In e eb a e Zoology, 2025, 22(1): 8–20 © INVERTEBRATE ZOOLOGY, 2025
Do eu a dig ades ha e a copula o y o gan?
The case o Halobio us c ispae K is ensen, 1982
(Ta dig ada: Halobio idae)
N.M. Bise o a1*, M.A. Banniko a1, O.V. Nikolae a2, V.V. Aleoshin2,3
1 In e eb a e Zoology Depa men , Lomonoso Moscow S a e Uni e si y, Leninskie Go y 1-12,
Moscow 119234 Russia.
2 Beloze sky Ins i u e o Physicochemical Biology, Lomonoso Moscow S a e Uni e si y, Leninskie
Go y 1-40, Moscow 119991 Russia.
3 Ins i u e o In o ma ion T ansmission P oblems (Kha ke ich Ins i u e), Russian Academy o Sci-
ences, Bolshoy Ka e ny pe . 19-1, Moscow, 127051 Russia.
Na alia Bise o a: nbise [email p o ec ed] ORCID h ps://o cid.o g/0000-0002-5481-0207
Ma ia Banniko a: [email p o ec ed] ORCID h ps://o cid.o g/0000-0002-5192-0873
Olga Nikolae a: [email p o ec ed] ORCID h ps://o cid.o g/0000-0003-2424-298X
Vladimi Aleoshin: [email p o ec ed] ORCID h ps://o cid.o g/0000-0002-3299-9950
* Co esponding au ho
ABSTRACT: Rep oduc i e s a egies o a dig ades a y conside ably among axa. Species
o He e o a dig ada ha e sepa a e gonopo e and anus, whe eas Eu a dig ada species ha e
a single cloacal opening, which is a sha ed exi om he cloaca, hindgu , and Malpighian
ubules. While in e nal e iliza ion has been hypo hesized o ce ain species based on he
p esence o spe ma ozoa in emale spe ma hecae, he mechanism o spe m ans e emains
undocumen ed. No ably, sexual dimo phism has no p e iously been epo ed in eu a dig ades.
In his s udy, males and emales o he eu a dig ade Halobio us c ispae K is ensen, 1982
we e collec ed om sand samples in he in e idal zone o Olene sky Island, Whi e Sea,
and analyzed using molecula , elec on-mic oscopy and immunocy ochemical me hods. Ou
indings ad ance he unde s anding o H. c ispae ep oduc i e biology, including he i s
documen ed e idence o sexual dimo phism in eu a dig ades. Males possess a p o usible
cloacal cylinde , hypo hesized o unc ion as a copula o y o gan. Dis inc mo phological
di e ences in cloacal s uc u e we e obse ed in wo sexes. Males ha e o al-shaped and
smalle cloaca wi h smalle cloacal pla es; emales ha e la ge , diamond-shaped cloaca,
wi h acu e- iangula cloacal pla es. Addi ionally, senso y o gans and muscula u e nea he
cloacal pla es and in he ou h pai o legs ha e been ound which sugges neu al egula ion
du ing ma ing, po en ially guiding copula o y in e ac ions. The p oposed ole o he male
cloacal cylinde as a copula o y o gan suppo s he hypo hesis o in e nal e iliza ion in his
g oup, changing he cu en unde s anding o ep oduc i e s a egies in a dig ades. These
indings unde sco e he need o deepe explo a ion o a dig ade ep oduc ion, pa icula ly
o cla i y he p e alence and mechanisms o in e nal e iliza ion ac oss species.
How o ci e his a icle: Bise o a N.M., Banniko a M.A., Nikolae a O.V., Aleoshin V.V.
2025. Do eu a dig ades ha e a copula o y o gan? The case o Halobio us c ispae K is-
ensen, 1982 (Ta dig ada: Halobio idae) // In e . Zool. Vol.22. No.1. P.8–20. doi: 10.15298/
in e zool.22.1.01
KEY WORDS: Ta dig ada, sexual dimo phism, ep oduc ion, e iliza ion, molecula
gene ics, scanning elec on mic oscopy, immunocy ochemis y.
De o ed o memo y o Claus Nielsen.
Do eu a dig ades ha e a copula o y o gan? 9
Обладают ли эутардиграды копулятивным органом?
Случай Halobio us c ispae K is ensen, 1982
(Ta dig ada: Halobio idae)
Н.М. Бисерова1*, М.А. Банникова1, О.В. Николаева2,
В.В. Алешин2,3
1 Кафедра зоологии беспозвоночных, Московский государственный университет им. М.В.
Ломоносова, Ленинские горы 1-12, Москва 119234 Россия.
2 Научно-исследовательский институт физико-химической биологии имени А. Н. Белозерского,
Ленинские горы 1-40, Москва 119991 Россия.
3 Институт проблем передачи информации имени А.А. Харкевича РАН, Большой Каретный
пер. 19-1, Москва 127994 Россия.
* Ответственный за переписку: nbise [email p o ec ed]
РЕЗЮМЕ: Репродуктивные стратегии тихоходок значительно различаются между
таксонами. Виды He e o a dig ada имеют отдельные гонопор и анус, а виды Eu a dig ada
имеют клоакальное отверстие, которое является общим выходом из клоаки, задней
кишки и Мальпигиевых сосудов. Для нескольких видов, у которых сперматозоиды были
обнаружены в семяприемниках самок, предполагается внутреннее оплодотворение,
но способ передачи спермы до сих пор неизвестен. Половой диморфизм ранее не
был отмечен у эутардиград.
Самцы и самки эутардиграды Halobio us c ispae K is ensen, 1982 были обнаружены в об-
разцах песчаного грунта в приливно-отливной зоне острова Оленевский Белого моря, и
изучены молекулярными, электронно-микроскопическими и иммуноцитохимическими
методами. Самцы обладают выдвижным клоакальным цилиндром, предположительно
функционирующим как копулятивный орган. Были обнаружены четкие различия в
морфологии клоаки у самок и самцов. У самцов клоака овальная, меньшего размера;
у самок клоака крупнее, ромбовидной формы, с остро-треугольными клоакаль-
ными пластинами. Это первый задокументированный случай внешнего полового
диморфизма у эутардиград. Были обнаружены мускулатура и сенсорные органы
клоакальных пластин четвертой пары ног, что предполагает нервную регуляцию в
процессе спаривания и копулятивных взаимодействий. Роль мужского клоакального
цилиндра как копулятивного органа предполагает внутреннее оплодотворение у
эутардиград, что меняет имеющиеся представления о репродуктивных стратегиях
этой группы тихоходок. Результаты подчеркивают необходимость более глубокого
изучения размножения тихоходок, в частности, для выяснения распространенности
и механизмов внутреннего оплодотворения у разных видов.
Как цитировать эту статью: Bise o a N.M., Banniko a M.A., Nikolae a O.V., Aleoshin
V.V. 2025. Do eu a dig ades ha e a copula o y o gan? The case o Halobio us c ispae
K is ensen, 1982 (Ta dig ada: Halobio idae) // In e . Zool. Vol.22. No.1. P.8–20. doi:
10.15298/in e zool.22.1.01
КЛЮЧЕВЫЕ СЛОВА: Ta dig ada, половой диморфизм, размножение, оплодотворение,
молекулярная генетика, сканирующая электронная микроскопия, иммуноцитохимия.
N.M. Bise o a e al.
10
In oduc ion
Halobio us c ispae K is ensen, 1982 is a
seconda ily ma ine a dig ade belonging o
he class Eu a dig ada, o de Pa achela, amily
Halobio idae (Gąsio ek e al., 2019; Degma e
al., 2024). The species was desc ibed in 1982
by Reinha d K is ensen, a Danish zoologis ,
and is he ype species o he genus Halobio us
(K is ensen, 1982). I inhabi s he in e idal and
sub idal zones wi hin algae hicke s (Møbje g
e al., 2007).
Ta dig ades a e known o exhibi ei he
gonocho ism (wi h sepa a e male and emale
indi iduals) o he maph odi ism. Rep oduc ion
occu s exclusi ely h ough game es (Be olani,
1983, 1990, 2001). Rep oduc i e s a egies
o a dig ades include amphimixis (c oss-
e iliza ion o sel - e iliza ion) and hely okous
pa henogenesis (apomixis o au omixis) (Be -
olani, Rebecchi, 1999; Be olani, 2001; Nelson
e al., 2015; Al ie o e al., 2018). In a dig ade
popula ions, sex a ios a y g ea ly (Rebecchi,
Be olani, 1994). Males a e mos o en ew,
ex emely a e, o comple ely unknown (Ramaz-
zo i, Maucci, 1983; Suzuki, 2008; Gąsio ek e
al., 2020). The e may also be seasonal di e -
ences in he p esence o males in popula ions
(Gąsio ek e al., 2020). In e nal e iliza ion has
only been supposed when a spe ma heca ull o
non-mo ile spe ma ozoa was ound wi hin he
emale (Be olani, Rebecchi, 1999; S ec e al.,
2021; Vecchi e al., 2022). Howe e , in o he
cases, in e nal e iliza ion has no ye been eli-
ably documen ed (Be olani, 1983, 1990, 2001;
Be olani, Rebecchi, 1999; Sugiu a, Ma sumo o,
2021a,b). The ep oduc i e sys em o a dig ades
consis s o an unpai ed gonad (o a y o es is, o
he maph odi ic gonad – o o es is) and geni al
duc s – wo in males and one in emales and
he maph odi es. Seminal esicles in males and
spe ma hecae in emales ha e been desc ibed
in some species (Be olani, Rebecchi, 1999).
Species o He e o a dig ada ha e sepa a e gono-
po e and anus, bu Eu a dig ada species lack
ex e nal gonopo es as hey ha e a cloaca whe e
he hindgu , Malpighian ubules and gonoduc s
e mina e (Nelson e al., 2015). In some ma ine
and e es ial he e o a dig ades, male gonopo e
opens a he op o a small, ex e nally p o uding
ube ha could acili a e copula ion (Be olani,
1990). Ven al cloaca o eu a dig ades is simila
in bo h sexes (Be olani, Rebecchi, 1999). Ex-
e nal sexual dimo phism o copula o y o gans
ha e ne e been obse ed in eu a dig ades.
Halobio us c ispae is cha ac e ized by cyclo-
mo phosis (K is ensen, 1982). The phenomenon
includes 4 main s ages: 1) pseudosimplex 1
(win e o m): he hibe na ion s age, cha ac e -
ized by he absence o ma u e gonads, a double
cu icle and con inuous placoids in he o m o
s ipes, long claws, he mou h and cloaca a e
closed wi h cu icula hickenings; 2) pseudo-
simplex 2 (sp ing o m): a sexually ma u ing
s age occu ing in sp ing a e shedding he old
cu icle. This s age is cha ac e ized by de eloping
gonads, ela i ely small claws, used placoids in
s ipes, and a small open mou h; 3) ac i e s age
(summe o m): gonads and claws comple ely
de eloped, he mou h and cloaca a e open, and
six pe ibuccal senso y o gans a e p esen ; he
pha yngeal placoids a e di ided in o mac o-
and mic oplacoids; 4) simplex (summe o m):
a mol ing s age, also cha ac e is ic o sexually
ma u e animals; i is ini ia ed by shedding o
he buccopha yngeal appa a us, which is newly
de eloped du ing di e en s ages o he simplex,
he mou h and cloaca a e closed, and pe ibuccal
senso y o gans a e absen (K is ensen, 1982,
Møbje g e al., 2007; Halbe g e al., 2009b).
The emales o Halobio us lay eggs in he
exu iae du ing moul ing (K is ensen, 1982).
Usually, eggs in he exu iae ha e smoo h shell
(Al ie o e al., 2018), o med inside he emale.
An oocy e and a gonad wall pa icipa e in his
p ocess (Pop awa, 2011). I was sugges ed ha
H. c ispae was cha ac e ized by ex e nal e iliza-
ion (K is ensen, 1982) since spe m mo phology
is o a “p imi i e” ype (no mal ac osome, heli-
coid nucleus and un ans o med mi ochond ia).
Also, no spe ma hecae o o he signs o in e nal
e iliza ion we e ound (K is ensen, 1982). To
da e, he e a e no addi ional da a in he li e a u e
abou e iliza ion p ocess in his species.
He e we p esen he i s obse a ion o sexual
dimo phism in cloacal s uc u e and p o ide he
i s desc ip ion o a cloacal cylinde in males o
Halobio us c ispae. Based on i s mo phology,
his s uc u e may unc ion as a copula o y o gan.
Ma e ial and me hods
Sampling
Sandy sedimen samples we e collec ed in June
2022 in he in e idal zone o Olene sky Island
(Kandalaksha Bay o he Whi e Sea) 66°31′19.6″N,
Do eu a dig ades ha e a copula o y o gan? 11
33°07′31.4″E. The samples con ained nume ous a di-
g ades. Animals we e so ed unde a s e eomic oscope
using a capilla y pipe e and placed in esh wa e o
1–2 min o elaxa ion. A o al o 18 samples we e
in es iga ed. Se e al indi iduals exhibi ing a ubula
p o usion a he caudal end be ween he 4 h pai o
legs we e selec ed o scanning elec on mic oscopy
examina ion.
Ligh mic oscopy (LM)
Specimens we e ixed in 4% o maldehyde in dis-
illed wa e neu alized wi h CaCO3, washed in dis illed
wa e and s o ed in Seinho s liquid (0.5% glyce in +
30% alcohol). The specimens we e examined using a
Leica CTR5000 ligh mic oscope.
Scanning elec on mic oscopy (SEM)
Fo scanning elec on mic oscopy, specimens
we e ixed in 2.5% glu a aldehyde on 0.1 M sodium
phospha e bu e (PBS) and pos ixed in 1% OsO4 on
PBS, pH 7.4, dehyd a ed in inc easing concen a ions
o e hanol, hen in a mix u e o 96% e hanol and 100%
ace one in an inc easing g adien o 10 min, and in
100% ace one wice o 10 min. The samples we e c i i-
cal poin d ied using liquid CO2 (HCP-2 C i ical Poin
D ye , Hi achi, 1980), spu e coa ed wi h a pla inum-
palladium mix u e (EIKO IB-3 Ion Coa e , 1980) and
examined using a The mo Fishe Scien i ic Qua o S
SEM. 6 specimens we e examined using SEM.
Con ocal lase scanning mic oscopy (CLSM)
Fo immunocy ochemical examina ion in a CLSM,
he samples we e ixed wi hin 2 hou s in 4% pa a o m
on PBS, pH 7.4, exposed o ul asound o 30 and 50
sec ul asound powe 120 W, equency 40 kHz, ol age
AC220 - 240 W, washed in PBS wice o 10 min and
kep in a solu ion o 7% T i on X100 in 0.1 M PBS wi h
he addi ion o 0.03% NaN3 on a shake a 4 °C o
wo days. Then samples we e incuba ed in a solu ion
o an ibodies agains α- ubulin. As p ima y an ibodies,
we used mouse monoclonal an i-ace yla ed- ubulin
(SIGMA, USA), clone 6 –11 B -1, p oduc numbe :
T6793 in a concen a ion o 1:1000 dilu ed in 0.1 M
PBS wi h he addi ion o 1% Bo ine Se um Albumin
(BSA), 1% Dime hyl sul oxide and 1% T i on X100
(TBP solu ion) o 3 days. A e se e al washes in TBP
solu ion (8 imes o 30 min), he samples we e incuba ed
in a solu ion con aining he seconda y an ibody: an i-
mouse AlexaFluo 488 om goa in a concen a ion o
1:1000 dilu ed in 0.1 M PBS wi h 1% DMSO o one
day and hen we e washed 2 imes o 30 min in he
same bu e . A e ha he samples we e s ained wi h
nuclea dye DAPI in 0.1 M PBS, a a concen a ion o
1:500 and washed wi h 0.1M PBS 10 imes o 20 min.
All p ocedu es we e ca ied ou on a shake a 4 °C.
The samples we e pu in g adien glyce ol se ies wi h
he addi ion o 2.5% DABCO (1,4-diazabicyclo[2.2.2]
oc ane): 30% glyce ol o e nigh , 60% 1,5 hou and 70%
1,5 hou . The p epa a ions we e examined in a Nikon
A1 con ocal mic oscope a wa eleng hs 405 nm and
488 nm. Two indi iduals we e examined. I should be
no ed ha he cu icle and issues o a dig ades ha e
high au o luo escence in wide ange.
DNA ex ac ion, PCR, and sequencing
Fou ixed in 96° e hanol indi iduals o Halobio us
c ispae we e ea ed wi h p o einase K a 50°С o 24
hou s. A e ha o al genomic DNA was ex ac ed
using he DIA om DNA P ep Ki (Isogen, Russia)
ollowing he p o ocol p o ided by he manu ac u e .
Two egions – nuclea ibosomal (con aining pa ial
18S and 28S RNA genes, comple e 5.8S RNA
gene, and ITS1 and ITS2 sequences) and mi ochon-
d ial (pa ial gene cox1) – we e ampli ied using
p ime s (5′-GTATCTGGTTGATCCTGCCAGT-3′
and 5′-ATATGCTTAARTTCAGCGGGT-3′ o
nuclea DNA and 5’-GGTCAACAAATCATAAA-
GATATTGG-3’ and 5’-TAAACTTCAGGGTGAC-
CAAAAAATCA-3’ o cox1) wi h Encyclo PCR
ki (E ogen, Russia). The PCR cycling condi ions
o DNA sequence included dena u a ion a 95 °C
o 5 min, ollowed by 40 cycles o dena u a ion a
95 °C o 30 s, annealing a 55 °C o 30 s, ex ension
a 72 °C o 3 min, and inal ex ension a 72 °C o
5 min. The PCR cycling condi ions o cox1 sequence
included dena u a ion a 95 °C o 5 min, ollowed by
35 cycles o dena u a ion a 95 °C o 30 s, anneal-
ing a 43 °C o 30 s, ex ension a 72 °C o 1 min,
and inal ex ension a 72 °C o 5 min. PCR p oduc s
we e sepa a ed wi h aga ose gel elec opho esis and
pu i ied using Cleanup Mini ki (E ogen, Russia).
Amplicons we e sequenced di ec ly wi h an Applied
Biosys ems 3730 DNA Analyze . The sequences o
H. c ispae we e deposi ed in GenBank wi h accession
numbe s PP537167 o nuclea DNA (3787 bp) and
PP535567 o cox1 gene agmen (686 bp).
Sequence alignmen
Ob ained nuclea DNA was aligned wi h h ee
sequence o H. c ispae om GenBank da abase
(Accession Numbe s EF620420, EF620421, and
EF620422; Møbje g e al., 2007) using MAFFT online
se ice (Ka oh e al., 2019) o accu a e p edic ion o
ITS2 egion localiza ion. The seconda y s uc u e o
nuclea ITS2 egions we e p edic ed using RNAs uc-
u e so wa e (Reu e , Ma hews, 2010) wi h de aul
alues. All p edic ed s uc u es o ITS2 we e manually
examined and compa ed o common s ems, loops,
and bulges. Compensa o y base changes (CBCs) in
di e en helices we e manually iden i ied o all ITS2
seconda y s uc u es.
Resul s
MOLECULAR ANALYSIS. 18S RNA and
cox1 gene sequences o he Whi e Sea H. c ispae
N.M. Bise o a e al.
12
Fig. 1. P edic ed seconda y s uc u e o ITS2 o Halobio us c ispae based on he nucleo ide sequence o H.
с ispae om Ba in Bay, he No h and Bal ic Seas, ed a ows indica e nucleo ide subs i u ions ound in he
Whi e Sea specimens. Helices a e numbe ed acco ding o Coleman (2000).
Do eu a dig ades ha e a copula o y o gan? 13
a e 100% iden ical wi h H. c ispae isola es om
GenBank da abase. ITS2 sequence o he Whi e
Sea H. c ispae is 93% iden ical wi h he h ee
iden ical o each o he H. c ispae isola es om
di e en loca ions (Ba in Bay, No h Sea, and
Bal ic Sea) (GenBank EF620420-2; Møbje g
e al., 2007). The p edic ed ITS2 seconda y
s uc u e o H. c ispae olded in o a ypical
s uc u e wi h ou common majo helices as
in mos euka yo es (Coleman, 2000, 2007),
wi h he longes helix III (Fig. 1). The Whi e
Sea isola e di e s om he o he isola es by
23 subs i u ions, howe e , he e is no a single
compensa o y base change (CBC) subs i u ion.
The absence o CBCs be ween Halobio us iso-
la es indica es he pu a i e absence o gene ic
isola ion be ween hese isola es which in u n
con i ms hei conspeci ici y (Coleman, 2000,
2007; Wol e al., 2013). Thus, he Whi e Sea
Halobio us isola e iden i ica ion o H. c ispae
was con i med wi h molecula da a.
EXTERNAL MORPHOLOGY. Specimens
o H. c ispae included adul s a he ac i e and
simplex s ages. Indi iduals a ied in size om
350 o 530 µm; he cu icle was smoo h wi hou
sculp u e; he claws we e la ge. LM examina ion
o whole moun s e ealed he p esence o males
and emales. Sex de e mina ion was based on he
p esence o game es o eggs wi hin gonads. Fe-
males con ained se e al la ge eggs (Fig. 2A–B).
Unde a ligh mic oscope in la e al p ojec ion, he
cloaca canal is isible, which is su ounded by
a sligh hickening o he cu icle (Fig. 2C). The
absence o pe ibuccal senso y o gans, a closed
mou h and cloaca, and newly syn hesized claws
indica ed ha he indi iduals we e in he simplex
(mol ing) s age. In ligh mic oscopy p epa a ions,
he a e age body leng h o emales was app oxi-
ma ely 530 µm. The leng h o males was simila
a ound 526 µm. Some indi iduals exhibi ed a
p o uding appendage a he caudal end be ween
he legs o he 4 h pai ; hese specimens measu ed
app oxima ely 350 µm long.
SCANNING ELECTRON MICROSCOPY
(SEM). SEM examina ion showed h ee a ian s
o he cloacal egion s uc u e in H. c ispae (Figs.
2D–G; 3). Some indi iduals had a p o uding
cu icula cylinde a he si e o he cloacal open-
ing be ween he 4 h pai o legs (Fig. 3A–D,
F–G). The dimensions o his s uc u e we e
app oxima ely 34 µm in leng h and abou 10
µm in diame e . In some specimens, he cloacal
cylinde was pa ially ex ended; a he base o
he incomple ely p o uded cylinde , semici cula
olds we e no iceable: an an e io old and a pai
o la e al olds, co esponding o o al-shaped
cloacal pla es (Fig. 3E–F). The diame e o he
cylinde wi h basal olds was 23 µm. The olds
a he base o he ex ended cloacal o gan indica e
a elescopic me hod o ex ension, a ea u e also
cha ac e is ic o a dig ade limbs. In he s udied
specimens wi h ex ended cloacal cylinde , we
obse ed comp ession o he pos e io body
egion ollowing he 3 d pai o legs, appa en ly
due o muscle con ac ion. The apical end o
he o gan has a pai ed opening, a ound which
ejacula ed ma e ial was obse ed (Fig. 3C–D).
The iden i ied emale wi h eggs in he gonad
(used o LM and subsequen ly o SEM) was
ound o ha e a diamond-shaped cloaca consis -
ing o acu ely angled iangula pla es enclosing
he opening (Fig. 2D–E). The dimensions o he
diamond-shaped cloaca we e 26.9 x 20.8 µm;
heigh o he uppe iangula pla e was 8.5 µm,
and he heigh o he lowe pai o pla es was
16.0 µm.
In addi ion, male indi iduals wi h an o al-
shaped cloaca we e obse ed. The dimensions
o he o al cloaca we e 21.7 x 13.8 µm; heigh
o he uppe o al pla e was 7.98 µm, and he
heigh o he lowe pai o pla es was 11.7 µm.
In he specimens wi h an o al cloaca, we ound
a pai o dis inc muscle a achmen si es and a
pai o p ominen s uc u es p esumably co -
esponding o senso y papillae, loca ed an e io
o he cloaca (Fig. 2H–J).
CONFOCAL LASER SCANNING MI-
CROSCOPY (CLSM). The wo indi iduals
(lacking dis inc eggs) we e examined o gonad
s uc u e using CLSM. The i s specimen was
a he ac i e s age, as e idenced by sepa a e
mac oplacoids in he pha ynx and he p esence
o pe ibuccal senso y o gans (no shown). The
second specimen was in he simplex (mol ing)
s age, wi h a double se o claws on he legs
(Fig. 4A). Using DAPI s aining, a sac-shaped
male gonad illed wi h male ep oduc i e cells
a a ious de elopmen al s ages was iden i ied
on he do sal side o bo h indi iduals, abo e he
in es ine (Fig. 4B). F om he caudal end o he
gonad o he pos e io end o he body, wo asa
de e en ia ex ended a ound he gu (Fig. 4C–D,
G). The gonoduc s con inued in o a usi o m ex-
ension illed wi h spe m — he seminal esicles
N.M. Bise o a e al.
14
Fig. 2. Gene al iew o he Halobio us c ispae indica ing ex e nal sexual dimo phism. A–E — emale wi h
eggs and diamond-shaped cloaca; F–J — p esumable males wi h closed o al cloaca. A–C — LM; D–J —
SEM. A — emale o Halobio us c ispae, habi us, la e al iew; B — pa o he gonad wi h eggs; C — cloaca
opening wi h a cu icula hickening (in a ci cle); D — emale, habi us, en al iew; E — he ul as uc u e
o he emale cloaca iangula pla es om he ig. D; F — specimen 1 wi h he o al cloaca (a owhead);
p esumable male, habi us, la e al iew; G — he ul as uc u e o he o al-shaped cloaca pla es om he
ig. F; H — specimen 2 wi h o al cloaca, en al iew; a owheads indica ing pai ed en al in e media e
a achmen si es “g” o skele al muscles; I — specimen 2 wi h he o al cloaca, habi us, an e io en al iew;
J — he senso y papilla loca ed nea he an e io pla e o he o al cloaca (a owhead).
Abb e ia ions: go — gonad; oCP — o al cloacal pla es; CP — iangula cloacal pla es.
Do eu a dig ades ha e a copula o y o gan? 15
Fig. 3. The s uc u e o he male cloacal o gan o Halobio us c ispae, SEM. А — gene al iew o he male
wi h a p o uding cloacal o gan (a ow); la e al iew, le side; B — he caudal end o he specimen on ig. A
wi h 4 h pai o legs and he pulled cloacal o gan (a ow); C–D — de ails o he male cloacal o gan in di e en
p ojec ions; showing he basal olds (a ows) and ejacula ed ma e ial a ound he opening; E — he 3 d pai o
legs o he specimen on ig. A showing double claws, p ima y b anches wi h accesso y poin s and wo si es o
he muscle a achmen (*) on he leg; F — he second specimen, habi us, an e io– en al iew; G — pa ially
p o uded cloacal o gan o he specimen om F (in ame) wi h h ee cloacal pla es a he base (a owheads).
Abb e ia ions: ex — ejacula ed ma e ial; o — ejacula o y canal opening.
N.M. Bise o a e al.
16
Fig. 4. The s uc u e o he male gonad Halobio us c ispae; immunocy ochemical s aining, CLSM; DAPI in
blue colo ; α-Tub in b igh g een; au o luo escence in o he colo s. A — gene al iew o a male wi h he gonad,
la e al iew, an e io end acing igh , LM; B — gene al iew o he isola ed gonad; wo seminal esicles a e
isible enci cling he midgu , la e al iew; C–D — pos e io pa o he male body; he igh (C) and le (D)
usi o m seminal esicles a e shown, one do sal and a pai o en al Malpighian ubules, pa o he midgu
and pa ially p o uded pa o he cloaca (a owheads) wi h α-Tub-IR in he wall (as e isk); E — muscle
bundles (au o luo escence) suppo ing he seminal esicles; F — he pos e io end o he body wi h he 4 h
pai o legs and pa ially ex ended cloacal o gan (a owhead), en al iew; seminal esicle, Malpighian
ubule and α-Tub-IR in he wall o cloacal cylinde (as e isk) a e indica ed; G — α-Tub-IR senso y ending in
he 4 h leg (a ow) and pa ially ex ended cloacal cylinde (a owhead) wi h α-Tub-IR in he wall (as e isk).
Abb e ia ions: go — gonad; m — muscle ibe ; mg — midgu ; m — Malpighian ubules; s — seminal esicle; 4 h —
he 4 h pai o legs.
