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
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Licensee MDPI, Basel, Swi ze land.
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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.
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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.
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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 .
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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.
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