and logit link function for the percentage of hatching. Overdispersion were checked for and corrected with Pearson Chi-square. All statistical analysis were performed using R Version 1.1.453 using survival and survminer packages and Statistica 13.3 software. RESULTS Experiments with corixid larvae. Association between mite infection and larval mortality None of the parasitized larvae (n = 60) reached the adult stage, compared to 43.1% of unparasitized larvae (n = 130), a highly significant difference (Fisher exact, P < 0.001). On four occasions the corixid individual died at stage V during a moult as shown in Fig. S1, although the mites completed development to the adult stage. On 4 occasions, infected larvae at stages II to IV did moult successfully to the next instar but did not reach the adult stage. Survival time in the laboratory was therefore shorter for parasitized larvae (mean ± S.E., 2.957 ± 0.244, range 1-8 days) than for unparasitized larvae (8.544 ± 0.564, range 1-15 days; Fig. 1). Cox proportional hazard regression analysis showed a negative significant correlation between infection and larval survival time (Table 1). There was also a significant effect of larva stage (Table 1), uninfected larval of instar V being more likely to survive than earlier instars (Table 1). The interaction parasitic status x larva stage was also statistically significant (Table 1). Chapter 4: Water mites, salinity and water bugs 150 Figure 1: Comparison of cumulative survival of different stages (II, III, IV and V) of larvae of Sigara lateralis between those parasitized by Hydrachna skorikowi and those unparasitized. Individuals were maintained in water from the collection site (Dulce pond, 0.8 g.l-1). Stages represented are those for the beginning of the experiment, e.g. if a larvae moulted from stage II to III before death, it is represented here as II. Table 1. Results of Cox proportional hazard regression analysis on S. lateralis larval survival based on different parasitic status and developmental stages. The table shows for each term in the design matrix the estimated coefficient βj (coef), the relative risk exp (βj) (exp (coef)), the standard error, the z-value and the corresponding P-value. Each P-value provides a test for the difference of each level with respect to the baseline. The overall P-value for factors with more than two levels (i.e. stage) and for the interaction stage*infection status, is obtained through the Wald test and is showed under the table. Effect Level of effect coef exp (coef) S.E. (coef) Zvalue P-value (>|z|) Infection status Unparasitized -3.045 0.047 0.678 -4.48 P<0.001 Stage III -1.627 0.196 0.558 -2.91 0.0035 IV -1.673 0.187 0.585 -2.85 0.0042 V -2.123 0.119 0.574 -3.69 P<0.001 Stage*infection status III*unparasitized 2.538 12.651 0.725 3.49 P<0.001 IV*unparasitized 1.645 5.184 0.746 2.20 0.027 V*unparasitized 1.430 4.180 0.731 1.96 0.047 Concordance= 0.717 (S.E. = 0.03). Rsquare= 0.379 (max possible= 1 ) Note: Overall P-value for Stage factor variable and Stage* infection status interaction. Wald test “Stage”; X2=14.1, df=3, P (> X2) = 0.0028 and “Stage* infection status”; X2 = 16, df = 3, P (> X2) = 0.0011. Plos One (2019) 14(1): e0209828 151 Experiments with adult corixids. Mortality rate and fecundity in relation with mite infection and salinity Cox regression analysis showed both mite infection by H. skorikowi and high salinities to be negatively associated with survival time of adult S. lateralis (Fig. 2a, Table 2). Survival time was significantly higher for females than males. There were also a significant interaction between salinity and infection status, indicating a relatively higher mortality of infected boatmen at infection higher salinities. Interations between infection status and sex and between salinity and sex were also significant, indicating a greater mortality in infected females, and in males exposed to high salinity, respectively (Table 2). Table 2: Effects of salinity treatments, infection status by Hydrachna skorikowi, sex (female and male) and their interactions on survival times in Sigara lateralis adults (Cox regression analysis). Salinity treatments were 0.5, 5, 10 and 15 g.l-1, and water from the collection site as a control (Ctrl: 0.8 g.l-1). The table shows for each term in the design matrix the estimated coefficient βj (coef), the relative risk exp (βj) (exp (coef)), the standard error, the z-value and the corresponding P-value. Each P-value provides a test for the difference of each level with respect to the baseline. The overall P-value for factors with more than two levels (i.e. salinity) and interactions infection status*sex, sex*salinity is obtained through the Wald test and is showed under the table. Level Level of effect coef exp (coef) S.E. (coef) Zvalue P-value (>|z|) Infection status Unparasitized -0.889 0.411 0.373 -2.388 0.016 Sex male 1.459 4.301 0.371 3.917 P<0.001 Salinity Ctrl 0.955 2.598 0.374 2.551 0.010 5 -0.218 0.804 0.417 -0.522 n.s. 10 0.565 1.759 0.418 1.350 n.s. 15 2.738 15.452 0.429 6.369 P<0.001 Infection status*sex Unparasitized * male 0.461 1.586 0.267 1.721 P<0.001 Infection status*salinity Unparasitized * Ctrl -0.719 0.538 0.403 -2.535 0.010 Unparasitized * 5 -0.619 1.388 0.465 0.705 n.s. Unparasitized * 10 0.782 1.957 0.466 1.994 0.049 Unparasitized * 15 0.672 0.826 0.489 -0.390 n.s. Sex*Salinity Male* Ctrl -1.638 0.194 0.423 -3.872 P<0.001 Male* 5 0.4003 1.492 0.461 0.870 n.s. Male* 10 0.2400 1.271 0.473 0.508 n.s. Male* 15 -1.121 0.326 0.493 -2.277 0.02 Concordance= 0.78 (S.E. = 0.026). Rsquare= 0.525(max possible= 1 ) Note: Overall P-value for Salinity factor variable and Infection status*Salinity and Sex*salinity interactions. Wald test “Salinity”; X2 = 26.3, df = 4, P (> X2) <0.0001; “Infection status*Salinity”; X2 = 14.8, df = 4, P (> X2) =0.0051; “Sex*Salinity”; X2 = 35.5, df = 4, P (> X2)) <0.0001. Chapter 4: Water mites, salinity and water bugs 152 Figure 2: (A) Survival time (cox regresion model), (B) number of eggs (GLM with a Posisson error and log link function) and (C) hatching percentage (GLM with a Binomial error and logit link function) for adult Sigara lateralis with and without mite parasites (Hydrachna skorikowi) under different salinity treatments. C.W. represents water from the collection site (0.8 g.l-1). Different letters above bars indicate significant differences (P ≤ 0.05 after Bonferroni correction) for unparasitized groups, numbers above bars indicate significant differences for parasitized groups and “*” between bars indicate significant differences for the interaction Infection status*salinity treatment.