Squalius platyceps (Actinopterygii: Cyprinidae) from skadar

International Journal of Biosciences IJB ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 11, p. 9-18, 2015 RESEARCH PAPER OPEN ACCESS Female reproductive cycle phases (oogenesis) of white chub - Squalius platyceps (Actinopterygii: Cyprinidae) from skadar lake (Montenegro) Marijana Krivokapic Faculty of Natural Sciences and Mathematics, Department for Biology, Dzordza Vasingtona, b.b., Podgorica 81000, Montenegro Key words: Female reproductive cycle, oogenesis, Squalius platyceps, Skadar Lake. http://dx.doi.org/10.12692/ijb/6.11.9-18 Article published on June 08, 2015 Abstract Female reproductive phases of Squalius platyceps from Skadar Lake, Montenegro are differentiated on the base of both, macroscopic observations and histological analyses of gonads. Temporal aspect of the maturity phases of male reproductive cycle are presented along with obtained data on oocytes size, as well. Spawning of White Chub from Skadar Lake starts by the end of March and last until mid of June. Research data for Squalius platyceps, as a newly recognized species, are discussed in the relation to completion all data for Squalius platyceps as a newly recognized species. * Corresponding Author: Marijana Krivokapic marijanak2011@gmail.com 9 Krivokapic

Introduction There is only one work for female reproductive cycle - oogenesis of White Chub - Squalius platyceps (Leuciscus cephalus albus, Bonaparte, 1838.) from the Skadar Lake (Sekulovic and Ivanovic 1969), so far. In this work oogenesis was differentiated on the base of macroscopic analyses only, so histological analyses of the oogenesis have not been done for this newly recognized species, so far. Leuciscus cephalus albus from Skadar Lake and Drin river basins is recognized as new species Squalius platyceps. This species is distinguished from other species of the genus Squalius in the Adriatic basin (Zupancic et al, 2010). In general, studies on gametogenesis in cyprinids started long time ago (Kulev, 1939., Mejen 1939. etc). Research on gametogenesis of fish species from Skadar lake started Ivanovic, in 1968 who analyzed reproductive cycle for many species and subspecies of fish over the time. His and other author researches of oogenesis were done for following species / subspecies from Skadar Lake: Alburnus albidus alborella, Alosa falax nilotica, Carassius auratus gibelio, Chondrostoma nasus ohridanus, Cyprinus carpio, Pachychilon pictum, Rutilius rubilio and Scardinius erythrophthalmus scardafa (Ivanovic, 1964, 1968, 1969, 1977, Sekulovic & Ivanovic 1969, 1971, Ivanovic & Knezevic 1975, Knezevic 1984, Soric 1979, 1984, Maric 1998). White Chub in Skadar Lake is a batch spawning species. In spawning period it gather in groups - smaller shoals at following locations in the north-west part of the Lake: Vucko Blato, Ploce and wider zone around islands Kamenik and Cakovica extended to Bjelak and Poseljani (Fig. 1.). Colour of fish is changing to more intensive nuances in the spawning period. More dark nuances can be seen on dorsal side that is dark olive colour as well as on lateral side with more intensive yellow line. Materials and methods Female reproductive cycle of White Chub from Skadar Lake is analyzed in the frame of reproductive study that author carried out on 205 female samples that are collected on following localities: (i) zone around islands Cakovica and Kamenik (Fig 2.), (ii) Vucko blato in front of Vranjina (Fig. 3.), (iii) Poseljani (Fig. 4) and (iv) shoreline zone of Ploca (Fig. 5.). Samples from sublacustrin springs (Radus, Bazagur, Karuc etc) were not collected because of different physical, chemical and biological conditions of their waters that are quiet different to other waters of the Lake. Overall results from the study are presented in authors doctoral thesis (Krivokapic, 2002). Fig. 1. Sampling locations in north-west part of Skadar Lake. 10 Krivokapic

Macroscopic and histological features of the phases of female reproductive cycle are basically described and determined on the base of Kiseljevic scale (Sekulic and Buckaja, 1963; Jovanovic Krsljanin, 1982, Jankovic, 1985) and then terminologically standardized in the reproductive phases given by Brown - Peterson (2011). this phase. Beside oogonias, primary and secondary oocytes (stages I and II) are present. Since data on oogenesis of White Chub Squalius platyceps from other locations in the basins of Skadar, Drin and Adriatic are missing, there was no possibility to compare data that are presented in this paper. Results Female reproductive cycle of White Chub from Skadar Lake (oogenesis) is firstly differentiated on six maturity phases that are described and determined on the base of macroscopic and histological features characteristic for the phases in Kiseljevic scale: I phase Designate juvenile, sexually immature fish. Ovaries are in the form of thin bands with no differences to testes. This phase characterize presence of oogonias in marginal zones of ovarian lobules as well as primary growth of young oocytes in the period of protoplasmic growth. Fig. 3. Zone of Vucko blato in front of Vranjina (Photo V. Buskovic). III phase Maturing begins and size of ovaries is suddenly increased. Roe is yellow colour and easy visible. Active growth of oocytes and their preparation for transformation in morphologically differentiated larger cells is characteristic for this phase. Oocytes are mostly in the process of vitellogenesis i.e. trophoplasmic growth. In the ovaries are also present cells of so-called reserve stocks of oogonias and oocytes that are in the process of pre-vitellogenesis, i.e. protoplasmic growth. Fig. 4. Locality Poseljani (Photo V. Buskovic). Fig. 2. Zone around island Kamenik and Cakovica (Photo V. Buskovic). II phase Ovaries are still small, transparent and pale. Blood vessel can be seen along the ovaries. Further development of sexual elements is characteristic for IV phase Females are sexually mature. Ovaries are large and occupy most of abdominal cavity. Maturing of the roe is finished. Roe has maximum size and round shape. In the ovaries are present numerous large size oocytes that are morphologically completely differentiated and completed trophoplasmic growth. Still are present oogonias and oocytes that are in the process 11 Krivokapic

of protoplasmic growth. Phases in the reproductive cycle of female White Chub from Skadar Lake are also differentiated from maturity and temporal (seasonal) aspects accommodated to newly standardized terminology (Brown - Peterson, 2011), as follows: Fig. 5. Shoreline zone of Ploca (Photo V. Buskovic). V phase Is also named as running since gentle pressure on gonads zone of abdomen causing roe run out. In ovaries are present scattered oogonias and oocytes in younger stages of their development (reserve stocks). VI phase: comes after spawning phase in which roe is released. Ovaries missed their previous large size and they are soft and purple red in colour due to small blood leak (haemorrhages). Presence of flaccid postovulatory follicles is characteristic for this phase. Process of the resorption of unreleased roe is followed by the process of restoration of ovaries. Younger oocytes are still in the development while white granule of new roe are visible. After a shorter period of time female gonads are turned to the maturity phase III that is also named as VI III. Fig. 7a. Histology cut of Skadar Chub gonad, April (spring period) individual of 5 + age group (en 100x). A. Maturity phases I. Immature phase: correspond to Kiseljevic I phase and designate juvenile, sexually immature - never spawned fish, ovaries are small and thin, in them are present oogonias. Fig. 7b. Histology cut of Skadar Chub gonad, April (spring period) individual of 6 + age group (en 100x). Fig. 6. Histology cut of Skadar Chub ovary, February (winter period) sexually immature individual of 1 + age group (en 100x). Short time upon this phase end up, ovaries are turned back to the phase II. II. Developing phase Belong to pre-spawning period and usually start at the beginning of March. Period before spawning depend on abiotic factors such as: sunlight, air pressure, water flow and quality, but primarily it depends on the temperature of waters (Jovanovic Krsljanin, 1982). III. Spawning capable phase 12 Krivokapic

When most favourable conditions presented, spawning can start. According to our data, spawning of White Chub from Skadar Lake is starting by the end of March beginning of April and last approximately 2.5 to 3 months, until first half of June. As a batch spawning species, White Chub release roe in portions. Fig. 7c. Histology cut of Skadar Chub gonad, May (spring period) individual of 7 + age group (en 100x). oocytes and lasts 3 months, in average. B. Temporal phases Winter phase Ovaries are in III phase of maturity, nourishment is decreased, but this is not a period ovaries are not active. Macroscopic analyses of the ovaries of White Chub from Skadar Lake from December and January confirm they are firm with compacted granules in the roe. Roe is changing its colour from opaque to light yellow. Histological analyses prove presence of oogonias and smaller oocytes. In the samples from February and first half of March roe had light orange colour (Fig. 6.). Coherent to the characteristics of prespawning period, gradual maturing is noted in the histological analyzes, due to presence of numerous large oocytes with increasing number of nucleuses. IV. Regressing phase Is coming after the spawning, as a restoration period that last for two months (July August). This phase is devoted to the resorption of unreleased roe and multiplication of young oocytes. Fig. 7e. Histology cut of Skadar Chub gonad, May (spring period) individual of 8 + age group (en 100x). Fig. 7d. Histology cut of Skadar Chub gonad, May (spring period) individual of 7 + age group (en 100x). IV. Regenerating phase: cover active oogenesis period that starting after the restoration period and it lasts approximately 3 months, in average (September, October, and November). Oocytes are passing differentiation process while ovaries are enlarged in this period. Period of active oogenesis continue to slow oogenesis period (December, January and February) that characterize slow increase of ovaries, poor nourishment and slow moving of the fish. This is also a period of maturing of already differentiated Spring period (Fig. 7.a-f.) - Females are growing intensively. Ovaries are in IV phase of maturity. Active preparation for spawning is based on intensive maturing of ovaries. By the end of March, in the ovaries of IV phase of maturity are present oocytes with the completed trophoplasmic growth. V phase of ovaries maturity take place in April and May when ovaries reach their maximum size. Ovaries are occupying most of abdominal cavity. This phase is named as running since gentle pressure on gonads zone of abdomen causing roe run out. Ovaries have light colours due to homogenization processes in the yolk of oocytes as well as changes in the nucleus (vitellus) and oocytes membrane. Macroscopically, roe is differentiated in 3 categories: (i) large size roe that is ready for first release, yellow orange in colour, situated in ventral 13 Krivokapic

side of ovary, mostly in its back part, while (ii) medium size roe and (iii) small size roe are light pale colour and situated in front part of ovary tissue that holds them grouped (classified). Histologicaly ovaries from this period indicate trophoplasmic growth, i.e. process of vitellogenesis. Comparing to winter period, oocytes are now significantly enlarged. Oogonias are present between oocytes. Among enlarged oocytes are present oocytes with heterogeneous and homogeneous cytoplasm. Imbalanced growth of oocytes cause various conditions for nutritive substances supply that is the reason why this period is assigned for trophoplasmic growth. In oocytes are visible vacuoles, drops of fat and granules of yolk. Process of vitellogenesis is not balanced that cause asynchrony growth and batch, i. e. portion release of roe. Maturing of oocytes is intensive until first spawning, i. e. first portions release of roe. Dimensions of mature roe that is ready for first spawning varying from 0.80 to 1.20 mm. Upon first portion release of roe, ovaries pass to maturity phase VI1 III2 i. e. second maturing phase for new portion of roe which release does not take long time. This phase characterize also presence of empty follicles, oocytes that setting yolk and ready to provide next portion of roe, as well as more young oocytes for next generation and unreleased oocytes in the resorption phase. Ovaries are purple red colour. and length with a shape similar to testes. Histological analyses confirmed presence of mature roe and a small number of large oocytes. In the cut are seen oocytes with homogeneous cytoplasm and a great number of empty follicles that are in the last phase of resorption process of unreleased roe. By the end of June, ovaries are in the post-spawning phase, maturity phase III (transition from VII to III), in most of population of White Chub from Skadar Lake. Ovaries are passing this phase upon resorption of unreleased roe finished. On histological cut of ovaries are also visible oogonias and oocytes with smaller dimensions. Great number of oocytes, with homogeneous cytoplasm dominate in histological cuts from July and beginning of August. Most of ovaries obtain firm structure in August. In summer, upon spawning finished, females of White Chub approach to intensive nourishment that consequently increase ovaries mass and accumulation of nutritive substances in them. This period represents a period of quick growth of oocytes, that is, a period of trophoplasmic growth. Fig. 8a. Histology cut of Skadar Chub gonad, June (summer period) individual of 5 + age group (en 100x). Fig. 7f. Histology cut of Skadar Chub gonad, May (spring period) individual of 9 + age group (en 100x). Summer period (Fig. 8. a-c.) - Encompass post-spawning period with reduced, loose ovaries. Granules of unreleased roe are present In the backside of ovaries. In the individuals which do not have unreleased roe, ovaries are thin Autumn period (Fig. 9. a-b.)-start in September when weight of ovaries is increasing. On histological cut of ovaries are seen numerous oogonias and oocytes in younger maturity phases. Process of resorption is mostly finished and oogenesis is very intensive. Oocytes with homogeneous cytoplasm dominate on histological cut in September while oocytes with heterogeneous cytoplasm dominate on histological cut of ovaries for samples from October. Oocytes in different stages of protoplasmic and trophoplasmic growth with starting 14 Krivokapic

vacuolization and vitellogenesis are stated at the end of October and beginning of November. Increase of the size of oocytes size and more evident vacuolization are seen in November and December. Macroscopically, oogonias and oocytes are stated as well as tiny granules of roe. Maturing cycle in ovaries starts again. elderliness. Slight female domination is noted in the samples from all seasons that support good reproductive balance. According to our macroscopic and histological analyses, spawning of White Chub from Skadar Lake starts by the end of March and last until mid of June. Discussion Previous analyses of the oogenesis of White Chub from Skadar Lake and Drin river basins has been done on the base of macroscopic descriptions, only (Sekulovic and Ivanovic 1969). Fig. 8b. Histology cut of Skadar Chub gonad, July (summer period) individual of 6 + age group (en 100x) Oocytes from all maturity phases were identified in the stages of previtellogenesis and vitellogenesis with different domination throughout maturity phases, respectively. Their size is measured seasonally on smaller number of samples. In winter (February), big oocytes of the individuals of age group 3 + have dimensions (Ob.10 Oc.10) 168.00 197.20 microns. Oocytes in younger phases have dimensions of 39.92 54.00 microns. In spring (April), oocytes of the individuals of age group 3 + have dimensions 161.18 172.30 microns. In summer (July), big oocytes of the individuals of age group 5 + have dimensions 460.80 590.50 microns while small ones have dimensions 40.10 54.00 microns. In autumn (October), big oocytes of the individuals of age group 4 + have dimensions 450.10 530.0 microns while small ones have dimensions 315.20 360.00 microns. Analyses of the oogenesis cycle of White Chub from Skadar Lake confirmed that females become reproductively mature and ready for spawning with first release of roe in the third (2 + ) that is fourth (3 + ) year of their life. Mature reproductive elements are also observed in the individuals in 12 + and 13 + year of live that indicate late arrival of physiologic Fig. 8c. Histology cut of Skadar Chub gonad of sexually immature individual of 1 + age group, August (summer period) (en 100x). Comparable histological analyses of the oogenesis of White Chub - Squalius platyceps from other locations in Skadar Lake are missing, so there was no possibility to compare presented data with adequate ones. Leuciscus cephalus albus from Skadar Lake basin is now recognized as new species Squalius platyceps, distinguished from other Squalius species in Adriatic basin, so completion of data for all aspects of this new species is required. Gametogenesis character as well as type of roe release and whole reproductive biology of fish species in Skadar Lake are species specific and closely connected to their living conditions (Ivanovic, 1969). Asynchrony growth of oocytes and portion release of roe are also adaptation of White Chub to the ecological conditions in Skadar Lake. Colour of fish is changing to more intensive nuances in the spawning period. More dark nuances can be seen on dorsal side 15 Krivokapic

that is dark olive colour as well as on lateral side with more intensive yellow line. from Skadar Lake and Drin river basins. References Brown Peterson N. 2011. A Standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science 3(1), 52-70. American Fisheries Society. Fig. 9a. Histology cut of Skadar Chub gonad, September (autumn period) (en 100x). Oogenesis cycle of White Chub from Skadar Lake confirmed that females become reproductively mature and ready for spawning with first release of roe in the third (2 + ) that is fourth (3 + ) year of their life. Spawning process of White Chub from Skadar Lake starts by the end of March and last until mid of June (Krivokapic, 2002 a; 2002 b., 2009.). Ivanovic B. 1969. Gametogeneza Pachychilon pictum (Heckel et Kner, 1843). Ichthyologia 4(1), 1-12. (Gametogenesis of Pachychilon pictum). Ivanovic B, Sekulovic T. 1971. Taksonomske karakteristike Leuciscus cephalus albus (Bonaparte, 1838) iz Skadarskog jezera. Ichthyologia 3(1), 27-36. ACTA Biologica Yugoslavica. Sarajevo. Yu (Taxonomic characteristics of Leuciscs cephalus albus from Skadar Lake). Ivanovic B, Knezevic B. 1978. Morfolosko ekoloske karakteristike oogeneze Chondrostoma nasus ohridanus iz Skadarskog jezera. Ekologija 13 (2), 173-179. Beograd, Yu. (Morphologic and ecologic characteristics of Chondrostoma nasus ohridanus oogenesis, from Skadar Lake). Fig. 9b. Histology cut of Skadar Chub gonad, September (autumn period) (en 100x). Ivanovic B, Knezevic B. 1975. Ooogeneza Scardinius erythrophthalmus (Bonaparte, 1832). Glasnik Republickog zavoda zastite prirode, Prirodnjackog muzeja u Podgorici 8, 43-49. (Oogenesis of Scardinius erythrophthalmus). That is similar to most of cyprinid species from the Lake (Ivanovic, 1964.; Knezevic, 1984.; Sekulovic & Ivanovic, 1971.; 2002 c) Its female reproductive cycle also consist five principal maturity phases. Female domination is noted in the samples from all seasons that support reproductive balance Krivokapic, 2001, 2002, 2004.). Histological analyses of the oogenesis are presented in parallel to the macroscopic descriptions above, as an essential contribution to the knowledge of White Chub - Squalius platyceps, newly recognized species Ivanovic B. 1977. Polno sazrijevanje i razmnozavanje Alosa falax nilotica u Skadarskom jezeru. Ichthyologia 9(1), 25-29. (Sex mature and spawning of Alosa falax nilotica from Skadar Lake). Jankovic D. 1958. Ekologija dunavske kecige (Acipenser ruthens L). Bioloski Institut Srbije. Posebno izdanje. Kniga 2, 1-145. Beograd. Yu. (Ecology of Danube sturgeon, Acipenser ruthens L). Jankovic D. 1971. Razmnozavanje sarana (Cyprinus carpio L) iz Skadarskog jezera. Arhiv bioloskih nauka, 16 Krivokapic

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