42 Anna DymekJakub Dymek

(1)

.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018

streszczenie

C

elem artykułu jest opisanie bu- dowy histologicznej spermateki traszki karpackiej (Lissotriton montandoni) w mikros- kopie świetlnym oraz porównanie z danymi dotyczącymi blisko spokrewnionej traszki zwyczajnej (L. vulgaris). Spermateka traszki karpackiej jest typu prostego, co oznacza, że składa się z licznych kanalików uchodzących do kloaki niezależnie od siebie. Kanaliki spermateki są wyścielone nabłonkiem jednowarst- wowym, natomiast kloaka nabłonkiem walcow- atym. W nabłonku są obecne liczne komórki wydzielnicze. Pomiędzy kanalikami spermateki występuje tkanka łączna zawierająca melano- fory. Struktura spermateki traszki karpackiej nie odbiega znacząco od struktury opisywanej u innych gatunków należących do rodziny Salamandridae; najwięcej podobieństw wyka- zuje do blisko spokrewnionej traszki zwyczajnej. Najistotniejszą różnicą pomiędzy L. montandoni i L. vulgaris jest odmienny skład wydzieliny komórek nabłonkowych, co może wpływać na obniżony sukces rozrodczy w przypadku kojarzeń międzygatunkowych.

abstract

T

his paper aimed to describe the histology of the Carpathian newt (Lissotriton montandoni) spermatheca by light microscopy, making comparisons with related species, es- pecially its sister species, the smooth newt (L.

vulgaris). The spermatheca of the Carpathian newt is of a simple type, hence it consists of numerous spermathecal tubules opening independently to the cloacal chamber. The tubules are lined with cuboidal epithelium, while the inner walls of the cloaca are lined with columnar epithelium. Numerous mucus-producing glandular cells are present in the epithelium.

The space between the tubules of the spermatheca is filled with connective tissue and contains melanophores. It was found that the structure of the Carpathian newt spermatheca does not differ much from those of other closely related Salamandridae family members.

Unsurprisingly, among the studied species, the spermatheca anatomy of the Carpathian newt was most similar to that of the closely related smooth newt. However, differences were found between these two species, including differences in the composition of the mucus secreted by the spermatheca tubules. This phenomenon could potentially affect the lower interspecies mating success between the Carpathian and

Anna Dymek Jakub Dymek

Department of Comparative Anatomy

Institute of Zoology and Biomedical Research Faculty of Biology

Jagiellonian University

ul. Gronostajowa 9, 30-387 Kraków e-mail: anna.tyrkalska@doctoral.uj.edu.pl

H i s to l o g y o f t H e C a r pat H i a n n e w t ( lissotriton montandoni )

(s a l a m a n d r i d a e, U r o d e l a) s p e r m at H e C a

Budowa histologiczna spermateki traszki karpackiej,

Lissotriton Montandoni

(Salamandridae, Urodela)

s

łowa kluczowe: rozród płazów ogonias- tych, kloaka, spermateka, Urodela, Lissotri- ton.

(2)

Introduction

T

he majority of species belonging to Urodela exhibit internal fertilization, although males do not possess copulatory organs and insemination is indirect. The males deposit packs of sperm, called spermatophores, which are collected by the females with their cloacal lips and stored in a special structure, the spermatheca (SEVER, 1992; PECIO AND RAFIŃSKI, 1985; SEVER, 2003). Only members of three primitive families, Sirenidae, Cryptobranchidae and Hynobiidae, lack this anatomical structure, instead exhibiting external fertilization (SEV- ER, 1991).

Two spermathecal types can be distin- guished in Urodela. The simple spermathecal type consists of numerous tubular glands, individually opening into the roof of the cloaca, and occurs in most Salamandroidea. The complex spermathecal type consists of tubular glands with distal bulbs opening to the common tube, connecting them with the cloacal chamber (e.g., in Plethodontidae) (SEVER, 2002).

SEVER ET AL. (1999, 2001) described the ultrastructural details of the spermatheca of the smooth newt, Lissotriton vulgaris (formerly Triturus vulgaris). This species is closely related to the Carpathian newt Lissotriton montandoni. L. vulgaris is widespread through most of Europe, while L. montandoni is endemic to the Carpathians and small part of the Sudetes Mountains. In areas where ranges of both species overlap, they easily hybridize, giving fertile offspring (BABIK ET AL., 2003). Despite the potential for hybridization, substantial pre-zy- gotic sexual isolation between these species is observed at the stage of courtship, most prob- ably emerging from differences in secondary

sexual traits, such as the differentiation of male peptide pheromones (MICHALAK ET AL., 1997; OSIKOWSKI, 2012). Other research (MICHALAK AND RAFIŃSKI, 1999) sug- gests that physiological and anatomical differences may account for the lower rates of successful sperm storage and fertilization in heterospecific matings. Thus, knowledge of the anatomy of female sperm storage organs of both species is crucial.

In this paper, we aimed to describe the anatomy of the Carpathian newt spermatheca by light microscopy and describe any interspecies variations between L. vulgaris and L.

montandoni.

Hi s t o l o g y o f t H e Ca r p a t H i a n n e w t (lissot r iton montandoni)

(salamandr idae, U^rodela) s p e r m a t H e C a s^tr. 42 - 49

K

^{ey words:} reproduction of salamanders, female cloaca, spermatheca, Urodela, Lisso- triton.

Materials and methods

T

he research was conducted on two mature females of L. montandoni with total body lengths of 86 and 93 mm. The animals were collected from the land during the beginning of the spring migration to the breeding pond. They were sacrificed by immersion in 0.1% MS-222 solution (Sigma®, St. Louis, MO, USA). The entire cloacal region was pre- served in Bouin fixative for 2 weeks, refresh- ing the solution every 3 days, before washing and storing in 75% ethanol. Subsequently, fixed cloacal regions were dehydrated in a graded se- ries of ethanol (70%, 96%, 100%), cleared with toluene and embedded in the tissue embed- ding medium Paraplast® Regular (Sigma®, St.

Louis, MO, USA). The material was then seri- ally sectioned (6 µm) on a microtome and af- fixed to glass slides. Selected slides were stained with hematoxylin and eosin (HE), Mallory’s trichrome, Passini stain and Alcian Blue and Periodic Acid Schiff (pH 2,5) stain. The slides were examined under a light microscope. The number of spermathecal tubules was counted in selected sections. All measurements were made using ImageJ software. The number of the tubules was counted in each section, as was

(3)

.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018 the number of openings into the cloaca. The

length (in µm) of the tubules was estimated by counting the sections in which the tubule was present. The slides were photographed using a Nikon COOLPIX P6000 camera. For each in- dividual, approximately 100 slides representing the entire cloacal region were prepared.

Results F

emale cloaca morphology

Both examined females lacked sperm in the spermathecal tubules. The spermatheca was of a simple type, consisting of spermathecal tubules opening independently to the walls of the cloaca (Fig. 1A-D).

The lumen of the cloacal tube is star- like in the cross-section (Fig. 1D), while in the more caudal part of the cloacal chamber the lumen is Y-shaped (Fig. 1B). In the cloacal tube two conspicuous folds (mid-dorsal and mid- ventral) project into the lumen and form a kind of partition (Fig. 1C). The mid-dorsal fold is present along the cloaca, while the mid-ventral fold occurs only in the cloacal tube and it is absent in the cloacal orifice (Fig. 1A-D).

F

emale spermatheca

Cross-sections of the spermatheca tubules revealed a round lumen (Fig. 1 A-D) that was typically filled with mucus connected with desquamation epithelium. Both studied females possessed 40–45 tubules in the spermatheca. The tubules were arranged unevenly between the right and left body part. Most of the cross-sectioned tubules and openings of tubules were located in the central region of the cloaca (Fig. 1B, C), while few were detected in the anterior and posterior regions (Fig. 1A, D).

The proximal part of every tubule was straight (Fig. 1B), while the distal part was sinuous (Fig.

1C). The tubule lengths ranged from 100–300 µm. The diameter of the lumen of a single tu-

ings were located perpendicular to the cloacal lumen (Fig. 1B, C). Tubules in the cephalic part of the cloaca were arranged in the forward di- rection, while tubules in the caudal part were arranged towards the back. Tubules in the central part of the cloaca were arranged in both directions.

The spermatheca tubules were lined with cuboidal epithelium, while the walls of the cloaca were lined with columnar epithelium (central part) and stratified (the most anterior and the most posterior part) (Fig. 2A). Cells of the spermatheca epithelium had large irregular- shaped nuclei and little cytoplasm. Those cells did not possess cilia. The epithelium cells of the cloaca possessed small nuclei with abundant cytoplasm (Fig. 2A).

Additionally, big, round mucus glandular cells with small nuclei and large cytoplasm were observed in the basis of the epithelium (Fig. 2A). Mucus covered the walls of the cloaca and formed clusters in the lumen of the cloaca (Fig. 2B). Additionally, mucus was connected with the desquamation epithelium cells (Fig.

2B). The secretory activity of the epithelium lining the inner cloaca walls was uniform. The mucus stained positive for carboxylated glycosaminoglycans (AB+) and negative for neutral carbohydrates (PAS-) (Fig. 2B).

The spaces between the tubules of the spermatheca were filled with connective tissue consisting of collagen fibers (Fig. 2C). In the connective tissue, numerous blood vessels and pigment cells (melanophores with granules of melanin) were observed (Fig. 2C).

Discussion

T

he data gathered provides the first anatomical description of L. montandoni spermatheca. As in other species of Salamandridae e.g. Lissotriton vulgaris, L. helveticus, Sala- mandrina terdigitata), this species possesses

(4)

F

ig. 1. Cloaca and spermatheca of the Carpathian newt from the most posterior (A) to most anterior (D) part. Hematoxylin and Eosin staining.

Abbreviations: cc: cloacal chamber; co: connective tissue; ct: cloacal tube; ds: distal part of spermathecal tubule; ep: epidermis; f: fold; mel: melanophore; ps: proximal part of sperma- thecal tubule; s: spermatheca; sm: smooth muscles. Scale bar: 200 µm.

(5)

F

ig. 2. Histological structure of the cloaca and the spermatheca of the Carpathian newt. (A): Tu- bules of spermatheca; (B): Secretions of the cloaca; (C): Connective tissue and the tubules of spermatheca. (A, C): Mallory`s trichrome staining; (B): Alcian blue and PAS staining. Abbreviations:

bv: blood vessel; cf: collagen fibers; co: connective tissue; ct: cloacal tube; e: epithelium; gc: glandular cell; lu: lumen of the spermathecal tubules; mel: melanophore; mu: mucus; s: spermatheca. Scale bar:

(A): 20 µm; (B): 50 µm; (C): 40 µm.

numerous spermathecal tubules opening independently into the roof of the posterior cloacal tube and the cloacal chamber (VERREL AND SEVER, 1988; BRIZZI AL., 1989, 2004).

The overall shape of the cloaca is similar to other related species studied to date. In the

cloacal tube, two conspicuous folds (mid-dorsal and mid-ventral) project into the lumen and form a kind of partition. The mid-dorsal fold is present along the cloaca, while the mid-ventral fold occurs only in the cloacal tube and is absent in the cloacal orifice. These findings are

(6)

similar to those described for Lissotriton helveticus. Folds in the cloaca can act as a kind of partition and help regulate the distribution of sperm in the spermatheca (BRIZZI ET AL., 2004). No ciliated epithelium was found in the cloaca of the Carpathian newt. This is unsurprising, since this trait seems to be an ancestral condition in salamandrids and has been lost in majority of genera, including all studied Lis- sotriton members (SEVER, 1991) .

The tubules of spermatheca are elongat- ed. The proximal part of the tubule is straight, while the distal part is sinuous. The straight part may facilitate the entry of sperm to the tubule and the sinuous part can increase sperm storage space.

Openings of the tubules are located perpendicular to the cloaca. Tubules in the cephalic, caudal and central parts of the cloaca are arranged in forward, reverse and bidirection- ally respectively, allowing a maximal number of spermathecal tubules within the animal body.

Both examined females lacked sperm in the spermatheca. This is unsurprising as the newts were collected at the beginning of the breeding season while immigrating to the breeding ponds. Overwinter sperm storage has not been reported in members of Lissotriton, including L. vulgaris (VERRELL AND SEVER, 1988).

In addition, females newts collected in spring during migration to breeding ponds and kept in water within a laboratory do not lay eggs, e.g. L. montandoni, L. vulgaris, Ichtyosaura alpestris (OSIKOWSKI AND RAFIŃSKI, 2001;OSIKOWSKI, 2007; PECIO, 1992).

Spermatheca is not the only female gland present in Urodele species. Dorsal glands can be found in members of Ambystomatidae and some species belonging to Plethodonti- dae. Female vent glands were also reported in some salamandrids, e.g. Pleurodeles waltl, Ty- lotriton verrucosus (SEVER, 1991) and Lisso- triton (Triturus) helveticus (BRIZZI ET AL., 2004). None of these additional glands were

found in this research, suggesting that they are absent in L. montandoni. Female vent glands were also absent from L. vulgaris (VERELL AND SEVER, 1988), confirming the hypoth- esis of SEVER (1991) that the female ventral glands are symplesiomorphic in urodeles and their absence is due to secondary loss. Dorsal and ventral glands are present in male newts, being found in both L. vulgaris (SEVER ET AL., 1990) and L. montandoni (OSIKOWSKI AND CIERNIAK-ZUZIA, 2013).

The spermatheca of the investigated mature L. montandoni females consisted of 40–55 tubules, which is similar to the results of VERRELL AND SEVER (1988) for L. vulgaris (40–60 tubules). Interestingly, the number of spermathecal tubules in L. helveticus females is much smaller and does not exceed 10 (BRIZZI ET AL., 2004). Generally, there are greater differences in cloacal anatomy between L. montandoni and L. helveticus than between L. montandoni and L. vulgaris.

Members of the family Salamandridae, despite having the spermatheca of the same, simple type, exhibit some differences in anatomy and morphology of this organ (SEVER, 2002). The spermathecas of L. montandoni are generally similar to those of L. vulgaris. Se- cretory activity is uniform, with an apocrine mode of secretion. Additionally, desquamation of the epithelium into the lumen occurs. These similarities are indicative of the close phyloge- netic relationship between the species of this genus. Despite the many similarities, there are differences between these two species, including in the composition of the secreted mucus. The mucus of the Carpathian newt stains positively for carboxylated glycosaminoglycans (AB+) but lacks neutral carbohydrates (PAS-), which is reversed in smooth newt mucus (AB-, PAS+) (SEVER, 2002). This difference may be significant as the composition of mucus is typically similar to the composition of the sperm cap (SEVER AND SIEGEL, 2006). Thus, the

Histology of the Carpathian newt (Lissotriton montandoni)

(Salamandridae, Urodela) spermatheca S^tr. 42 - 49

(7)

.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018 WWW.NAUKOWCY.ORG.PL 2 (20)/2018 mucus composition could act as a natural phys-

iological barrier between the Carpathian and the smooth newts (MiChalak ET al., 1997).

The presence of female sperm storage and common polyandry in newts (BiRkhEaD aND MØllER, 1998; OSikOWSki aND RaFiŃSki, 2001; OSikOWSki, 2003) cre- ate conditions for the occurrence of sperm competition and cryptic female choice (BiRk- hEaD aND MØllER, 1998). The simple type spermatheca provide an opportunity to divide sperm from various males. in such a sit- uation, sperm from the first insemination may fill the most posterior glands, with sperm from the next mating filling more anterior glands.

Eggs, when passing from the oviduct through the cloaca, first meet sperm from the last male.

This phenomenon could lead to the last male advantage in progeny of polyandrous females, as reported by SEVER (2002).

acknowledgments

W

e would like to thank a. Osikowski for useful comments on the manuscript and D.

Podkowa for her help in making glass slides.

The work was partly supported by PhD Stu- dents Society of Jagiellonian University for anna Dymek.

References

BaBik W., SzyMURa J. M., RaFiŃSki J. 2003. Nuclear markers, mitochondrial DNa and male secondary traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni). Molecular Ecology. 12, 1913–1930.

BiRkhEaD T. R., MØllER a. P. 1998.

Sperm Competition and Sexual Selection. ac- ademic Press. Uk.

BRizzi R., DElFiNO G., CallONi C.

1989. Female cloacal anatomy in the spectacled salamander, Salamandrina terdigitata (amphibia: Salamandridae). herpetologica. 45, 310–322.

BRizzi R., DElFiNO G., TaNTERi G.

2004. Cloacal anatomy of the palmate newt, Triturus helveticus (amphibia, Salamandri- dae). amphibia-Reptilia. 25, 233–245.

MiChalak P., GRzESik J., RaFiŃSki J. 1997. Tests for sexual incompatibility between two newt species, Triturus vulgaris and T. montandoni: No choice mating design. Evo- lution. 51, 2045–2050.

MiChalak P., RaFiŃSki J. 1999. Sexual isolation between two newt species, Triturus vulgaris and T. montandoni (amphibia, Uro- dela, Salamandridae). Biological Journal of linnean Society. 67(3), 343–352.

OSikOWSki a. 2003. Biologiczne konse- kwencje poliandrii u samic traszek z rodzaju Triturus (Biological consequences of polyandry in female newts of genus Triturus). PhD The- sis. kraków. Poland.

OSikOWSki a. 2007. Sperm transport after insemination in the alpine newt (Triturus alpestris, Caudata, Salamandridae). Folia bio- logica. krakow. 55, 109–114.

OSikOWSki a. 2012. asymmetric female preferences for courtship pheromones in two closely-related newt species, the smooth newt (lissotriton vulgaris) and the Carpathian newt (l. montandoni) (Salamandridae). zoological Science. 29(6), 390–395.

OSikOWSki a., CiERNiak-zUzia k.

2013. Cloacal anatomy of the male Carpath- ian newt, lissotriton montandoni (amphibia, Salamandridae), in the breeding season. zoo-

(8)

logical Science. 30(9), 748–753.

OSikOwSki A., RAfińSki J. 2001. Mul- tiple insemination increases reproductive success of female Montandon’s newt (Triturus montandoni, Caudata, Salamandridae^{). Behav-} ioral Ecology and Sociobiology. 49, 145–149.

PECiO A. 1992. insemination and egg laying dynamics in the smooth newt, Triturus vulgaris, in the laboratory. Journal of Herpetology. 2, 5–7.

PECiO A., RAfińSki J. 1985. Sexual be- haviour of the Montandon’s newt, Triturus montandoni (Boulenger) (Caudata: Salaman- dridae). Amphibia-Reptilia. 6, 11–22.

SEvER D. M. 1991. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata i).i. Evolution at the family level. Herpetologica. 47, 165–193.

SEvER D. M. 1992. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). iv. Salamandridae. The Anatomical Records. 232, 229–244.

SEvER D. M. 2002. female sperm storage in amphibians. Journal of Experimental Zoology.

292, 165–179.

SEvER D. M. 2003. Reproductive Biology and Phylogeny of Urodela. Science Publishers, Enfield, NH.

SEvER D. M., HAlliDAy T., MORiAR- Ty E. C., ARAñO B. 2001. Sperm storage in females of the smooth newt (Triturus v. vulgaris l.): ii. Ultrastructure of the spermathecae after the breeding season. Acta Zoologica.

82(1), 49–56.

SEvER D. M., HAlliDAy T., wAigHTS

v., BROwN J., DAviES H. A., MORiAR- Ty E. C. 1999. Sperm storage in females of the smooth newt (Triturus v. vulgaris l.): i.

Ultrastructure of the spermathecae during the breeding season. Journal of Experimental Zo- ology. 283, 51–70.

SEvER D. M., SiEgEl D. S. 2006. Sperm aggregations in the spermatheca of the red back salamander (Plethodon cinereus). Acta Zoologica. 87, 331–340.

SEvER D. M., vERREll P. A., HAlli- DAy T. R., gRiffiTHS M., wAigHTS v.

1990. The cloaca and cloacal glands of the male smooth newt, Triturus vulgaris vulgaris (lin- naeus), with especial emphasis on the dorsal gland. Herpetologica. 46(2), 160–168.

vERREll P. A., SEvER D. M. 1988. The cloaca and spermatheca of the female smooth newt, Triturus vulgaris l. (Amphibia: Urodela:

Salamandridae). Acta Zoologica. 69, 65–70.