STR. 42 - 49 Anna Dymek (20)/2018 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: [email protected] HISTOLOGY OF T H E C A R PAT H I A N N E W T (L ISSOTRITON MONTANDONI) (S ALAMANDRIDAE, URODELA) SPERMAT H E C A Budowa histologiczna spermateki traszki karpackiej, Lissotriton Montandoni (Salamandridae, Urodela) streszczenie abstract Celem artykułu jest opisanie bu- This paper aimed to describe the his- (20)/2018 WWW.NAUKOWCY.ORG.PL 2 dowy histologicznej spermateki traszki kar- tology of the Carpathian newt (Lissotriton packiej (Lissotriton montandoni) w mikros- montandoni) spermatheca by light microscopy, kopie świetlnym oraz porównanie z danymi making comparisons with related species, es- dotyczącymi blisko spokrewnionej traszki pecially its sister species, the smooth newt (L. zwyczajnej (L. vulgaris). Spermateka traszki vulgaris). The spermatheca of the Carpathian karpackiej jest typu prostego, co oznacza, że newt is of a simple type, hence it consists of składa się z licznych kanalików uchodzących numerous spermathecal tubules opening inde- do kloaki niezależnie od siebie. Kanaliki sper- pendently to the cloacal chamber. The tubules mateki są wyścielone nabłonkiem jednowarst- are lined with cuboidal epithelium, while the wowym, natomiast kloaka nabłonkiem walcow- inner walls of the cloaca are lined with colum- atym. W nabłonku są obecne liczne komórki nar epithelium. Numerous mucus-producing wydzielnicze. Pomiędzy kanalikami spermateki glandular cells are present in the epithelium. występuje tkanka łączna zawierająca melano- The space between the tubules of the sper- fory. Struktura spermateki traszki karpackiej matheca is filled with connective tissue and nie odbiega znacząco od struktury opisywanej contains melanophores. It was found that the u innych gatunków należących do rodziny structure of the Carpathian newt spermathe- (20)/2018 WWW.NAUKOWCY.ORG.PL 2 Salamandridae; najwięcej podobieństw wyka- ca does not differ much from those of other zuje do blisko spokrewnionej traszki zw- closely related Salamandridae family members. yczajnej. Najistotniejszą różnicą pomiędzy Unsurprisingly, among the studied species, the L. montandoni i L. vulgaris jest odmienny spermatheca anatomy of the Carpathian newt skład wydzieliny komórek nabłonkowych, co was most similar to that of the closely related może wpływać na obniżony sukces rozrodczy smooth newt. However, differences were found w przypadku kojarzeń międzygatunkowych. between these two species, including differ- ences in the composition of the mucus secreted Słowa kluczowe: rozród płazów ogonias- by the spermatheca tubules. This phenomenon tych, kloaka, spermateka, Urodela, Lissotri- could potentially affect the lower interspecies ton. mating success between the Carpathian and smooth newts. WWW.NAUKOWCY.ORG.PL 2 42 NR 2(20)/2018 NAUKI PRZYRODNICZE H ISTOLOGY OF T H E C ARPAT H IAN NEWT (L ISSOTRITON MONTANDONI ) (S ALAMANDRIDAE , U RODELA ) SPERMAT H E C A STR. 42 - 49 Key words: reproduction of salamanders, sexual traits, such as the differentiation of male (20)/2018 female cloaca, spermatheca, Urodela, Lisso- peptide pheromones (MICHALAK ET AL., triton. 1997; OSIKOWSKI, 2012). Other research (MICHALAK AND RAFIŃSKI, 1999) sug- Introduction gests that physiological and anatomical dif- ferences may account for the lower rates of The majority of species belonging to successful sperm storage and fertilization in Urodela exhibit internal fertilization, although heterospecific matings. Thus, knowledge of the males do not possess copulatory organs and in- anatomy of female sperm storage organs of semination is indirect. The males deposit packs both species is crucial. of sperm, called spermatophores, which are col- In this paper, we aimed to describe the lected by the females with their cloacal lips and anatomy of the Carpathian newt spermatheca stored in a special structure, the spermatheca by light microscopy and describe any inter- (SEVER, 1992; PECIO AND RAFIŃSKI, species variations between L. vulgaris and L. 1985; SEVER, 2003). Only members of three montandoni. primitive families, Sirenidae, Cryptobranchidae and Hynobiidae, lack this anatomical structure, (20)/2018 WWW.NAUKOWCY.ORG.PL 2 Materials and methods instead exhibiting external fertilization (SEV- ER, 1991). The research was conducted on two Two spermathecal types can be distin- mature females of L. montandoni with total guished in Urodela. The simple spermathe- body lengths of 86 and 93 mm. The animals cal type consists of numerous tubular glands, were collected from the land during the be- individually opening into the roof of the clo- ginning of the spring migration to the breed- aca, and occurs in most Salamandroidea. The ing pond. They were sacrificed by immersion complex spermathecal type consists of tubular in 0.1% MS-222 solution (Sigma®, St. Louis, glands with distal bulbs opening to the com- MO, USA). The entire cloacal region was pre- mon tube, connecting them with the cloacal served in Bouin fixative for 2 weeks, refresh- chamber (e.g., in Plethodontidae) (SEVER, ing the solution every 3 days, before washing 2002). and storing in 75% ethanol. Subsequently, fixed SEVER ET AL. (1999, 2001) described cloacal regions were dehydrated in a graded se- the ultrastructural details of the spermatheca of ries of ethanol (70%, 96%, 100%), cleared with the smooth newt, Lissotriton vulgaris (formerly toluene and embedded in the tissue embed- Triturus vulgaris). This species is closely related ding medium Paraplast® Regular (Sigma®, St. (20)/2018 WWW.NAUKOWCY.ORG.PL 2 to the Carpathian newt Lissotriton montan- Louis, MO, USA). The material was then seri- doni. L. vulgaris is widespread through most ally sectioned (6 µm) on a microtome and af- of Europe, while L. montandoni is endemic to fixed to glass slides. Selected slides were stained the Carpathians and small part of the Sudetes with hematoxylin and eosin (HE), Mallory’s Mountains. In areas where ranges of both spe- trichrome, Passini stain and Alcian Blue and cies overlap, they easily hybridize, giving fertile Periodic Acid Schiff (pH 2,5) stain. The slides offspring (BABIK ET AL., 2003). Despite the were examined under a light microscope. The potential for hybridization, substantial pre-zy- number of spermathecal tubules was counted gotic sexual isolation between these species is in selected sections. All measurements were observed at the stage of courtship, most prob- made using ImageJ software. The number of ably emerging from differences in secondary the tubules was counted in each section, as was WWW.NAUKOWCY.ORG.PL 2 NR 2(20)/2018 NAUKI PRZYRODNICZE 43 Anna Dymek, Jakub Dymek STR. 42 - 49 the number of openings into the cloaca. The ings were located perpendicular to the cloacal length (in µm) of the tubules was estimated by lumen (Fig. 1B, C). Tubules in the cephalic part (20)/2018 counting the sections in which the tubule was of the cloaca were arranged in the forward di- present. The slides were photographed using a rection, while tubules in the caudal part were Nikon COOLPIX P6000 camera. For each in- arranged towards the back. Tubules in the cen- dividual, approximately 100 slides representing tral part of the cloaca were arranged in both the entire cloacal region were prepared. directions. The spermatheca tubules were lined with cuboidal epithelium, while the walls of the clo- Results aca were lined with columnar epithelium (cen- Female cloaca morphology tral part) and stratified (the most anterior and Both examined females lacked sperm in the most posterior part) (Fig. 2A). Cells of the the spermathecal tubules. The spermatheca was spermatheca epithelium had large irregular- of a simple type, consisting of spermathecal tu- shaped nuclei and little cytoplasm. Those cells bules opening independently to the walls of the did not possess cilia. The epithelium cells of the cloaca (Fig. 1A-D). cloaca possessed small nuclei with abundant The lumen of the cloacal tube is star- cytoplasm (Fig. 2A). (20)/2018 WWW.NAUKOWCY.ORG.PL 2 like in the cross-section (Fig. 1D), while in the Additionally, big, round mucus glandu- more caudal part of the cloacal chamber the lu- lar cells with small nuclei and large cytoplasm men is Y-shaped (Fig. 1B). In the cloacal tube were observed in the basis of the epithelium two conspicuous folds (mid-dorsal and mid- (Fig. 2A). Mucus covered the walls of the cloaca ventral) project into the lumen and form a kind and formed clusters in the lumen of the cloaca of partition (Fig. 1C). The mid-dorsal fold is (Fig. 2B). Additionally, mucus was connected present along the cloaca, while the mid-ventral with the desquamation epithelium cells (Fig. fold occurs only in the cloacal tube and it is 2B). The secretory activity of the epithelium absent in the cloacal orifice (Fig. 1A-D). lining the inner cloaca walls was uniform. The mucus stained positive for carboxylated glycos- aminoglycans (AB+) and negative for neutral emale spermatheca F carbohydrates (PAS-) (Fig. 2B). Cross-sections of the spermatheca tu- The spaces between the tubules of the bules revealed a round lumen (Fig. 1 A-D) spermatheca were filled with connective tissue that was typically filled with mucus connected consisting of collagen fibers (Fig. 2C). In the with desquamation epithelium. Both studied connective tissue, numerous blood vessels and (20)/2018 WWW.NAUKOWCY.ORG.PL 2 females possessed 40–45 tubules in the sper- pigment cells (melanophores with granules of matheca. The tubules were arranged unevenly melanin) were observed (Fig. 2C). between the right and left body part. Most of the cross-sectioned tubules and openings of tu- bules were located in the central region of the cloaca (Fig. 1B, C), while few were detected in Discussion the anterior and posterior regions (Fig. 1A, D). The data gathered provides the first The proximal part of every tubule was straight anatomical description of L. montandoni sper- (Fig. 1B), while the distal part was sinuous (Fig. matheca. As in other species of Salamandridae 1C). The tubule lengths ranged from 100–300 e.g.