Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner,1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast

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International Journal of Research in Pharmacy and Biosciences Volume 2, Issue 6, July 2015, PP 16-23 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)

1Anne Marie Ndiaye 1Reproductive Biology Laboratory, Department of Animal Biology, Faculty of Science and Technology, Cheikh Anta Diop-Dakar University, BP 5005 Dakar Fann, Senegal.

ABSTRACT Diplodus bellottii is a teleostean fish which belongs to the Sparidea family. Diplodus bellottii can be found almost everywhere all along senegalese coast, what makes it a usual fish. After their reproduction mechanism achieved, some male subjects turn female by a switching sex process. Diplodus bellottii culinary rank shows how this fish is well appreciated. Consequently the Diplodus bellottii also has a significant economic interest as it is part of fish products that Senegal exports.In order to study the reproductive biology of Diplodus bellottii we conducted monthly sampling of D. bellottii at the beaches of Dakar ( Soumbédioum, Ouakam, Yoff, Yarakh) and on the Petite côte at Joal precisely. We followed monthly maturation of male and female gonads observing histological sections of those aforementioned sexual organs to phothonic and electronic microscopes to help us determine their sexual cycle. Monthly variations in average of gonadosomatic ratio (RGS), hepatosomatic ratio (RHS) and condition factor (Kv) confirm that the sexual cycle has two time periods of reproduction which are of unequal importances. The first one lasts from January to June and the second one from August to December. Our study's item follows a sexual rest of a month in July. The length at the first sexual maturity of Diplodus bellottii is 14.02 cm for females 14.07 cm for males. Keywords: reproduction periods, gonadal maturation, length at first sexual maturity, Diplodus bellottii, Sparidae, Senegal.

INTRODUCTION Senegal is located at the West African side. Its coastline measures 550km on the Atlantic ocean. Fishing has long been practiced in Senegal. In Senegal, 16 species of Sparidae are exported which include Diplodus bellotti. It lives on the Senegalese coast on rocky and sandy bottoms at depths of up to 100m. It is most usual between 30 and 50m. In Senegal it can be found everywhere. It is usually fished from the beach or on the open seas with a seine or by angling. Diplodus bellottii is hermaphrodite protandrous. However sexual inversion does not affect the entire population [1].It represents a significant share of the landings of Sparidae. It makes 20% of the catch of sargus.Diplodus bellottii is exported to Asian countries. The revenues from its fisheries contribute significantly to improving the living conditions of ship owners. Despite this financial contribution little research focusing on the reproductive biology of Diplodus bellottii were conducted in Senegal. The main purpose of this work was to fill this gap. MATERIALS AND METHODS Sampling of this study consists of individuals of D. bellottii including 536 females, 305 males from the landings of the artisanal fishing at the beaches of Ouakam Yarakh, Soumbédioum and Joal. Monthly sampling was carried out in two stages from March 1992 to June 1993 and from February to July 2007. Since 2007 until 2013 we continue to histological sections to check if there is no changeS that would affect the gonadsThe specimens are between 10cm and 26.5cm, their weight varies between 37.92 g and 478.25g. Each fish taken, the standard length (Lst) is measured to the millimeter, the empty weight (WE), gonad weight (WG) and the liver (WL) are weighed to the tenth of a gram. The gonado-somatic index (GSI) is the ratio of gonad weight (wG) to the eviscerated weight of the animal. Variations in the GSI reflect the weight changes of the gonads during gonadal maturation.

*Address for correspondence [email protected] International Journal of Research in Pharmacy and Biosciences V2 ● I6 ● July2015 16 Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast”

The hepato-somatic index (HSI) is the ratio of liver weight (WL) to the eviscerated weight of the animal. Variations in the HSI reflect periodically the weight changes of the liver. The condition factor (Kv) is the ratio of eviscerated weight of the animal to the cube of the standard length of the fish. These three parameters are calculated as a percentage using the following formulas

GSI = WG/WE × 100 (1)

HSI = WL/ WE × 100 (2) 3 Kv = [WE/(Lst) ] ×100 (3) The length at first sexual maturity is the size at which 50% of the population show signs of sexual activities (from stage III). These individuals are split by gender and by size class. The estimate of this parameter is made on samples taken during the reproduction period. The size at first sexual maturity is calculated using the following formula: P= 100/(1+exp[-a(Lst-L50)] (4) Thanks to macroscopic observation gender of each person taken is determinated. For individuals with undetermined gonad, sex is known after a microscopic examination. The color, texture and density of the superficial vasculature of the gonads are also mentioned. To study gonadal maturation we used a scale with seven stages based on Conand's one [2].To achieve microscopic observations we collected fragments of the middle region of gonads in different stages of maturation. They are fixed in a Bouain- Holland liquid. After dehydration and embedding in paraffin, the 7μm sections are made with a microtome and stained of Stassnie Masson trichrome according to Gabe technique [3].The treated sections are placed between slide and cover slip in a Canada balsam. Observations are made using light microscopy MoticBA 310 and a SMZ-168 TL stereo microscope with image analysis systems. For electronic microscopy, small pieces of fresh fish gonads were fixed in 2.5 glutaraldhehyde in sodium cacodylate buffer 0.1m CaCl2 0.2 to pH 7.2. Rinsing lasts 24 hours in a phosphate buffer. The material is postfixed for one hour. The inclusion is made in Epon after dehydrated it with ethanol and propylene oxide. The slides are made with the ultramicrotome Porter Blum MTI. They are contrasted with uranyl acetate and lead citrate. They were observed with a Jeol100CXII microscop from electronic microscopy service of the Faculty of Sciences and Technology of UCAD. RESULTS Ovarian Maturation The organization of the ovary of D. bellottii is similar to the other Sparidae one. The ovaries are even, elongated and suspended by the mesovarium to the wall of the abdomen. They can be opened to the outside by the urogenital orifice. The ovary is a organ made of cross ovarian strips to the long axis. They protrude into the cavity and ovaries are the seat of ovarian maturation. Each ovarian follicle consists of an oocyte surrounded by the oocyte envelopes that are primary envelope which comes just after the cytoplasm and the outer casing. The latter is formed by the thecal layer separed from the follicular cell layer by the basal membrane. Stage I The ovaries are thread-like, firm consistency. Ovarian lamellae contain only small ovarian follicles (Figure 1). The ovarian follicle consists of a cytoplasmic mass surrounding a large nucleus with a single large nucleolus. This stage covers the months of August and September for the first reproduction period. Stage II The light pink ovaries are transparent. At the periphery of the nuclear membrane are several nucleoli. The outer coating remains flat and difficult to observe (Figure 2). This stage extends from October to November. Stage III The ovaries are of a more pronounced pink. Vascularization becomes visible. The ovaries have a finely granular appearance. Small oocytes are identifiable through the membrane of the ovary. The various structures constituting the ovarian follicle are in place. It appears from the outside towards the

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Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast” inside of the oocyte an outer coating compound the theca, the basal membrane, the follicular layer, the primary envelope and the oocyte itself. This stage is marked by the presence of more numerous cortical alveoli in the average cytoplasm. It is the beginning of vitellogenesis.The oolemme develops numerous microvilli surrounding the oocyte surface. This stage takes place from December to January. Stage IV The ovaries are colored bright yellow and are larger. The Ovarian follicle has its cytoplasm filled of yolk vesicles. The cortical alveoli form a ring around the nucleus. Theca and follicular layer are thickened as the primary oocyte envelope. The microvilli become long.This stage is observed from February to March. Stage V Dark yellow ovaries are highly vascularized. We notice more yolk vesicles that occupy the cytoplasm which becomes increasingly opaque (Figure 3). The thecal , follicular layers, and especially primary envelope have almost completed their edification. The yolk vesicles start to fuse (Figure4). The primary envelope counts two zones. The one lying adjacent the cytoplasm is lamellar. The outer is homogeneous (fig5).This stage is observed in April. Stage VI It covers the end of the maturation and spawning of oocytes. The very large and vascularized ovaries occupy two thirds of the abdominal cavity. The ovaries have a yellow-orange coloring. Well individualized, the oocytes are identifiable by transparency through the ovary wall. They escape from the ovary at the touch of the abdominal cavity. Examination of histological sections showed that the cortical alveoli migrate to the periphery of the cytoplasm to form there the fertilization membrane. The yolk vesicles merge themselves giving more imposing masses throughout the whole cytoplasm. The nucleus is deported to a pole. We also notice at this stage a significant increase in oocyte volume. The oocyte is hydrated. The flattened primary envelope detaches from follicular and thecal layers. The microvilli withdraw and the homogeneous party becomes continuous (Fig. 6). This stage is observed from May to June for the first reproduction period then from October to December for the second one. Stage VII This corresponds to sexual rest. The gonads are flabby and very vascularized. They are dark brown colored and have red spots corresponding to necrosed zones. Histological sections contain only follicles at the early previtellogenesis and atretic oocytes. This stage is observed in July. The Maturation of the Testicles The testicles are elongated and attached to the abdominal wall by a mesorchium. As the female maturation the male's one happens in seven stages. Stage I The testicles are threadlike; it is difficult to identify them among the viscera. On histological sections, spermatogonia occupy the periphery of cysts (Figure.7). The cytes are delimited by the prolongation of the Sertoli cells (Figure.8). Spermatogonia are also present in test throughout the year. Stages II and III The testicles are white, their section is rounded. Examination of histological sections shows that the spermatocytes I and II are more numerous in the cysts (Figure 6). They are present in the testicles from October to November. Stages IV and V The testicles are pearly white. Their section becomes ovoid. They make up three quarters of the viscera. These two stages are observed in the middle of spermiogenesis .The spermatocytes occupy the periphery of cysts. Their number is constantly decreasing as they differentiate into spermatids (Figure. 9) .Young spermatids (stage IV) are larger than the older spermatids (stage V) which appear thin. Spermatids begin their spermiogenesis in cysts which is opened at the end of stage IV to release

International Journal of Research in Pharmacy and Biosciences V2 ● I5 ● June 2015 18 Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast” older spermatids in the lumen of the testicle where they complete their development. These stages are observed from December to March. Stage VI at this stage the testicles are very large and milky white. The semen escapes by a simple push of the abdominal cavity. The sperm histological sections have a grain appearance but much thinner. They are in the testicles lumen (Figure.10). This stage is observed during the months from April to June Stage VII testicles have lost much of their strength of their volume. They have become flabby. They contain few spermatogonia and spermatocytes. It covers the month of July. Sexual Inversion Examination of histological sections showed some young oocytes progressively invading the testicular tissue. It contains only spermatogonia. After the issuance of the sperm, the males change sex (Figure 11). Only part of the population changes sex after the expulsion of sperm. Sexual inversion is very usual in individuals which standard length (Lst) is over than 14.5cm The Size at First Maturity The size at first sexual maturity is the standard length at which 50% of the population is mature. It is 14.02 cm with a = 1.091 for females. It is reached in males who have a standard length equal to 14.07 cm with a = 0.98. Average Monthly Variations In Gonado-Somatic Index (GSI) Hepato-Somatic Index(HSI) And Condition Factor (KV). Monthly monitoring of these three parameters confirms the evolution of gonadal maturation in both sexes. The analysis of mean monthly variations in the ratio shows that the gonado-somatic index (GSI) is low from January to March. It gradually increases to reach its maximum values in females in May (3.71) then in October (2.09) and in males in April (4.44) then in November (1.87). The GSI regresses in females from May to June then from October to December and for males from April to June, then from November to December. In both sexes the sexual rest is observed in July. Examination of the average monthly variations of hepato-somatic index (HSI) shows that in females in both reproduction periods, the HSI values are lower than those of GSI. They reach their peak values in September (1.72) for females and in July (1.6) for males. The values of the condition factor (Kv) for females and males are superior to those of the HSI all year except during the first reproduction period. The greatest values of Kv are reached in August (2.37) for females and July (3.23) for males.The minimum fall in April (1.98) for females and December (1.57) for males.

Monthly variations of gonado-somatic index Monthly variation of gonado-somatic 'GSI), hemato-somatic index (HSI)and condition factor 4 (GSI), hematosomatic (HSI) and to males of D. bellottii condition factor (Kv) to females of D. bellottii 3.5 3 GSI GSI 2.5 2 5 HSI HSI 1.5 4.5 4 Kv 1 Kv 3.5 0.5 3 0

2.5

september

january june august october

febuary march april november december

july may 2 1.5 1 0.5

september

january june august october

febuary march april november december

july may

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Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast”

120 120 %cum 100 L50solv 100 %cum

80 80 L50solv 60 60

Matur males Matur 40

20 20

Mature Mature fmales 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 0 5 10 15 20 standard length (cm) standard length (cm)

Fig1. Gr: 40 start of previtellogenesis, oocyte: OC

Fig2. Gr:10, End of previtellogenesis, nucleus: N

Fig3. Cytoplasm oocyte filled of yolk vesicles Gr.: 10

Fig4. End of vitellogenesis, yolk vesicle fusioGR. : 40,yolk vesicle : YV

International Journal of Research in Pharmacy and Biosciences V2 ● I5 ● June 2015 20 Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast”

Fig5. Primary envelope of follicle ovarian detaches from follicular layer. Gr.: 5000. Follicle cell: FC, primary envelope: PE, primary envelope Lamellar: PEL, primary envelope homogenous: PEH

Fig6. follicule cell (FC) decrase in size, microvilli (MV) projecting from oocyte (O) . Gr : 5000.

Fig7. Test of D. bellottii. Gr.:40. Start of spermatogenesis. Spermatogony : SPG

Fig8 : Gr.: 5000. Sertoli cells delimited cyts. Sertoli cell: SC, spermatocytes : SPC

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Fig10. Gr.:10 End of spermatogenesis, spermatozoides are the most present in the cytes.

Fig11. Gr.10. Test of D. bellottii changing sex.Oocyte: OC, Spermatocyte: SPG DISCUSSION The study of gonadal maturation and monthly monitoring of gonadosomatic index hepatosomatic index and condition factor showed that D. bellottii has two reproduction periods. The first period is from January to June and the second one is registered from August to December. After the first period of reproduction, the animal observes a sexual rest during the month of July. It is the most important. The values of the condition factor (Kv) are lower than those of GSI and HSI. It is explained by the high energy mobilization for matruaration gonadal products specifically in the female germ line. In Sparidae and particularly in seabream all species do not have two reproduction periods. Some seabream have only one breeding period whose duration varies widely. D. sargus cadenati reproduces only once all year [4] .In Algeria the reproduction period of D. sargus extends from January to May [5] .In Tunisia it reproduces the same period [6 ]. D. vulgaris has a sexual cycle which count only one single reproduction period. [7] It extends from November to February in Tunisia. [8] It lasts from October to February in Algeria [9]). In female gonado-somatic index increases from January marking the beginning of vitellogenesis. It reaches its maximum in May and then gradually decreases until July. The value of the GSI in this month is the lowest. Meanwhile in females and males the hepatosomatic index and the condition factor increase significantly to reach their highest value after the first reproduction period, when food is abundant. The availability of food results in an accumulation of reserves in the muscles and at the mesentery. These reserves flow through the liver which explains the increase in the HSI this period. They borrow the bloodstream before being released in the gonads or they will be used in the development of genital products especially in females during vitellogenesis. During the maturation the primary envelope becomes thick. It is constituted by a zone homogeneous and an inner layer formed by 9 lamella. In male subjets, sperm emission is from March to June. Diplodus bellottii is a protandrous hermaphrodite. But the sex switch affects only a tiny part of the population. Some males after the issue of male genital products change sex and become females and wait for the next reproduction period. They will contribute at the next reproduction period as females. Sexual rest lasts only for the month of July for both sexes. The second reproduction period is very limited in time and in intensity. It begins in August with stages III and IV in females, in males with spermatocytes and spermatids. The maximum value of GSI is lower than the one of the first reproduction period. It could result from genital fatigue.

International Journal of Research in Pharmacy and Biosciences V2 ● I5 ● June 2015 22 Anne Marie Ndiaye “Study of the Sexual Cycle and Sexual Inversion of Diplodus Bellottii (Steindachner, 1882; Teleosteans: Sparidae) in Atlantic Ocean Waters on Senegalese Coast” The size at first sexual maturity has a little difference between D. bellottii's females and males. As is the case in some sea breams. It is 20.788cm in females and 21.467cm in males for D. sargus cadenati [4]. For D. vulgaris, it is 13.84cm in females and 13.64cm in males [8]. In Algeria it is 20.8 cm for females and 19.6 cm for male [9]). D. sargus females reach their first maturity at 20cm and for males at 20.2 cm [9] .The size at first maturity is reached in eastern Algeria with D. cervinus for a total length of 25 cm in both sexes[10]. D.annularis requires a total length of 10.5 cm to reach maturity in both sexes [11] .We noticed that the smaller female of D. bellottii in vitellogenesis measures 12cm. [1] CONCLUSION In Senegalese waters D. bellottii has annual sexual cycle including two reproduction periods. The first one lasts from January to June and the second one from August to December. The second period is less important than the first one. The sexual rest takes place during July which coincides with a food abundance time. ACKNOWLEDGEMENTS This work was well done thanks to the support of: Mamour Touré research professor at the Laboratory of Reproductive Biology Department of Animal at the U.C.A.D Pape Ndiaye Ibnou research professor at the Biology Department of Animal at the U.C.A.D Aliou Faye from the Graduate Institute of fishery and Aquaculture of the U.C.A.D All technicians of the Animal Biology Department of the U.C.A.D REFERENCES

[1] Ndiaye A.M.Contribution à la connaisssance de la biologie de la reproduction de deux espèces de Sparidae (poisson téléostéen) des cotes senegalaises.D.E.A.Biol.Anim.1995 44pp. [2] Conand C. Maturité sexuelle et fécondité du tassergal, Pomatomus saltator(L., 1766) poisson.Pomatomidae Bull. IFAN vol.37, 1975 395-466. [3] Gabe M. Techniques histologiques. Ed. Masson & Cie1968, 113pp. [4] Dia M., Wague A. et Ghorbel M. (2009). Etude de la reproduction chez Diplodus sargus cadenati(Sparidae) de la cote de Nouadhibou (Mauritanie). Bulletin na. – natn. Tech. Mer de Salammbo, vol.36, p.21-30. [5] Benchale .W et Hichem Kara M. Age, croissance et reproduction du sar commun Diplodus sargus sargus (Sparidae). Des cotes de l’Est algérien. Rapp. Comm. int. Mer Médit., 39, 2010 [6] Mouine N., Francour P., ktari M. and Chakroun – Marzouk N. The reproductive biology of Diplodus sargus sargus in the gulf of Tunis (central Mediterranean). Scientia Marina 7(13) 2007. [7] Mouine N., Mohamed-Hédi Ktari .& Nadia Chakroun-Marzouk (2013). Age and growth of Diplodus vulgaris (Sparidae) in the Gulf of Tunis. Cybium 34(1) : 37-45 [8] Taieb Aymen Hadj, Ghorbel Mohmed, Hamida Nader Ben Hadj and Jarboui Othman. (2012).Reproductive biology of Diplodus vulgaris(Teleostei,Sparidae) in the southern Tunisian water(Central Mediterranean). Acta Adriat., 53(3) : 437 – 446. [9] DJOUAHRA C. & K. HARCHOUCHE. Reproduction de Diplodus vulgaris (Poisson Sparidés) des côtes Algériennes. USTH – FBS-4th International congress of the population & Animal communities « Dynamics & Biodiversity of terrestrial & ecosystems « « CIPCA4 » Taghit(Bechar) Algerie 19-21 November,2013. [10] Derbal F. et M. Hichem Kara. Âge, croissance et reproduction du sar tambour Diplodus [11] cervinus cervinus (Sparidae) des côtes de l’Est algérien. Cybium 2013, 37(4) : 247-254. [12] Chaouch H, O.Hamid – Ben Abdallah, Ghorbel M. and Jarboui O.Journal Appli. Ichthyol. 2013, 29: 796-800.

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