Leg Attachment Devices of Tiger Beetles (Coleoptera, Cicindelidae) and Their Relationship to Their Habitat Preferences
Abstract
Adherence to smooth substrates is closely related to the morphology and distribution of adhesive structures on insects’ legs, so it is hypothesized that the adhesive structures have been evolved as an adaption to smooth substrates in specific environments. However, the factors that promote the evolution of adhesive structures are still unclear. Using scanning electron microscopy, we compared the microstructure of the tarsi of five tiger beetle species, both male and female, belonging to two tribes living in arboreal and non-arboreal environments. We found that the different types of adhesive setae, including elongated spoon-like setae, elliptical setae, branched setae, filament-like setae, discoidal setae, spatulate setae and tapered setae, varied in different environments and genders. The adaptive evolution of these adhesive structures was probably driven by the selective pressures of both mating behavior and the presence of smooth substrates in the respective environments.
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Citation: Liu, Z.; Gorb, S.N.; Liang, H.; Bai, M.; Lu, Y. Leg Attachment Devices of Tiger Beetles (Coleoptera, Cicindelidae) and Their Relationship to Their Habitat Preferences. Insects 2024,15, 650. https://doi.org/ 10.3390/insects15090650 Academic Editor: Hong Pang Received: 29 July 2024 Revised: 20 August 2024 Accepted: 26 August 2024 Published: 29 August 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). insects Article Leg Attachment Devices of Tiger Beetles (Coleoptera, Cicindelidae) and Their Relationship to Their Habitat Preferences Zheng Liu 1,2, Stanislav N. Gorb 3, Hongbin Liang 1, Ming Bai 1,4 and Yuanyuan Lu 1,* 1 Key Laboratory of Animal Biodiversity Conservation and Integrated Pest Management, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (Z.L.); [email protected] (H.L.); [email protected] (M.B.) 2Hebei Key Laboratory of Animal Diversity, Langfang Normal University, Langfang 065000, China 3Department of Functional Morphology and Biomechanics, Institute of Zoology, Christian-Albrechts-University of Kiel, D-24118 Kiel, Germany; [email protected] 4University of Chinese Academy of Sciences, Beijing 100049, China *Correspondence: [email protected] Simple Summary: Adherence to smooth substrates is closely related to the morphology and distribution of adhesive structures on insects’ legs, so it is hypothesized that the adhesive structures have been evolved as an adaption to smooth substrates in specific environments. However, the factors that promote the evolution of adhesive structures are still unclear. Using scanning electron microscopy, we compared the microstructure of the tarsi of five tiger beetle species, both male and female, belonging to two tribes living in arboreal and non-arboreal environments. We found that the different types of adhesive setae, including elongated spoon-like setae, elliptical setae, branched setae, filament-like setae, discoidal setae, spatulate setae and tapered setae, varied in different environments and genders. The adaptive evolution of these adhesive structures was probably driven by the selective pressures of both mating behavior and the presence of smooth substrates in the respective environments. Abstract: The ability of many insects to adhere vertically or even upside down to smooth substrates is closely related to the morphology and distribution of the adhesive structures on their legs. During locomotion, the legs are in direct contact with different substrates, and it is hypothesized that the adhesive structures have been evolved as an adaption to smooth substrates in specific environments. To investigate whether there is a relationship between the presence of adhesive structures and the combined effects of different environments and mating behavior, we compared five species of tiger beetles belonging to two tribes living in arboreal and non-arboreal environments, respectively. In three non-arboreal species, we found a specific type of adhesive structure consisting of elongated spoon-like setae present on the protarsi of males but absent on the male mesoand metatarsi and on females. In Tricondyla pulchripes, an arboreal species living on stems, we found three types of adhesive setae on male protarsi, while only two types of setae were found on male mesoand metatarsi and on females. In Neocollyris linearis, an arboreal species living on leaves, we found three types of adhesive setae on male pro-, mesoand meta-tarsi but only two types of adhesive setae on females. The adaptive evolution of these adhesive structures was probably driven by the selective pressures of both mating behavior and the presence of smooth substrates in the respective environments. It is discussed that the adhesive structures in tiger beetles may be an adaptive evolutionary response to the plant surfaces and may play an important role in species differentiation. Keywords: microstructure; adhesive setae; tarsus; Cicindelidae; scanning electron microscopy 1. Introduction The morphological diversity and function of the adhesive structures have long been of interest to scientists [ 1 – 4 ]. Many insects have the ability to adhere to smooth surfaces, and their adhesive ability has been attributed to the attachment structures on their leg segments, Insects 2024,15, 650. https://doi.org/10.3390/insects15090650 https://www.mdpi.com/journal/insects
Insects 2024,15, 650 2 of 16 such as tarsi, pretarsi or the distal end of tibia, which are often in direct contact with substrates during locomotion [ 5 ]. For example, ants use a soft, smooth arolium between the claws for attachment [ 6 – 8 ], flies have two hairy pulvilli under the claws [ 9 – 13 ], and leaf beetles rely on a large area of adhesive setae on the ventral side of the tarsomeres [14–19]. Morphological diversity in adhesive structures has been found in several insects [ 7 , 14 , 20 – 24 ], but the relationship between different adhesive microstructures and different environments has only been studied in a few selected species [ 4 , 25 – 27 ]. Generally, adhesive structures are more developed in the groups in close contact with plants [ 4 , 5 , 28 – 30 ]. We speculate that many phytophagous groups or some arboreal predatory groups may have experienced the selective pressure of smooth plant substrates in their environment, but more in-depth analyses and systematic studies are relatively scarce [31,32]. In the present paper, we explore the correlation between adhesive microstructures and specific environments, and we attempt to figure out the driving forces behind the emergence of adhesive structures in the adaptive evolutionary process of tiger beetles (Coleoptera: Adephaga: Cicindelidae), a group of predatory insects, which occupy diverse habitats (Figure 1). There are 130 genera and more than 2900 species of tiger beetles in the world’s fauna [ 33 – 35 ]. They are predators of small arthropods and are known for their extremely high running speed and highly developed visual system [ 36 ]. Based on the consensus of all modern phylogenomic studies, Duran and Gough recognized six tribes (Manticorini, Megacephalini, Collyridini, Ctenostomatini, Cicindelini and Oxycheilini) in the family Cicindelidae [ 37 , 38 ]. Amblycheilini has been recognized as an independent tribe in some studies [ 39 ], or as part of the tribe Megacephalini. Most species of the tribes Manticorini, Megacephalini, Cicindelini and Oxycheilini live on gravel ground and run fast, while other tribes, such as Collyridini and Ctenostomatini, are arboreal and live on trees, e.g., the genus Neocollyris [ 37 ]. The habitats of tiger beetles are more diverse than those of most families of Coleoptera. Insects2024,15,xFORPEERREVIEW2of17 1.Introduction Themorphologicaldiversityandfunctionoftheadhesivestructureshavelongbeen ofinteresttoscientists[1–4].Manyinsectshavetheabilitytoadheretosmoothsurfaces, andtheiradhesiveabilityhasbeenattributedtotheattachmentstructuresontheirleg segments,suchastarsi,pretarsiorthedistalendoftibia,whichareoftenindirectcontact withsubstratesduringlocomotion[5].Forexample,antsuseasoft,smootharoliumbetweentheclawsforattachment[6–8],flieshavetwohairypulvilliundertheclaws[9–13], andleafbeetlesrelyonalargeareaofadhesivesetaeontheventralsideofthetarsomeres [14–19]. Morphologicaldiversityinadhesivestructureshasbeenfoundinseveralinsects [7,14,20–24],buttherelationshipbetweendifferentadhesivemicrostructuresanddifferent environmentshasonlybeenstudiedinafewselectedspecies[4,25–27].Generally,adhesivestructuresaremoredevelopedinthegroupsinclosecontactwithplants[4,5,28–30]. Wespeculatethatmanyphytophagousgroupsorsomearborealpredatorygroupsmay haveexperiencedtheselectivepressureofsmoothplantsubstratesintheirenvironment, butmorein-depthanalysesandsystematicstudiesarerelativelyscarce[31,32]. Inthepresentpaper,weexplorethecorrelationbetweenadhesivemicrostructures andspecificenvironments,andweattempttofigureoutthedrivingforcesbehindthe emergenceofadhesivestructuresintheadaptiveevolutionaryprocessoftigerbeetles (Coleoptera:Adephaga:Cicindelidae),agroupofpredatoryinsects,whichoccupydiverse habitats(Figure1).Thereare130generaandmorethan2900speciesoftigerbeetlesinthe world’sfauna[33–35].Theyarepredatorsofsmallarthropodsandareknownfortheir extremelyhighrunningspeedandhighlydevelopedvisualsystem[36].Basedontheconsensusofallmodernphylogenomicstudies,DuranandGoughrecognizedsixtribes(Manticorini,Megacephalini,Collyridini,Ctenostomatini,CicindeliniandOxycheilini)inthe familyCicindelidae[37,38].Amblycheilinihasbeenrecognizedasanindependenttribein somestudies[39],oraspartofthetribeMegacephalini.MostspeciesofthetribesManticorini,Megacephalini,CicindeliniandOxycheiliniliveongravelgroundandrunfast, whileothertribes,suchasCollyridiniandCtenostomatini,arearborealandliveontrees, e.g.,thegenusNeocollyris[37].Thehabitatsoftigerbeetlesaremorediversethanthoseof mostfamiliesofColeoptera. Figure1.Arborealandnon-arborealtigerbeetlegroupsinhabitingdifferentenvironments. Figure 1. Arboreal and non-arboreal tiger beetle groups inhabiting different environments. The adhesive structures of tiger beetle legs have rarely been studied. Stork [ 14 ] examined one male tiger beetle (Cicindela campestris) by scanning electron microscopy (SEM) and found many adhesive setae on the three proximal tarsomeres of the protarsi. Pearson found hairy pads in some species [ 36 ], but the details of their microstructure have not been studied. Until now, there has been no comparative morphological study of the adhesive structures in more species, especially in tiger beetles occupying different habitats.
Insects 2024,15, 650 3 of 16 We used comparative morphological methods in our study. The tarsi of five tiger beetle species, living in two very different habitats (arboreal versus non-arboreal), were examined by SEM. It was hypothesized that the arboreal species have developed adhesive structures as an adaptation to the smooth leaf surfaces of plants. Since, in some beetle groups, the males use adhesive pads on their legs to attach themselves to the female during mating, we also compared the sexes of the species studied. 2. Materials and Methods 2.1. Materials The adult specimens of five tiger beetle species (Cicindela sachalinensis,Cosmodela separata,Cylindera kaleea,Tricondyla pulchripes and Neocollyris linearis) were collected in China between 2008 and 2022. The specimens of Ci. sachalinensis were collected in Beijing, 2022. The specimens of Co. separata and Cy. kaleea were collected in Zhejiang Province, 2016. The specimens of T. pulchripes were collected in Hong Kong, 2017. The specimens of N. linearis were collected in Yunnan Province, 2008. All the specimens were from the National Animal Collection Resource Center of the Institute of Zoology, Chinese Academy of Sciences (IZCAS, Beijing, China). The specimens of T. pulchripes were dry preserved, whereas the others were preserved in 95% ethanol. Details of the studied specimens are provided in Table 1. Table 1. Species of Cicindelidae and their collection locations. Tribe Species Collection Site Habitat 1 Cicindelini Cicindela sachalinensis Wuling Mountain, Beijing Non-arboreal 2 Cicindelini Cosmodela separata Jiangpu County, Zhejiang Non-arboreal 3 Cicindelini Cylindera kaleea Siming Mountain, Zhejiang Non-arboreal 4 Collyridini Tricondyla pulchripes Hong Kong Arboreal, tree stem 5 Collyridini Neocollyris linearis Xishuangbanna, Yunnan Arboreal, tree leaf The five species were examined under a scanning electron microscope. The three non-arboreal species belong to tribe Cicindelini [ 36 ], whereas the two arboreal species belong to tribe Collyridini [ 37 ]. The tarsi (pro-, mesoand metatarsus) of one male and one female adult specimen of each species were examined and measured. The cuticle of the female elytra and ventral side of metasternum were examined. 2.2. Terminology The morphological terminology mostly follows that of Stork (1980) [ 14 ], Beutel and Gorb (2001) [5] and Betz (2003) [40]. 2.3. Photographic System Observations were carried out under an Olympus SZ61 stereomicroscope. The digital images were taken with a Canon 5D digital camera in conjunction with a Canon MP-E 65 mm f/2.8 1-5X Macro Lens (Canon Inc., Tokyo, Japan). The digital camera flash system was composed of Nikon wireless remote speedlight SB-R200 (Nikon Inc., Tokyo, Japan) and Nikon wireless speedlight commander SU-800 (Nikon Inc., Tokyo, Japan). The Macro Lens was fitted to a StackShot macro rail (Cognisys Inc., Traverse, USA), then the images were stacked by Helicon Focus v.7.6.1. All the images were adjusted in Adobe Photoshop (Adobe Inc., San Jose, CA, USA). 2.4. Scanning Electron Microscopy (SEM) The tarsi of the forelegs were removed from the body, cleaned with 2% phosphate buffered saline, stepwise dehydrated in ethanol (75%, 85%, 95%, 3 × 100%), CO 2 criticalpoint-dried, coated with platinum, and then examined and photographed with a HITACHI SU8010 field emission scanning electron microscope (HITACHI Co. Ltd., Tokyo, Japan). The SEM images were post-processed with Adobe Photoshop (Adobe Inc.)
Insects 2024,15, 650 4 of 16 Morphometry of the Attachment System The length and width of the setae were measured from the SEM images using Image-J 1.53 (National Institutes of Health, Bethesda, MD, USA) and displayed in the Results. To quantify the density of the setae on the tarsi, a 50 × 50 µ m frame was applied on different areas of the SEM images using Image-J 1.53 (National Institutes of Health, USA). All the setae within the frame (50 × 50 µ m = 2500 µ m 2 ) were counted and the calculated mean value (number of setae divided by 25) represents the density of setae per 100 µ m 2 . Each part was measured three times and averaged (n= 3). 3. Results The adhesive setae of males and females from all five species studied were different and sexually dimorphic (see the details below). 3.1. Cicindelini Cicindela sachalinensis and Cosmodela separata are non-arboreal species rushing rapidly on the ground, and Cylindera kaleea usually lives on the ground or in low shrubs. Only males of these species have developed adhesive setae on the protarsi. There are five tarsomeres in each tarsus. In males, the first to third tarsomeres of the protarsus are enlarged and widened (Figure 2A,G,M). They are covered with elongated spoon-like adhesive setae on the ventral side (Figure 2B,H,N). The fourth and fifth tarsomeres are nearly bald. The male mesoand metatarsi are without adhesive setae. The tarsomeres are slender, with thick and short setae on the ventral side (Figure 2C,D,I,O,P). In females, the tarsomeres are slender, without adhesive setae on all five tarsomeres of the pro-, mesoand metatarsi (Figure 2E,F,J,L,Q,R). A few short and thick setae are present on each tarsomere. Insects2024,15,xFORPEERREVIEW5of17 Figure2.Theventralviewofthetarsiandadhesivesetaeinnon-arborealspeciesofCicindelidae. (A–F).Cicindelasachalinensis.(A).Maleprotarsus.(B).Elongatedspoon-likesetaeonthemaleprotarsus.(C).Malemesotarsus.(D).Malemetatarsus.(E).Femaleprotarsus.(F).Femalemeso-and Figure 2. Cont.
Insects 2024,15, 650 5 of 16 Insects2024,15,xFORPEERREVIEW5of17 Figure2.Theventralviewofthetarsiandadhesivesetaeinnon-arborealspeciesofCicindelidae. (A–F).Cicindelasachalinensis.(A).Maleprotarsus.(B).Elongatedspoon-likesetaeonthemaleprotarsus.(C).Malemesotarsus.(D).Malemetatarsus.(E).Femaleprotarsus.(F).Femalemeso-and Figure 2. The ventral view of the tarsi and adhesive setae in non-arboreal species of Cicindelidae. (A–F). Cicindela sachalinensis. (A). Male protarsus. (B). Elongated spoon-like setae on the male protarsus. (C). Male mesotarsus. (D). Male metatarsus. (E). Female protarsus. (F). Female mesoand metatarsus. (G–L). Cosmodela separata. (G). Male protarsus. (H). Elongated spoon-like setae on the male protarsus. (I). Male mesotarsus. (J). Female protarsus. (K). Female mesotarsus. (L). Female metatarsus. ( M–R ). Cylindera kaleea. (M). Male protarsus. (N). Elongated spoon-like setae on the male protarsus. (O). Male mesotarsus. (P). Male metatarsus. (Q). Female protarsus. R. Female mesoand metatarsus. Abbreviations: UN, unguis (claw); Tar I, the 1st tarsomere; Tar II, the 2nd tarsomere; Tar III, the 3rd tarsomere; Tar IV, the 4th tarsomere; Tar V, the 5th tarsomere; sh, setal shaft; tp, terminal plate. 3.1.1. Cicindela sachalinensis Morawitz, 1862 The fourth and fifth tarsomeres of males are nearly bald, with five pairs of short setae on the fourth tarsomeres and three on the fifth tarsomeres. Elongated spoon-like setae (Figure 2B). Elongated spoon-like setae are the only type of seta on the ventral surface of the male protarsi. These setae were found on the first, second and third tarsomeres. The setal shaft is straight, about 89.50 ± 2.80 µ m (n= 3) in length and 5.14 ± 0.33 µ m (n= 3) in width (at the base). Each seta has an elongated spoon-like terminal plate at the tip. The ventral side of the terminal plate is regularly transversely ribbed, approximately 33.43 ± 2.77 µ m (n= 3) in length and 8.19 ± 0.29 µ m (n= 3) in width. The density of the elongated spoon-like setae is low: 0.32 setae per 100 µm2(n= 3). 3.1.2. Cosmodela separata (Fleutiaux, 1894) The fourth and fifth tarsomeres of males are nearly bald, with five pairs of short setae on the fourth tarsomeres and three on the fifth tarsomeres. Elongated spoon-like setae (Figure 2H). Elongated spoon-like setae are the only type of seta on the ventral surface of the male protarsi. These setae were found on the first, second and third tarsomeres. The setal shaft is straight, about 78.19 ± 6.71 µ m (n= 3) in length
Insects 2024,15, 650 6 of 16 and 4.97 ± 0.17 µ m (n= 3) in width (at the base). Each seta has an elongated spoon-like terminal plate at the tip. The ventral side of the terminal plate is regularly transversely ribbed, approximately 43.66 ± 1.00 µ m (n= 3) in length and 7.42 ± 0.30 µ m (n= 3) in width. The density of the elongated spoon-like setae is rather low: 0.24 setae per 100 µm2(n= 3). 3.1.3. Cylindera kaleea (Bates, 1866) The fourth and fifth tarsomeres of males are nearly bald, with five pairs of short and thick setae on the fourth and fifth tarsomeres separately. Elongated spoon-like setae (Figure 2N). Elongated spoon-like setae are the only type of seta on the ventral surface of the male protarsi. These setae were found on the first, second and third tarsomeres. The setal shaft is straight, about 59.42 ± 3.69 µ m (n= 3) in length and 5.57 ± 0.07 µ m (n= 3) in width (at the base). Each seta has an elongated spoon-like terminal plate at the tip. The ventral surface of the terminal plate is regularly transversely ribbed, approximately 18.56 ± 1.90 µ m (n= 3) in length and 7.65 ± 0.83 µ m (n= 3) in width. The density of the elongated spoon-like setae is rather low: 0.24 setae per 100 µm2(n= 3). 3.2. Collyridini 3.2.1. Subtribe Tricondylina: Tricondyla pulchripes White, 1844 As an arboreal species living on tree stems, T. pulchripes has well-developed adhesive setae on all the tarsi of males and females, but the types of setae and their location are different in males and females. In both genders, the general morphology of the tarsomeres is as follows. The first tarsomere (Tar I) is elongated. The second tarsomere is slightly enlarged and widened. The third and fourth tarsomeres are asymmetrical and larger on the lateral side (Figure. 3A,E,H,I,M). The ventral surfaces of the first, second, third and fourth tarsomeres are densely covered with adhesive setae. The fifth tarsomere (Tar V) is slender and covered with scattered thick and long setae on the ventral, lateral and dorsal surfaces (Figure 3J). In males, there are three types of adhesive setae: elliptical setae, branched setae and filament-like setae. In females, there are two kinds of adhesive setae: branched setae and filament-like setae. The thick and long setae on the fifth tarsomere are presumably not adhesive. Elliptical setae. These setae are situated on the ventral surfaces of the first, second and third tarsomeres of the male protarsi. The setal shaft is straight, about 54.61 ± 1.06 µ m ( n= 3 ) in length and 4.13 ± 0.12 µ m (n= 3) in width (at the base). Each seta has an elliptical terminal plate at the end. The terminal plate is approximately 19.62 ± 0.88 µ m (n= 3) in long diameter and 8.74 ± 0.43 µ m (n= 3) in short diameter (Figure 3C). The elliptical setae have a relatively low density and there are only 0.48 setae per 100 µ m 2 (n= 3). There are transversal stripes on the ventral side of the setae. Branched setae. These setae are situated on the ventral surface of the fourth tarsomere and on the surrounding edge of the first to third tarsomeres of the male protarsi (Figure 3D). Also, they are present on the ventral surfaces of the third and fourth tarsomeres of the male mesoand metatarsi (Figure 3F) and on the tarsi of all female legs (Figure 3K,O). These setae are elongate, slender, with small protuberances, about 148.12 ± 9.17 µ m (n= 3) in length and 3.82 ± 0.04 µ m (n= 3) in width (at the base). The apex of the seta is curved and acute. The average density of the branched setae is 1.68 setae per 100 µm2(n= 3). Filament-like setae. These setae are situated on the distal ventral surfaces of the first and second tarsomeres of all female legs and male mesoand metatarsi. On the setal shafts, these setae have grooves oriented at some angle to the setal axis. Their tips are gradually tapered (Figure 3G,L,P). The setae are about 90.17 ± 4.12 µ m (n= 3) in length and 6.66 ±0.41 µm (n= 3) in width (at the basis). The average density of the setae is 0.4 setae per 100 µm2(n= 3).
Insects 2024,15, 650 7 of 16 Insects2024,15,xFORPEERREVIEW7of17 Inmales,therearethreetypesofadhesivesetae:ellipticalsetae,branchedsetaeand filament-likesetae.Infemales,therearetwokindsofadhesivesetae:branchedsetaeand filament-likesetae.Thethickandlongsetaeonthefifthtarsomerearepresumablynot adhesive. Ellipticalsetae.Thesesetaearesituatedontheventralsurfacesofthefirst,second andthirdtarsomeresofthemaleprotarsi.Thesetalshaftisstraight,about54.61±1.06µm (n=3)inlengthand4.13±0.12µm(n=3)inwidth(atthebase).Eachsetahasanelliptical terminalplateattheend.Theterminalplateisapproximately19.62±0.88µm(n=3)in longdiameterand8.74±0.43µm(n=3)inshortdiameter(Figure3C).Theellipticalsetae havearelativelylowdensityandthereareonly0.48setaeper100µm2(n=3).Thereare transversalstripesontheventralsideofthesetae. Branchedsetae.Thesesetaearesituatedontheventralsurfaceofthefourthtarsomere andonthesurroundingedgeofthefirsttothirdtarsomeresofthemaleprotarsi(Figure 3D).Also,theyarepresentontheventralsurfacesofthethirdandfourthtarsomeresof themalemeso-andmetatarsi(Figure3F)andonthetarsiofallfemalelegs(Figure3K,O). Thesesetaeareelongate,slender,withsmallprotuberances,about148.12±9.17µm(n= 3)inlengthand3.82±0.04µm(n=3)inwidth(atthebase).Theapexofthesetaiscurved andacute.Theaveragedensityofthebranchedsetaeis1.68setaeper100µm2(n=3). Filament-likesetae.Thesesetaearesituatedonthedistalventralsurfacesofthefirst andsecondtarsomeresofallfemalelegsandmalemeso-andmetatarsi.Onthesetal shafts,thesesetaehavegroovesorientedatsomeangletothesetalaxis.Theirtipsare graduallytapered(Figure3G,L,P).Thesetaeareabout90.17±4.12µm(n=3)inlength and6.66±0.41µm(n=3)inwidth(atthebasis).Theaveragedensityofthesetaeis0.4 setaeper100µm2(n=3). Figure3.ThetarsiandadhesivesetaeofTricondylapulchripes(arborealspecies).(A–H).Male.(A). Protarsus,ventralview.(B).Unguis(claw)ofprotarsus.(C).Ellipticalsetaeonprotarsus.(D). Branchedsetaeonprotarsus.(E).Mesotarsus,ventralview.(F).Branchedsetaeonmesotarsus.(G). Filament-likesetaeonmesotarsus.(H).Metatarsus,ventralview.(I–P).Female.(I).Protarsus, Figure 3. The tarsi and adhesive setae of Tricondyla pulchripes (arboreal species). (A–H). Male. (A). Protarsus, ventral view. (B). Unguis (claw) of protarsus. (C). Elliptical setae on protarsus. (D). Branched setae on protarsus. (E). Mesotarsus, ventral view. (F). Branched setae on mesotarsus. (G). Filament-like setae on mesotarsus. (H). Metatarsus, ventral view. (I–P). Female. (I). Protarsus, ventral view. (J). Unguis (claw) of protarsus. (K). Branched setae on protarsus. (L). Filament-like setae on protarsus. (M). Metatarsus, ventral view. (N). Unguis (claw) of metatarsus. (O). Branched setae on metatarsus. (P). Filament-like setae on metatarsus. Abbreviations: UN, unguis (claw); Tar I, the 1st tarsomere; Tar II, the 2nd tarsomere; Tar III, the 3rd tarsomere; Tar IV, the 4th tarsomere; Tar V, the 5th tarsomere; sh, setal shaft; tp, terminal plate. 3.2.2. Subtribe Collyridina: Neocollyris linearis (Schmidt-Göbel, 1846) Neocollyris linearis is an arboreal species living on trees. It has well-developed adhesive setae on the male and female tarsi, but the setal types and locations are different in males and females. In both genders, the general morphology of tarsomeres is as follows. The first and second tarsomeres (Tar I and Tar II) are elongate. The third and fourth tarsomeres (Tar III and Tar IV) are slightly enlarged and wide (Figure 4A,E,G,I,O). The ventral surfaces of the first, second, third and fourth tarsomeres are densely covered with adhesive setae, which consist of two parts: setal shaft (sh) and specialized tip. The fifth tarsomere (Tar V) is wide and covered with scattered thick and short setae on the ventral surface (Figure 4D,J). In males, there are three types of adhesive setae: discoidal, spatulate, and tapered setae. In females, there are only two types of adhesive setae: spatulate and tapered setae. The thick short setae on the fifth tarsomere are presumably not adhesive.
Insects 2024,15, 650 8 of 16 Insects2024,15,xFORPEERREVIEW9of17 Figure4.ThetarsiandadhesivesetaeofNeocollyrislinearis(arborealspecies).(A–H).Male.(A). Protarsus,ventralview.(B).Unguis(claw)ofprotarsus.(C).Discoidalsetaeonprotarsus.(D).Spatulatesetaeonprotarsus.(E).Mesotarsus,ventralview.(F).Spatulatesetaeonmesotarsus.(G).Metatarsus,ventralview.(H).Discoidalsetaeonmetatarsus.(I–P).Female.(I).Protarsus,ventralview. (J).Unguis(claw)ofprotarsus.(K).Spatulatesetaeonprotarsus.(L).Taperedsetaeonprotarsus. (M).Spatulatesetaeonmesotarsus.(N).Taperedsetaeonmesotarsus.(O).Metatarsus,ventralview. (P).Spatulatesetaeonmetatarsus.Abbreviations:UN,unguis(claw);TarI,the1sttarsomere;TarII, the2ndtarsomere;TarIII,the3rdtarsomere;TarIV,the4thtarsomere;TarV,the5thtarsomere;sh, setalshaft;tp,terminalplate. 3.3.MicrostructureoftheFemaleCuticleSurfaceandtheMaleBehaviorduringMating 3.3.1.Cicindelini CicindelasachalinensisandCosmodelaseparata Duringmating,themalesofspeciesthatbelongtothistribealwaysusethedeveloped mandibletoclaspthebasicareaofthepterothroaxofthefemales(Figure5A,G–I).The frontlegsdonotalwayscontactwiththefemaleelytraorherbodyside(Figure5A,G). ByobservingthecuticlesurfaceofthefemaleelytraandmetasternumusingSEM,it wasfoundthatthereareregularlyarrangedhexagonalsurfacestructuresonthefemale elytra,withtheconcavesurfaceinthehexagons(Figure6A–C).Thecuticleofthemetasternumalsohasahexagonalsurfacepattern,notconcave,butratherasheet-likewitha lowerheight(Figure6D–F).ThesurfaceoffemaleCosmodelaseparatahassomeroughness atthemicro-scale.ThewidthofthehexagonsofCi.sachalinensisis15.63±0.49µm(n=3) intheelytraand14.98±0.29µm(n=3)inthemetasternum,respectively.Thewidthofthe hexagonsofCo.separatais13.47±1.29µm(n=3)intheelytraand12.56±0.19µm(n=3) inthemetasternum,respectively.BothCi.sachalinensisandCo.separatahavesomekindof roughnessontheelytraandCo.separataalsosomeroughnessonthemetasternum. Figure 4. The tarsi and adhesive setae of Neocollyris linearis (arboreal species). (A–H). Male. (A). Protarsus, ventral view. (B). Unguis (claw) of protarsus. (C). Discoidal setae on protarsus. (D). Spatulate setae on protarsus. (E). Mesotarsus, ventral view. (F). Spatulate setae on mesotarsus. (G). Metatarsus, ventral view. (H). Discoidal setae on metatarsus. (I–P). Female. (I). Protarsus, ventral view. (J). Unguis (claw) of protarsus. (K). Spatulate setae on protarsus. (L). Tapered setae on protarsus. (M). Spatulate setae on mesotarsus. (N). Tapered setae on mesotarsus. (O). Metatarsus, ventral view. (P). Spatulate setae on metatarsus. Abbreviations: UN, unguis (claw); Tar I, the 1st tarsomere; Tar II, the 2nd tarsomere; Tar III, the 3rd tarsomere; Tar IV, the 4th tarsomere; Tar V, the 5th tarsomere; sh, setal shaft; tp, terminal plate. Discoidal setae. These setae are situated on the ventral surfaces of the first, second and third tarsomeres of the male tarsi (Figure 4C,H). The setal shaft is straight, with two or three grooves across the junction to the setal terminal plate, about 40.61 ± 1.15 µ m ( n= 3 ) in length and 3.56 ± 0.20 µ m (n= 3) in width. Each seta has a discoidal terminal plate with 1–2 tips at the proximal part. The terminal plate is approximately 8.56 ± 0.54 µ m (n= 3) in diameter (Figure 4C). The discoidal setae have a relatively low density: 0.6 setae per 100 µm2(n= 3). Spatulate setae. These setae are situated on the ventral surface of the fourth tarsomere in both male and female tarsi (Figure 4D,F,K,M,P). The setal shaft is straight, slightly bent, 64.71 ± 3.71 µ m (n= 3) in length and 2.35 ± 0.14 µ m (n= 3) in width. Each seta has a spatulate terminal plate. The widest part of the plate is 10.64 ± 0.48 µ m (n= 3) in width (Figure 4D,K). The ventral and dorsal surfaces of the terminal plate are smooth, without obvious substructure. The spatulate setae have a high density: 1.8 setae per 100 µ m 2 ( n= 3 ). These spatulate setae are very similar with those of leaf beetles [ 14 , 16 , 28 ] and ladybird beetles [14]. Tapered setae. These setae are situated on the ventral surfaces of the second and third tarsomeres of female legs (Figure 4L,N) and the surrounding edge of the first to third tarsomeres’ ventral surfaces of the male tarsi (Figure 4H). These setae are elongated, slender,
Insects 2024,15, 650 9 of 16 about 66.42 ± 5.18 µ m (n= 3) in length and 4.12 ± 0.09 µ m (n= 3) in width (at the base). The setal shaft is straight at the base and tapered toward the curved acute tip. The density of the tapered setae is 1.12 setae per 100 µm2(n= 3). 3.3. Microstructure of the Female Cuticle Surface and the Male Behavior during Mating 3.3.1. Cicindelini Cicindela sachalinensis and Cosmodela separata During mating, the males of species that belong to this tribe always use the developed mandible to clasp the basic area of the pterothroax of the females (Figure 5A,G–I). The front legs do not always contact with the female elytra or her body side (Figure 5A,G). Insects2024,15,xFORPEERREVIEW10of17 Figure5.Thecontactpositionofthelegsduringmating.(A).MatingofRopaloteresdesgodinsii(Fairmaire,1887)(Sichuan,photographedbyLiHe).(B).MatingofTricondylapulchripes(HongKong, photographedbySiuyeungHo).(C).MatingofTricondylapulchripes(HongKong,photographedby AlfredCheung).(D).MatingofNeocollyrisparvula(Chaudoir,1848)(NaglaBlock,Palghar,Maharashtra,India,photographedbyDineshSharma).(E,F).MatingofNeocollyrissp.(YeoorHills,Thane West,Thane,Maharashtra,India,photographedbyAnilKumarVerma).(G–I).Screenshotfroma videoofthematingprocessofCosmodelajuxtata(AcciavattiandPearson,1989).(G).At15s(red arrowsshowthattheprotarsiofthemaledoesnotcontactthebodyofthefemale).(H).At20s(red arrowsshowtheprotarsiofthemaletryingtocontactthebodyofthefemale,andthemaleclasps thefemalewithhismandibles).(I).At22s(redarrowsshowthesameasin(H),theprotarsiofthe malecontactwiththebodyofthefemalemoretightly). Figure 5. The contact position of the legs during mating. (A). Mating of Ropaloteres desgodinsii (Fairmaire, 1887) (Sichuan, photographed by Li He). (B). Mating of Tricondyla pulchripes (Hong Kong, photographed by Siuyeung Ho). (C). Mating of Tricondyla pulchripes (Hong Kong, photographed by Alfred Cheung). (D). Mating of Neocollyris parvula (Chaudoir, 1848) (Nagla Block, Palghar, Maharashtra, India, photographed by Dinesh Sharma). (E,F). Mating of Neocollyris sp. (Yeoor Hills, Thane West, Thane, Maharashtra, India, photographed by Anil Kumar Verma). (G–I). Screenshot from a video of the mating process of Cosmodela juxtata (Acciavatti and Pearson, 1989). (G). At 15 s (red arrows show that the protarsi of the male does not contact the body of the female). (H). At 20 s (red arrows show the protarsi of the male trying to contact the body of the female, and the male clasps the female with his mandibles). (I). At 22 s (red arrows show the same as in (H), the protarsi of the male contact with the body of the female more tightly). By observing the cuticle surface of the female elytra and metasternum using SEM, it was found that there are regularly arranged hexagonal surface structures on the female elytra, with the concave surface in the hexagons (Figure 6A–C). The cuticle of the metasternum also has a hexagonal surface pattern, not concave, but rather a sheet-like with a lower height (Figure 6D–F). The surface of female Cosmodela separata has some roughness at the micro-scale. The width of the hexagons of Ci. sachalinensis is 15.63 ± 0.49 µ m (n= 3) in the elytra and 14.98 ± 0.29 µ m (n= 3) in the metasternum, respectively. The width of the hexagons of Co. separata is 13.47 ± 1.29 µ m (n= 3) in the elytra and 12.56 ± 0.19 µ m (n= 3) in the metasternum, respectively. Both Ci.sachalinensis and Co.separata have some kind of roughness on the elytra and Co.separata also some roughness on the metasternum.
Insects 2024,15, 650 16 of 16 48. Wolff, J.O.; Gorb, S.N. Comparative morphology of pretarsal scopulae in eleven spider families. Arthropod Struct. Dev. 2012,41, 419–433. [CrossRef] 49. Frost, F.; Gorb, S.N.; Wolff, J.O. Adhesion and friction in hunting spiders: The effect of contact splitting on their attachment ability. Zool. Anz. 2018,273, 231–239. [CrossRef] 50. Autumn, K.; Liang, Y.A.; Hsieh, S.T.; Zesch, W.; Chan, W.P.; Kenny, T.W.; Fearing, R.; Full, R.J. Adhesive force of a single gecko foot-hair. Nature 2000,405, 681–685. [CrossRef] [PubMed] 51. Heepe, L.; Höft, S.; Michels, J.; Gorb, S.N. Material gradients in fibrillar insect attachment systems: The role of joint-like elements. Soft Matter 2018,14, 7026–7033. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.