Leg Attachment Devices of Tiger Beetles (Coleoptera, Cicindelidae) and Their Relationship to Their Habitat Preferences

Ci a ion: Liu, Z.; Go b, S.N.; Liang, H.; Bai, M.; Lu, Y. Leg A achmen De ices o Tige Bee les (Coleop e a, Cicindelidae) and Thei Rela ionship o Thei Habi a P e e ences. Insec s 2024,15, 650. h ps://doi.o g/ 10.3390/insec s15090650 Academic Edi o : Hong Pang Recei ed: 29 July 2024 Re ised: 20 Augus 2024 Accep ed: 26 Augus 2024 Published: 29 Augus 2024 Copy igh : © 2024 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). insec s A icle Leg A achmen De ices o Tige Bee les (Coleop e a, Cicindelidae) and Thei Rela ionship o Thei Habi a P e e ences Zheng Liu 1,2, S anisla N. Go b 3, Hongbin Liang 1, Ming Bai 1,4 and Yuanyuan Lu 1,* 1 Key Labo a o y o Animal Biodi e si y Conse a ion and In eg a ed Pes Managemen , Ins i u e o Zoology, Chinese Academy o Sciences, Beijing 100101, China; [email p o ec ed] (Z.L.); [email p o ec ed] (H.L.); [email p o ec ed] (M.B.) 2Hebei Key Labo a o y o Animal Di e si y, Lang ang No mal Uni e si y, Lang ang 065000, China 3Depa men o Func ional Mo phology and Biomechanics, Ins i u e o Zoology, Ch is ian-Alb ech s-Uni e si y o Kiel, D-24118 Kiel, Ge many; [email p o ec ed] 4Uni e si y o Chinese Academy o Sciences, Beijing 100049, China *Co espondence: [email p o ec ed] Simple Summa y: Adhe ence o smoo h subs a es is closely ela ed o he mo phology and dis ibu- ion o adhesi e s uc u es on insec s’ legs, so i is hypo hesized ha he adhesi e s uc u es ha e been e ol ed as an adap ion o smoo h subs a es in speci ic en i onmen s. Howe e , he ac o s ha p omo e he e olu ion o adhesi e s uc u es a e s ill unclea . Using scanning elec on mic oscopy, we compa ed he mic os uc u e o he a si o i e ige bee le species, bo h male and emale, belonging o wo ibes li ing in a bo eal and non-a bo eal en i onmen s. We ound ha he di e en ypes o adhesi e se ae, including elonga ed spoon-like se ae, ellip ical se ae, b anched se ae, ilamen -like se ae, discoidal se ae, spa ula e se ae and ape ed se ae, a ied in di e en en i onmen s and gende s. The adap i e e olu ion o hese adhesi e s uc u es was p obably d i en by he selec i e p essu es o bo h ma ing beha io and he p esence o smoo h subs a es in he espec i e en i onmen s. Abs ac : The abili y o many insec s o adhe e e ically o e en upside down o smoo h subs a es is closely ela ed o he mo phology and dis ibu ion o he adhesi e s uc u es on hei legs. Du ing locomo ion, he legs a e in di ec con ac wi h di e en subs a es, and i is hypo hesized ha he adhesi e s uc u es ha e been e ol ed as an adap ion o smoo h subs a es in speci ic en i onmen s. To in es iga e whe he he e is a ela ionship be ween he p esence o adhesi e s uc u es and he combined e ec s o di e en en i onmen s and ma ing beha io , we compa ed i e species o ige bee les belonging o wo ibes li ing in a bo eal and non-a bo eal en i onmen s, espec i ely. In h ee non-a bo eal species, we ound a speci ic ype o adhesi e s uc u e consis ing o elonga ed spoon-like se ae p esen on he p o a si o males bu absen on he male meso- and me a a si and on emales. In T icondyla pulch ipes, an a bo eal species li ing on s ems, we ound h ee ypes o adhesi e se ae on male p o a si, while only wo ypes o se ae we e ound on male meso- and me a a si and on emales. In Neocolly is linea is, an a bo eal species li ing on lea es, we ound h ee ypes o adhesi e se ae on male p o-, meso- and me a- a si bu only wo ypes o adhesi e se ae on emales. The adap i e e olu ion o hese adhesi e s uc u es was p obably d i en by he selec i e p essu es o bo h ma ing beha io and he p esence o smoo h subs a es in he espec i e en i onmen s. I is discussed ha he adhesi e s uc u es in ige bee les may be an adap i e e olu iona y esponse o he plan su aces and may play an impo an ole in species di e en ia ion. Keywo ds: mic os uc u e; adhesi e se ae; a sus; Cicindelidae; scanning elec on mic oscopy 1. In oduc ion The mo phological di e si y and unc ion o he adhesi e s uc u es ha e long been o in e es o scien is s [ 1 – 4 ]. Many insec s ha e he abili y o adhe e o smoo h su aces, and hei adhesi e abili y has been a ibu ed o he a achmen s uc u es on hei leg segmen s, Insec s 2024,15, 650. h ps://doi.o g/10.3390/insec s15090650 h ps://www.mdpi.com/jou nal/insec s Insec s 2024,15, 650 2 o 16 such as a si, p e a si o he dis al end o ibia, which a e o en in di ec con ac wi h subs a es du ing locomo ion [ 5 ]. Fo example, an s use a so , smoo h a olium be ween he claws o a achmen [ 6 – 8 ], lies ha e wo hai y pul illi unde he claws [ 9 – 13 ], and lea bee les ely on a la ge a ea o adhesi e se ae on he en al side o he a some es [14–19]. Mo phological di e si y in adhesi e s uc u es has been ound in se e al insec s [ 7 , 14 , 20 – 24 ], bu he ela ionship be ween di e en adhesi e mic os uc u es and di e en en i onmen s has only been s udied in a ew selec ed species [ 4 , 25 – 27 ]. Gene ally, adhesi e s uc u es a e mo e de eloped in he g oups in close con ac wi h plan s [ 4 , 5 , 28 – 30 ]. We specula e ha many phy ophagous g oups o some a bo eal p eda o y g oups may ha e expe ienced he selec i e p essu e o smoo h plan subs a es in hei en i onmen , bu mo e in-dep h analyses and sys ema ic s udies a e ela i ely sca ce [31,32]. In he p esen pape , we explo e he co ela ion be ween adhesi e mic os uc u es and speci ic en i onmen s, and we a emp o igu e ou he d i ing o ces behind he eme gence o adhesi e s uc u es in he adap i e e olu iona y p ocess o ige bee les (Coleop e a: Adephaga: Cicindelidae), a g oup o p eda o y insec s, which occupy di e se habi a s (Figu e 1). The e a e 130 gene a and mo e han 2900 species o ige bee les in he wo ld’s auna [ 33 – 35 ]. They a e p eda o s o small a h opods and a e known o hei ex emely high unning speed and highly de eloped isual sys em [ 36 ]. Based on he consensus o all mode n phylogenomic s udies, Du an and Gough ecognized six ibes (Man ico ini, Megacephalini, Colly idini, C enos oma ini, Cicindelini and Oxycheilini) in he amily Cicindelidae [ 37 , 38 ]. Amblycheilini has been ecognized as an independen ibe in some s udies [ 39 ], o as pa o he ibe Megacephalini. Mos species o he ibes Man ico ini, Megacephalini, Cicindelini and Oxycheilini li e on g a el g ound and un as , while o he ibes, such as Colly idini and C enos oma ini, a e a bo eal and li e on ees, e.g., he genus Neocolly is [ 37 ]. The habi a s o ige bee les a e mo e di e se han hose o mos amilies o Coleop e a. Insec s2024,15,xFORPEERREVIEW2o 17   1.In oduc ion Themo phologicaldi e si yand unc iono  headhesi es uc u esha elongbeen o in e es  oscien is s[1–4].Manyinsec sha e heabili y oadhe e osmoo hsu aces, and hei adhesi eabili yhasbeena ibu ed o hea achmen s uc u eson hei leg segmen s,suchas a si,p e a sio  hedis alendo  ibia,whicha eo enindi ec con ac  wi hsubs a esdu inglocomo ion[5].Fo example,an suseaso ,smoo ha oliumbe- ween heclaws o a achmen [6–8], liesha e wohai ypul illiunde  heclaws[9–13], andlea bee les elyonala gea eao adhesi ese aeon he en alsideo  he a some es [14–19]. Mo phologicaldi e si yinadhesi es uc u eshasbeen oundinse e alinsec s [7,14,20–24],bu  he ela ionshipbe weendiffe en adhesi emic os uc u esanddiffe en  en i onmen shasonlybeens udiedina ewselec edspecies[4,25–27].Gene ally,adhe- si es uc u esa emo ede elopedin heg oupsinclosecon ac wi hplan s[4,5,28–30]. Wespecula e ha manyphy ophagousg oupso somea bo ealp eda o yg oupsmay ha eexpe ienced heselec i ep essu eo smoo hplan subs a esin hei en i onmen , bu mo ein-dep hanalysesandsys ema ics udiesa e ela i elysca ce[31,32]. In hep esen pape ,weexplo e heco ela ionbe weenadhesi emic os uc u es andspeci icen i onmen s,andwea emp  o igu eou  hed i ing o cesbehind he eme genceo adhesi es uc u esin headap i ee olu iona yp ocesso  ige bee les (Coleop e a:Adephaga:Cicindelidae),ag oupo p eda o yinsec s,whichoccupydi e se habi a s(Figu e1).The ea e130gene aandmo e han2900specieso  ige bee lesin he wo ld’s auna[33–35].Theya ep eda o so smalla h opodsanda eknown o  hei  ex emelyhigh unningspeedandhighlyde eloped isualsys em[36].Basedon hecon- sensuso allmode nphylogenomics udies,Du anandGough ecognizedsix ibes(Man- ico ini,Megacephalini,Colly idini,C enos oma ini,CicindeliniandOxycheilini)in he amilyCicindelidae[37,38].Amblycheilinihasbeen ecognizedasanindependen  ibein somes udies[39],o aspa o  he ibeMegacephalini.Mos specieso  he ibesMan- ico ini,Megacephalini,CicindeliniandOxycheilinili eong a elg oundand un as , whileo he  ibes,suchasColly idiniandC enos oma ini,a ea bo ealandli eon ees, e.g., hegenusNeocolly is[37].Thehabi a so  ige bee lesa emo edi e se han hoseo  mos  amilieso Coleop e a.  Figu e1.A bo ealandnon-a bo eal ige bee leg oupsinhabi ingdiffe en en i onmen s. Figu e 1. A bo eal and non-a bo eal ige bee le g oups inhabi ing di e en en i onmen s. The adhesi e s uc u es o ige bee le legs ha e a ely been s udied. S o k [ 14 ] examined one male ige bee le (Cicindela campes is) by scanning elec on mic oscopy (SEM) and ound many adhesi e se ae on he h ee p oximal a some es o he p o a si. Pea son ound hai y pads in some species [ 36 ], bu he de ails o hei mic os uc u e ha e no been s udied. Un il now, he e has been no compa a i e mo phological s udy o he adhesi e s uc u es in mo e species, especially in ige bee les occupying di e en habi a s. Insec s 2024,15, 650 3 o 16 We used compa a i e mo phological me hods in ou s udy. The a si o i e ige bee le species, li ing in wo e y di e en habi a s (a bo eal e sus non-a bo eal), we e examined by SEM. I was hypo hesized ha he a bo eal species ha e de eloped adhesi e s uc u es as an adap a ion o he smoo h lea su aces o plan s. Since, in some bee le g oups, he males use adhesi e pads on hei legs o a ach hemsel es o he emale du ing ma ing, we also compa ed he sexes o he species s udied. 2. Ma e ials and Me hods 2.1. Ma e ials The adul specimens o i e ige bee le species (Cicindela sachalinensis,Cosmodela sepa a a,Cylinde a kaleea,T icondyla pulch ipes and Neocolly is linea is) we e collec ed in China be ween 2008 and 2022. The specimens o Ci. sachalinensis we e collec ed in Beijing, 2022. The specimens o Co. sepa a a and Cy. kaleea we e collec ed in Zhejiang P o ince, 2016. The specimens o T. pulch ipes we e collec ed in Hong Kong, 2017. The specimens o N. linea is we e collec ed in Yunnan P o ince, 2008. All he specimens we e om he Na ional Animal Collec ion Resou ce Cen e o he Ins i u e o Zoology, Chinese Academy o Sciences (IZCAS, Beijing, China). The specimens o T. pulch ipes we e d y p ese ed, whe eas he o he s we e p ese ed in 95% e hanol. De ails o he s udied specimens a e p o ided in Table 1. Table 1. Species o Cicindelidae and hei collec ion loca ions. T ibe Species Collec ion Si e Habi a 1 Cicindelini Cicindela sachalinensis Wuling Moun ain, Beijing Non-a bo eal 2 Cicindelini Cosmodela sepa a a Jiangpu Coun y, Zhejiang Non-a bo eal 3 Cicindelini Cylinde a kaleea Siming Moun ain, Zhejiang Non-a bo eal 4 Colly idini T icondyla pulch ipes Hong Kong A bo eal, ee s em 5 Colly idini Neocolly is linea is Xishuangbanna, Yunnan A bo eal, ee lea The i e species we e examined unde a scanning elec on mic oscope. The h ee non-a bo eal species belong o ibe Cicindelini [ 36 ], whe eas he wo a bo eal species belong o ibe Colly idini [ 37 ]. The a si (p o-, meso- and me a a sus) o one male and one emale adul specimen o each species we e examined and measu ed. The cu icle o he emale ely a and en al side o me as e num we e examined. 2.2. Te minology The mo phological e minology mos ly ollows ha o S o k (1980) [ 14 ], Beu el and Go b (2001) [5] and Be z (2003) [40]. 2.3. Pho og aphic Sys em Obse a ions we e ca ied ou unde an Olympus SZ61 s e eomic oscope. The digi al images we e aken wi h a Canon 5D digi al came a in conjunc ion wi h a Canon MP-E 65 mm /2.8 1-5X Mac o Lens (Canon Inc., Tokyo, Japan). The digi al came a lash sys em was composed o Nikon wi eless emo e speedligh SB-R200 (Nikon Inc., Tokyo, Japan) and Nikon wi eless speedligh commande SU-800 (Nikon Inc., Tokyo, Japan). The Mac o Lens was i ed o a S ackSho mac o ail (Cognisys Inc., T a e se, USA), hen he images we e s acked by Helicon Focus .7.6.1. All he images we e adjus ed in Adobe Pho oshop (Adobe Inc., San Jose, CA, USA). 2.4. Scanning Elec on Mic oscopy (SEM) The a si o he o elegs we e emo ed om he body, cleaned wi h 2% phospha e bu e ed saline, s epwise dehyd a ed in e hanol (75%, 85%, 95%, 3 × 100%), CO 2 c i ical- poin -d ied, coa ed wi h pla inum, and hen examined and pho og aphed wi h a HITACHI SU8010 ield emission scanning elec on mic oscope (HITACHI Co. L d., Tokyo, Japan). The SEM images we e pos -p ocessed wi h Adobe Pho oshop (Adobe Inc.) Insec s 2024,15, 650 4 o 16 Mo phome y o he A achmen Sys em The leng h and wid h o he se ae we e measu ed om he SEM images using Image-J 1.53 (Na ional Ins i u es o Heal h, Be hesda, MD, USA) and displayed in he Resul s. To quan i y he densi y o he se ae on he a si, a 50 × 50 µ m ame was applied on di e en a eas o he SEM images using Image-J 1.53 (Na ional Ins i u es o Heal h, USA). All he se ae wi hin he ame (50 × 50 µ m = 2500 µ m 2 ) we e coun ed and he calcula ed mean alue (numbe o se ae di ided by 25) ep esen s he densi y o se ae pe 100 µ m 2 . Each pa was measu ed h ee imes and a e aged (n= 3). 3. Resul s The adhesi e se ae o males and emales om all i e species s udied we e di e en and sexually dimo phic (see he de ails below). 3.1. Cicindelini Cicindela sachalinensis and Cosmodela sepa a a a e non-a bo eal species ushing apidly on he g ound, and Cylinde a kaleea usually li es on he g ound o in low sh ubs. Only males o hese species ha e de eloped adhesi e se ae on he p o a si. The e a e i e a some es in each a sus. In males, he i s o hi d a some es o he p o a sus a e enla ged and widened (Figu e 2A,G,M). They a e co e ed wi h elonga ed spoon-like adhesi e se ae on he en al side (Figu e 2B,H,N). The ou h and i h a - some es a e nea ly bald. The male meso- and me a a si a e wi hou adhesi e se ae. The a some es a e slende , wi h hick and sho se ae on he en al side (Figu e 2C,D,I,O,P). In emales, he a some es a e slende , wi hou adhesi e se ae on all i e a some es o he p o-, meso- and me a a si (Figu e 2E,F,J,L,Q,R). A ew sho and hick se ae a e p esen on each a some e. Insec s2024,15,xFORPEERREVIEW5o 17    Figu e2.The en al iewo  he a siandadhesi ese aeinnon-a bo ealspecieso Cicindelidae. (A–F).Cicindelasachalinensis.(A).Malep o a sus.(B).Elonga edspoon-likese aeon hemalep o a - sus.(C).Malemeso a sus.(D).Maleme a a sus.(E).Femalep o a sus.(F).Femalemeso-and Figu e 2. Con . Insec s 2024,15, 650 5 o 16 Insec s2024,15,xFORPEERREVIEW5o 17    Figu e2.The en al iewo  he a siandadhesi ese aeinnon-a bo ealspecieso Cicindelidae. (A–F).Cicindelasachalinensis.(A).Malep o a sus.(B).Elonga edspoon-likese aeon hemalep o a - sus.(C).Malemeso a sus.(D).Maleme a a sus.(E).Femalep o a sus.(F).Femalemeso-and Figu e 2. The en al iew o he a si and adhesi e se ae in non-a bo eal species o Cicindelidae. (A–F). Cicindela sachalinensis. (A). Male p o a sus. (B). Elonga ed spoon-like se ae on he male p o a sus. (C). Male meso a sus. (D). Male me a a sus. (E). Female p o a sus. (F). Female meso- and me a a sus. (G–L). Cosmodela sepa a a. (G). Male p o a sus. (H). Elonga ed spoon-like se ae on he male p o a sus. (I). Male meso a sus. (J). Female p o a sus. (K). Female meso a sus. (L). Female me a a sus. ( M–R ). Cylinde a kaleea. (M). Male p o a sus. (N). Elonga ed spoon-like se ae on he male p o a sus. (O). Male meso a sus. (P). Male me a a sus. (Q). Female p o a sus. R. Female meso- and me a a sus. Abb e ia ions: UN, unguis (claw); Ta I, he 1s a some e; Ta II, he 2nd a some e; Ta III, he 3 d a some e; Ta IV, he 4 h a some e; Ta V, he 5 h a some e; sh, se al sha ; p, e minal pla e. 3.1.1. Cicindela sachalinensis Mo awi z, 1862 The ou h and i h a some es o males a e nea ly bald, wi h i e pai s o sho se ae on he ou h a some es and h ee on he i h a some es. Elonga ed spoon-like se ae (Figu e 2B). Elonga ed spoon-like se ae a e he only ype o se a on he en al su ace o he male p o a si. These se ae we e ound on he i s , second and hi d a some es. The se al sha is s aigh , abou 89.50 ± 2.80 µ m (n= 3) in leng h and 5.14 ± 0.33 µ m (n= 3) in wid h (a he base). Each se a has an elonga ed spoon-like e minal pla e a he ip. The en al side o he e minal pla e is egula ly ans e sely ibbed, app oxima ely 33.43 ± 2.77 µ m (n= 3) in leng h and 8.19 ± 0.29 µ m (n= 3) in wid h. The densi y o he elonga ed spoon-like se ae is low: 0.32 se ae pe 100 µm2(n= 3). 3.1.2. Cosmodela sepa a a (Fleu iaux, 1894) The ou h and i h a some es o males a e nea ly bald, wi h i e pai s o sho se ae on he ou h a some es and h ee on he i h a some es. Elonga ed spoon-like se ae (Figu e 2H). Elonga ed spoon-like se ae a e he only ype o se a on he en al su ace o he male p o a si. These se ae we e ound on he i s , second and hi d a some es. The se al sha is s aigh , abou 78.19 ± 6.71 µ m (n= 3) in leng h Insec s 2024,15, 650 6 o 16 and 4.97 ± 0.17 µ m (n= 3) in wid h (a he base). Each se a has an elonga ed spoon-like e minal pla e a he ip. The en al side o he e minal pla e is egula ly ans e sely ibbed, app oxima ely 43.66 ± 1.00 µ m (n= 3) in leng h and 7.42 ± 0.30 µ m (n= 3) in wid h. The densi y o he elonga ed spoon-like se ae is a he low: 0.24 se ae pe 100 µm2(n= 3). 3.1.3. Cylinde a kaleea (Ba es, 1866) The ou h and i h a some es o males a e nea ly bald, wi h i e pai s o sho and hick se ae on he ou h and i h a some es sepa a ely. Elonga ed spoon-like se ae (Figu e 2N). Elonga ed spoon-like se ae a e he only ype o se a on he en al su ace o he male p o a si. These se ae we e ound on he i s , second and hi d a some es. The se al sha is s aigh , abou 59.42 ± 3.69 µ m (n= 3) in leng h and 5.57 ± 0.07 µ m (n= 3) in wid h (a he base). Each se a has an elonga ed spoon-like e minal pla e a he ip. The en al su ace o he e minal pla e is egula ly ans e sely ibbed, app oxima ely 18.56 ± 1.90 µ m (n= 3) in leng h and 7.65 ± 0.83 µ m (n= 3) in wid h. The densi y o he elonga ed spoon-like se ae is a he low: 0.24 se ae pe 100 µm2(n= 3). 3.2. Colly idini 3.2.1. Sub ibe T icondylina: T icondyla pulch ipes Whi e, 1844 As an a bo eal species li ing on ee s ems, T. pulch ipes has well-de eloped adhesi e se ae on all he a si o males and emales, bu he ypes o se ae and hei loca ion a e di e en in males and emales. In bo h gende s, he gene al mo phology o he a some es is as ollows. The i s a some e (Ta I) is elonga ed. The second a some e is sligh ly enla ged and widened. The hi d and ou h a some es a e asymme ical and la ge on he la e al side (Figu e. 3A,E,H,I,M). The en al su aces o he i s , second, hi d and ou h a some es a e densely co e ed wi h adhesi e se ae. The i h a some e (Ta V) is slende and co e ed wi h sca e ed hick and long se ae on he en al, la e al and do sal su aces (Figu e 3J). In males, he e a e h ee ypes o adhesi e se ae: ellip ical se ae, b anched se ae and ilamen -like se ae. In emales, he e a e wo kinds o adhesi e se ae: b anched se ae and ilamen -like se ae. The hick and long se ae on he i h a some e a e p esumably no adhesi e. Ellip ical se ae. These se ae a e si ua ed on he en al su aces o he i s , second and hi d a some es o he male p o a si. The se al sha is s aigh , abou 54.61 ± 1.06 µ m ( n= 3 ) in leng h and 4.13 ± 0.12 µ m (n= 3) in wid h (a he base). Each se a has an ellip ical e minal pla e a he end. The e minal pla e is app oxima ely 19.62 ± 0.88 µ m (n= 3) in long diame e and 8.74 ± 0.43 µ m (n= 3) in sho diame e (Figu e 3C). The ellip ical se ae ha e a ela i ely low densi y and he e a e only 0.48 se ae pe 100 µ m 2 (n= 3). The e a e ans e sal s ipes on he en al side o he se ae. B anched se ae. These se ae a e si ua ed on he en al su ace o he ou h a some e and on he su ounding edge o he i s o hi d a some es o he male p o a si (Figu e 3D). Also, hey a e p esen on he en al su aces o he hi d and ou h a some es o he male meso- and me a a si (Figu e 3F) and on he a si o all emale legs (Figu e 3K,O). These se ae a e elonga e, slende , wi h small p o ube ances, abou 148.12 ± 9.17 µ m (n= 3) in leng h and 3.82 ± 0.04 µ m (n= 3) in wid h (a he base). The apex o he se a is cu ed and acu e. The a e age densi y o he b anched se ae is 1.68 se ae pe 100 µm2(n= 3). Filamen -like se ae. These se ae a e si ua ed on he dis al en al su aces o he i s and second a some es o all emale legs and male meso- and me a a si. On he se al sha s, hese se ae ha e g oo es o ien ed a some angle o he se al axis. Thei ips a e g adually ape ed (Figu e 3G,L,P). The se ae a e abou 90.17 ± 4.12 µ m (n= 3) in leng h and 6.66 ±0.41 µm (n= 3) in wid h (a he basis). The a e age densi y o he se ae is 0.4 se ae pe 100 µm2(n= 3). Insec s 2024,15, 650 7 o 16 Insec s2024,15,xFORPEERREVIEW7o 17   Inmales, he ea e h ee ypeso adhesi ese ae:ellip icalse ae,b anchedse aeand ilamen -likese ae.In emales, he ea e wokindso adhesi ese ae:b anchedse aeand ilamen -likese ae.The hickandlongse aeon he i h a some ea ep esumablyno  adhesi e. Ellip icalse ae.Thesese aea esi ua edon he en alsu aceso  he i s ,second and hi d a some eso  hemalep o a si.These alsha iss aigh ,abou 54.61±1.06µm (n=3)inleng hand4.13±0.12µm(n=3)inwid h(a  hebase).Eachse ahasanellip ical e minalpla ea  heend.The e minalpla eisapp oxima ely19.62±0.88µm(n=3)in longdiame e and8.74±0.43µm(n=3)insho diame e (Figu e3C).Theellip icalse ae ha ea ela i elylowdensi yand he ea eonly0.48se aepe 100µm2(n=3).The ea e ans e sals ipeson he en alsideo  hese ae. B anchedse ae.Thesese aea esi ua edon he en alsu aceo  he ou h a some e andon hesu oundingedgeo  he i s  o hi d a some eso  hemalep o a si(Figu e 3D).Also, heya ep esen on he en alsu aceso  he hi dand ou h a some eso  hemalemeso-andme a a si(Figu e3F)andon he a sio all emalelegs(Figu e3K,O). Thesese aea eelonga e,slende ,wi hsmallp o ube ances,abou 148.12±9.17µm(n= 3)inleng hand3.82±0.04µm(n=3)inwid h(a  hebase).Theapexo  hese aiscu ed andacu e.Thea e agedensi yo  heb anchedse aeis1.68se aepe 100µm2(n=3). Filamen -likese ae.Thesese aea esi ua edon hedis al en alsu aceso  he i s  andsecond a some eso all emalelegsandmalemeso-andme a a si.On hese al sha s, hesese aeha eg oo eso ien eda someangle o hese alaxis.Thei  ipsa e g adually ape ed(Figu e3G,L,P).These aea eabou 90.17±4.12µm(n=3)inleng h and6.66±0.41µm(n=3)inwid h(a  hebasis).Thea e agedensi yo  hese aeis0.4 se aepe 100µm2(n=3).  Figu e3.The a siandadhesi ese aeo T icondylapulch ipes(a bo ealspecies).(A–H).Male.(A). P o a sus, en al iew.(B).Unguis(claw)o p o a sus.(C).Ellip icalse aeonp o a sus.(D). B anchedse aeonp o a sus.(E).Meso a sus, en al iew.(F).B anchedse aeonmeso a sus.(G). Filamen -likese aeonmeso a sus.(H).Me a a sus, en al iew.(I–P).Female.(I).P o a sus, Figu e 3. The a si and adhesi e se ae o T icondyla pulch ipes (a bo eal species). (A–H). Male. (A). P o a sus, en al iew. (B). Unguis (claw) o p o a sus. (C). Ellip ical se ae on p o a sus. (D). B anched se ae on p o a sus. (E). Meso a sus, en al iew. (F). B anched se ae on meso a sus. (G). Filamen -like se ae on meso a sus. (H). Me a a sus, en al iew. (I–P). Female. (I). P o a sus, en al iew. (J). Unguis (claw) o p o a sus. (K). B anched se ae on p o a sus. (L). Filamen -like se ae on p o a sus. (M). Me a a sus, en al iew. (N). Unguis (claw) o me a a sus. (O). B anched se ae on me a a sus. (P). Filamen -like se ae on me a a sus. Abb e ia ions: UN, unguis (claw); Ta I, he 1s a some e; Ta II, he 2nd a some e; Ta III, he 3 d a some e; Ta IV, he 4 h a some e; Ta V, he 5 h a some e; sh, se al sha ; p, e minal pla e. 3.2.2. Sub ibe Colly idina: Neocolly is linea is (Schmid -Göbel, 1846) Neocolly is linea is is an a bo eal species li ing on ees. I has well-de eloped adhesi e se ae on he male and emale a si, bu he se al ypes and loca ions a e di e en in males and emales. In bo h gende s, he gene al mo phology o a some es is as ollows. The i s and second a some es (Ta I and Ta II) a e elonga e. The hi d and ou h a some es (Ta III and Ta IV) a e sligh ly enla ged and wide (Figu e 4A,E,G,I,O). The en al su aces o he i s , second, hi d and ou h a some es a e densely co e ed wi h adhesi e se ae, which consis o wo pa s: se al sha (sh) and specialized ip. The i h a some e (Ta V) is wide and co e ed wi h sca e ed hick and sho se ae on he en al su ace (Figu e 4D,J). In males, he e a e h ee ypes o adhesi e se ae: discoidal, spa ula e, and ape ed se ae. In emales, he e a e only wo ypes o adhesi e se ae: spa ula e and ape ed se ae. The hick sho se ae on he i h a some e a e p esumably no adhesi e. Insec s 2024,15, 650 8 o 16 Insec s2024,15,xFORPEERREVIEW9o 17    Figu e4.The a siandadhesi ese aeo Neocolly islinea is(a bo ealspecies).(A–H).Male.(A). P o a sus, en al iew.(B).Unguis(claw)o p o a sus.(C).Discoidalse aeonp o a sus.(D).Spa - ula ese aeonp o a sus.(E).Meso a sus, en al iew.(F).Spa ula ese aeonmeso a sus.(G).Me - a a sus, en al iew.(H).Discoidalse aeonme a a sus.(I–P).Female.(I).P o a sus, en al iew. (J).Unguis(claw)o p o a sus.(K).Spa ula ese aeonp o a sus.(L).Tape edse aeonp o a sus. (M).Spa ula ese aeonmeso a sus.(N).Tape edse aeonmeso a sus.(O).Me a a sus, en al iew. (P).Spa ula ese aeonme a a sus.Abb e ia ions:UN,unguis(claw);Ta I, he1s  a some e;Ta II, he2nd a some e;Ta III, he3 d a some e;Ta IV, he4 h a some e;Ta V, he5 h a some e;sh, se alsha ; p, e minalpla e. 3.3.Mic os uc u eo  heFemaleCu icleSu aceand heMaleBeha io du ingMa ing 3.3.1.Cicindelini CicindelasachalinensisandCosmodelasepa a a Du ingma ing, hemaleso species ha belong o his ibealwaysuse hede eloped mandible oclasp hebasica eao  hep e o h oaxo  he emales(Figu e5A,G–I).The on legsdono alwayscon ac wi h he emaleely ao he bodyside(Figu e5A,G). Byobse ing hecu iclesu aceo  he emaleely aandme as e numusingSEM,i  was ound ha  he ea e egula lya angedhexagonalsu aces uc u eson he emale ely a,wi h heconca esu acein hehexagons(Figu e6A–C).Thecu icleo  heme as- e numalsohasahexagonalsu acepa e n,no conca e,bu  a he ashee -likewi ha lowe heigh (Figu e6D–F).Thesu aceo  emaleCosmodelasepa a ahassome oughness a  hemic o-scale.Thewid ho  hehexagonso Ci.sachalinensisis15.63±0.49µm(n=3) in heely aand14.98±0.29µm(n=3)in heme as e num, espec i ely.Thewid ho  he hexagonso Co.sepa a ais13.47±1.29µm(n=3)in heely aand12.56±0.19µm(n=3) in heme as e num, espec i ely.Bo hCi.sachalinensisandCo.sepa a aha esomekindo  oughnesson heely aandCo.sepa a aalsosome oughnesson heme as e num. Figu e 4. The a si and adhesi e se ae o Neocolly is linea is (a bo eal species). (A–H). Male. (A). P o- a sus, en al iew. (B). Unguis (claw) o p o a sus. (C). Discoidal se ae on p o a sus. (D). Spa ula e se ae on p o a sus. (E). Meso a sus, en al iew. (F). Spa ula e se ae on meso a sus. (G). Me a a - sus, en al iew. (H). Discoidal se ae on me a a sus. (I–P). Female. (I). P o a sus, en al iew. (J). Unguis (claw) o p o a sus. (K). Spa ula e se ae on p o a sus. (L). Tape ed se ae on p o a sus. (M). Spa ula e se ae on meso a sus. (N). Tape ed se ae on meso a sus. (O). Me a a sus, en al iew. (P). Spa ula e se ae on me a a sus. Abb e ia ions: UN, unguis (claw); Ta I, he 1s a some e; Ta II, he 2nd a some e; Ta III, he 3 d a some e; Ta IV, he 4 h a some e; Ta V, he 5 h a some e; sh, se al sha ; p, e minal pla e. Discoidal se ae. These se ae a e si ua ed on he en al su aces o he i s , second and hi d a some es o he male a si (Figu e 4C,H). The se al sha is s aigh , wi h wo o h ee g oo es ac oss he junc ion o he se al e minal pla e, abou 40.61 ± 1.15 µ m ( n= 3 ) in leng h and 3.56 ± 0.20 µ m (n= 3) in wid h. Each se a has a discoidal e minal pla e wi h 1–2 ips a he p oximal pa . The e minal pla e is app oxima ely 8.56 ± 0.54 µ m (n= 3) in diame e (Figu e 4C). The discoidal se ae ha e a ela i ely low densi y: 0.6 se ae pe 100 µm2(n= 3). Spa ula e se ae. These se ae a e si ua ed on he en al su ace o he ou h a some e in bo h male and emale a si (Figu e 4D,F,K,M,P). The se al sha is s aigh , sligh ly ben , 64.71 ± 3.71 µ m (n= 3) in leng h and 2.35 ± 0.14 µ m (n= 3) in wid h. Each se a has a spa ula e e minal pla e. The wides pa o he pla e is 10.64 ± 0.48 µ m (n= 3) in wid h (Figu e 4D,K). The en al and do sal su aces o he e minal pla e a e smoo h, wi hou ob ious subs uc u e. The spa ula e se ae ha e a high densi y: 1.8 se ae pe 100 µ m 2 ( n= 3 ). These spa ula e se ae a e e y simila wi h hose o lea bee les [ 14 , 16 , 28 ] and ladybi d bee les [14]. Tape ed se ae. These se ae a e si ua ed on he en al su aces o he second and hi d a some es o emale legs (Figu e 4L,N) and he su ounding edge o he i s o hi d a some es’ en al su aces o he male a si (Figu e 4H). These se ae a e elonga ed, slende , Insec s 2024,15, 650 9 o 16 abou 66.42 ± 5.18 µ m (n= 3) in leng h and 4.12 ± 0.09 µ m (n= 3) in wid h (a he base). The se al sha is s aigh a he base and ape ed owa d he cu ed acu e ip. The densi y o he ape ed se ae is 1.12 se ae pe 100 µm2(n= 3). 3.3. Mic os uc u e o he Female Cu icle Su ace and he Male Beha io du ing Ma ing 3.3.1. Cicindelini Cicindela sachalinensis and Cosmodela sepa a a Du ing ma ing, he males o species ha belong o his ibe always use he de eloped mandible o clasp he basic a ea o he p e o h oax o he emales (Figu e 5A,G–I). The on legs do no always con ac wi h he emale ely a o he body side (Figu e 5A,G). Insec s2024,15,xFORPEERREVIEW10o 17    Figu e5.Thecon ac posi iono  helegsdu ingma ing.(A).Ma ingo Ropalo e esdesgodinsii(Fai - mai e,1887)(Sichuan,pho og aphedbyLiHe).(B).Ma ingo T icondylapulch ipes(HongKong, pho og aphedbySiuyeungHo).(C).Ma ingo T icondylapulch ipes(HongKong,pho og aphedby Al edCheung).(D).Ma ingo Neocolly ispa ula(Chaudoi ,1848)(NaglaBlock,Palgha ,Maha- ash a,India,pho og aphedbyDineshSha ma).(E,F).Ma ingo Neocolly issp.(Yeoo Hills,Thane Wes ,Thane,Maha ash a,India,pho og aphedbyAnilKuma Ve ma).(G–I).Sc eensho  oma ideoo  hema ingp ocesso Cosmodelajux a a(Accia a iandPea son,1989).(G).A 15s( ed a owsshow ha  hep o a sio  hemaledoesno con ac  hebodyo  he emale).(H).A 20s( ed a owsshow hep o a sio  hemale ying ocon ac  hebodyo  he emale,and hemaleclasps he emalewi hhismandibles).(I).A 22s( eda owsshow hesameasin(H), hep o a sio  he malecon ac wi h hebodyo  he emalemo e igh ly). Figu e 5. The con ac posi ion o he legs du ing ma ing. (A). Ma ing o Ropalo e es desgodinsii (Fai mai e, 1887) (Sichuan, pho og aphed by Li He). (B). Ma ing o T icondyla pulch ipes (Hong Kong, pho og aphed by Siuyeung Ho). (C). Ma ing o T icondyla pulch ipes (Hong Kong, pho og aphed by Al ed Cheung). (D). Ma ing o Neocolly is pa ula (Chaudoi , 1848) (Nagla Block, Palgha , Maha ash a, India, pho og aphed by Dinesh Sha ma). (E,F). Ma ing o Neocolly is sp. (Yeoo Hills, Thane Wes , Thane, Maha ash a, India, pho og aphed by Anil Kuma Ve ma). (G–I). Sc eensho om a ideo o he ma ing p ocess o Cosmodela jux a a (Accia a i and Pea son, 1989). (G). A 15 s ( ed a ows show ha he p o a si o he male does no con ac he body o he emale). (H). A 20 s ( ed a ows show he p o a si o he male ying o con ac he body o he emale, and he male clasps he emale wi h his mandibles). (I). A 22 s ( ed a ows show he same as in (H), he p o a si o he male con ac wi h he body o he emale mo e igh ly). By obse ing he cu icle su ace o he emale ely a and me as e num using SEM, i was ound ha he e a e egula ly a anged hexagonal su ace s uc u es on he emale ely a, wi h he conca e su ace in he hexagons (Figu e 6A–C). The cu icle o he me as e - num also has a hexagonal su ace pa e n, no conca e, bu a he a shee -like wi h a lowe heigh (Figu e 6D–F). The su ace o emale Cosmodela sepa a a has some oughness a he mic o-scale. The wid h o he hexagons o Ci. sachalinensis is 15.63 ± 0.49 µ m (n= 3) in he ely a and 14.98 ± 0.29 µ m (n= 3) in he me as e num, espec i ely. The wid h o he hexagons o Co. sepa a a is 13.47 ± 1.29 µ m (n= 3) in he ely a and 12.56 ± 0.19 µ m (n= 3) in he me as e num, espec i ely. Bo h Ci.sachalinensis and Co.sepa a a ha e some kind o oughness on he ely a and Co.sepa a a also some oughness on he me as e num. Insec s 2024,15, 650 16 o 16 48. Wol , J.O.; Go b, S.N. Compa a i e mo phology o p e a sal scopulae in ele en spide amilies. A h opod S uc . De . 2012,41, 419–433. [C ossRe ] 49. F os , F.; Go b, S.N.; Wol , J.O. Adhesion and ic ion in hun ing spide s: The e ec o con ac spli ing on hei a achmen abili y. Zool. Anz. 2018,273, 231–239. [C ossRe ] 50. Au umn, K.; Liang, Y.A.; Hsieh, S.T.; Zesch, W.; Chan, W.P.; Kenny, T.W.; Fea ing, R.; Full, R.J. Adhesi e o ce o a single gecko oo -hai . Na u e 2000,405, 681–685. [C ossRe ] [PubMed] 51. Heepe, L.; Hö , S.; Michels, J.; Go b, S.N. Ma e ial g adien s in ib illa insec a achmen sys ems: The ole o join -like elemen s. So Ma e 2018,14, 7026–7033. [C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .