Reproduction of the internal inseminator Trachelyopterus galeatus (Linnaeus,

Reproduction of the internal inseminator Trachelyopterus galeatus (Linnaeus, 1766) (Pisces: Auchenipteridae). Daniel Moreira Roriz Lemes,¹ Bianco Vizioli,¹ Lucas Marcon¹ and Nilo Bazzoli¹ ¹Programa de Pós-Graduação em Biologia de Vertebrados, Pontifícia Universidade Católica de Minas Gerais. Av. Dom José Gaspar 500-30535-610, Belo Horizonte, Minas Gerais, Brazil. Correspondence: Nilo Bazzoli, Programa de Pós-Graduação em Biologia de Vertebrados, PUC Minas-Av. Dom José Gaspar 500-30535-610, Belo Horizonte- MG, Brazil. E-mail: bazzoli@pucminas.br Abstract To study the reproductive biology of Trachelyopterus galeatus in Irapé reservoir in the Jequitinhonha River basin, Minas Gerais, Brazil, a total of 372 females and 254 males were captured in bimonthly sampling from May 2012 to April 2013. The sex ratio showed a predominance of females. The stages of gonadal maturation and spawning type were determined through macro- and microscopic analysis of the gonads as well as by variation in the gonadosomatic indices. The reproduction peak of females and males is between November and February during which the biological indices showed higher values for females. Trachelyopterus galeatus is a partial spawner and reproduction throughout the year, when physico-chemical factors of the water are favourable for reproduction

of fish in Neotropical environments. The biological indices indicated that reproduction does not affect the physiological condition and fish health. In vitellogenic follicles were observed a large diameter (1017.53 ± 207.87 µm), thin zona pelúcida (2.71 ± 0.52 µm), and high follicular cells (45.41 ± 11.88 µm), which are characteristics of Siluriformes. Mucosubstances were detected in the follicular cells of vitellogenic follicles for egg adhesiveness. In the ovarian lamellae of mature ovaries, spermatozeugmatas were observed and in the testes, spermatozoa were present with an elongated head, characteristics of internally inseminating fish. Keywords: ovaries, testes, biological indices, histochemical, spermatozeugmata, follicular atresia. Introduction Trachelyopterus galeatus belongs to the Siluriformes Order and Auchenipteridae family that is widely distributed in South America and this family is comprised of an endemic catfish group of the Neotropical region with at least 20 genera (Ferraris, 2003). Different to many Silurifomes, all the Auchenipteridae family presents sexual dimorphism and internal insemination, and adult males have a modified anal fin called the gonopodium that transfers sperm to females as well as acting as an accessory seminal vesicle of the testicles (Loir et al., 1989; Meisner et al., 2000; Parreira et al., 2009). Females can keep unfertilised oocytes and spermatozeugmatas in epithelial folds for up to six months before fertilisation and only release eggs when they find ideal locations to deposit these oocytes (Ferraris, 2003; Melo et al., 2011).

The evaluation of gonadal maturation stages and frequency during the year is important to understand the reproductive behaviour of a species within its habitat. Analysis of biological indices related to reproduction allows researchers to discover the way in which the fish use environmental resources and energy (Carvalho et al., 2009). The Irapé reservoir located in the Jequitinhonha River basin, Minas Gerais, Brazil, and covers an area of 137 km 2 with depths greater than 200 metres close to the damn. The reservoir is considered young in which the assembly of fish found in colonization. The colonization of the reservoir is characterised by high biological production that results in the release of various nutrients during the decomposition of organic matter that has been submerged after the filling of the reservoir (Agostinho et al., 2007). Considering the lack of information on the reproduction of T. galeatus in the Jequitinhonha River basin, this study examined the reproductive biology of this species in the Irapé reservoir for the first time, using different morphological techniques with the aim to provide information for species conservation. Material and methods Sampling, biological indices, and biometry Trachelyopterus galeatus specimens were collected bimonthly from the Irapé reservoir, on the Jequitinhonha River (15º30 and 18º15 S, 38º30 and 44º00 W), Minas Gerais, Brazil, from May 2012 to April 2013, using gillnets with meshes of various sizes. A total of 626 fish (372 females and 254 males) were captured. After dissection, the following biometric parameters were determined for each specimen: total length (TL), body weight (BW), gonad weight (GW),

liver weight (LW), stomach weight (SW), and coelomic fat weight (CFW). From the biometric measurements taken, we calculated the following indices: gonadosomatic (GSI = GW x 100/BW), hepatosomatic (HSI= LW x 100/BW), stomach repletion (SRI = SW x 100/BW), celomic fat (CFI = CFW x 100/BW), and Fulton condition factor (K = BW x 100/TL³). Soon after, were collected gonads fragments males and females for histology. Gonadal maturation stages and spawning type The gonadal maturation stages and spawning type were determined through macro- and microscopic characteristics of the gonads as well as variation of the GSI and the frequency distribution of the gonadal maturation stages (Paschoalini et al., 2013). Histology, histochemistry and histometry For histology, gonad fragments previously fixed in Bouin s fluid for 8-12h were embedded in paraffin, sectioned at 5 µm thickness, and stained with haematoxylin-eosin (HE). To detect carbohydrates, proteins, and lipids in the ovarian follicles the following histochemical techniques were used: periodic acid-schiff (PAS), Alcian Blue (AB) 2.5 and 0.5 ph, and Sudan black B (SB). The diameters of 50 intact spherical follicles with little shrinkage at each stage of development were determined from histological slides using an Olympus BX 50 light microscope with Olympus CellSens Standard 1.9 software. The thickness of the zona pellucida and height of the follicular cells in pre-vitellogenic and vitellogenic follicles were also measured.

Physical-chemical variables of the water The temperature, dissolved oxygen concentration, ph, turbidity, and electrical conductivity of the water were measured bimonthly during the collections carried out in the reproductive peak period in the most Neotropical fish, from October 2012 to February 2013. Statistical analysis Average values of the biological indices and gonadal maturation stage for each bimester as well as oocyte diameter, thickness of the zona pellucida, and the height of the follicular cells were assessed using Statistica 7.0 software by analysis of variance. After a normal distribution test, the data were compared using one-way ANOVA analysis of variance followed by a Duncan test with a significance level of p < 0.05. Results Trachelyopterus galeatus ovaries were sacciform and had yellowing when ripe (Fig. 1A). The testes had a central portion from which projections originated to form filiform lobes. The cranial region of mature testes presented long and thin whitish lobes while the caudal region presented seminal vesicles and thicker, shorter lobes, which were pink in colour (Fig. 1B). Folliculogenesis began with the proliferation of primordial germ cells, oogonia, originating the follicles: Initial perinucleolar, advanced perinucleolar, pre-vitellogenic and vitellogenic. Pre-vitellogenic follicles exhibited a thin zona pellucida and cubic follicular cells. In initial pre-vitellogenic follicles, the cortical

alveoli formed a continuous collar in the peripheral ooplasma while for advanced pre-vitellogenic follicles, cortical alveoli almost filled the ooplasma. The vitellogenic follicles presented cortical alveoli composed of large vesicles, spherical yolk globules, a thin zona pellucida, and highly prismatic follicular cells (Fig. 2 A-F and Table 1). In the follicular cells of vitellogenic follicles, neutral glycoproteins, sulphated acid and carboxylic acid glycoconjugates were detected, from the positive reactions observed from PAS and AB techniques at ph 2.5 and 0.5. Glycoproteins and carboxylic acid glycoconjugates were found in the zona pellucida from the positive reactions to PAS and AB at ph 2.5. In the cortical alveoli and yolk globules, neutral glycoproteins were observed from the positive reaction in the PAS technique. In yolk globules lipids were also revealed by the positive reaction to Sudan black B (Fig. 3 A-F). The following maturation stages were determined for females: F1 = Resting, F2 = Initial maturation, F3 = mature, and F4 = spawned. In ovarian lamellae of mature fish, sperm packages called spermatozeugmata were observed parallel to each other. Unovulated follicles that had started to undergo regression or atresia were categorised into three stages: initial atresia, intermediate atresia, and advanced atresia (Fig. 4 A-H). Histologically, the seminiferous tubules of the mature testicles were full of sperm with elongated head. The seminiferous tubules communicate with each other, forming an anastomosing tubular system. The following maturation stages were determined for males: M1 = Resting, M2 = Initial maturation, M 3 = mature, and M 4 = spent (Fig. 5 A-D).

Females ranged in length from 11.0 to 21.5 cm and for males the length range was 10.5 to 21.0 cm. The body weight of the females ranged from 25.24 to 234.83 g while for males it was 17.5 to 183.11 g. Females predominated in the population structure compared to males. The largest absolute frequency bimonthly by gonadal maturation stage was recorded for females in the months of November and December and most fish captured was in the resting stage (Table 2). The GSI for females followed the maturation stage, increasing gradually from stage 1 to stage 3, before then decreasing in stage 4. The HSI also increased gradually with the maturation stage. The SRI and CFI showed higher values in stage 4. The Fulton condition factor (K) showed higher values during stages 2 and 3 (Table 3). The female reproductive peak occurred in the November/December and January/February bimesters, when the GSI, HSI SRI, CFI, and K had higher values (Table 4). The GSI in males also accompanied the maturation stage, increasing gradually from stage 1 to stage 3, and then decreasing in stage 4. The HSI, SRI, CFI, and K showed slight variations (P > 0.05) (Table 3). Similarly to females, the male reproductive peak occurred in the November/December and January/February periods, when the GSI values were higher (Table 4). The average values of the physical and chemical parameters of the water during the reproductive peak are in Table 5. Discussion This study shows the information about the reproductive biology of the internal inseminator Trachelyopterus galeatus such as the presence of spermatozeugmatas in ovarian lamellae of mature fish, as observed by Meisner et

al. (2000). It also has the characteristics of internally inseminating fish and spermatozoa with an elongated head. Its follicular cells contain vitellogenic follicles with mucosubstances that can be responsible for the adhesiveness of the eggs. The biological indices indicated that reproduction does not affect the physiological condition and fish health. The reproductive peak of T. galeatus coincides with the period in the which the physical and chemical parameters of the water are favourable for reproduction. It shows reproductive activity throughout the year, indicating that the species is a partial spawner. The sex ratio showed a predominance of females and this may be related to differences between the sexes, selectivity of sampling apparatus, and population stratification (Hojo et al., 2004; Carvalho et al., 2009). Macroscopic and microscopic morphology of the ovaries and testes of T. galeatus is similar to that described by Melo et al. (2011). Generally, fish with external fertilisation have spermatozoon with spherical or ovoid head, a fact that is considered evolutionarily ancient (Jamieson and Leung, 1991). Furthermore, fish with internal insemination have an elongated head spermatozoon, as observed in this study, which is a characteristic of fish from the Auchenipteridae family (Parreira et al., 2009). An elongated head in spermatozoon facilitates its passage through the gonopodium of males and labyrinthine structures of the females and facilitates the formation of spermatozeugmata (Burns et al., 2002; Melo et al., 2011). Several studies of inseminating fish show that spermatozegmatas are adhered one to another through membrane junctions; sperm are found only in the ovaries of females in the maturation or mature stages; fertilised eggs and embryos are not found in the ovaries; there is prior contact between sperm and oocytes in

the female genital tract before spawning; fertilisation occurs only when follicles/sperm are released into the external environment (Javonillo et al., 2009). Folliculogenesis of T. galeatus followed a basic pattern found in most teleost fish, with a primary stage of intense synthesis of RNA and proteins, and a second stage of growth due to the formation of cortical alveoli and yolk globules (Lubzens et al., 2010; Martins et al., 2010; Marcon et al., 2015). The diameter of vitellogenic oocytes may vary between families and populations of the same species due to environmental variations (Kolm and Ahnesjö, 2005). For T. galeatus, vitellogenic follicles were observed with a large diameter, thin zona pellucida, and high follicular cells, which are characteristics common to vitellogenic follicles of Siluriformes (Melo et al., 2011). After spawning, the unreleased vitellogenic follicles enter regression or atresia, which was classified into three stages in this study, exhibiting similar morphological characteristics to those described by Miranda et al. (1999), Santos et al. (2005), Normando et al. (2014) and Marcon et al. (2015). The yellow bodies observed in the final atresia have no endocrine function and are rapidly reabsorbed (Miranda et al., 1999; Santos et al., 2005; Arantes et al., 2011). Glycoproteins found in the cortical alveoli are released into the perivitelline space during fertilisation constituting blockade on polyspermy as well as acting in hardening the chorion (Hart, 1990). In the zona pellucida of T. galeatus neutral glycoproteins were also detected, probably due to the presence of coriogenina, the main glycoproteic component of the zona pellucida (Hamazaki et al., 1989). The presence of neutral glycoproteins in the zona pellucida is common in most teleosts (Bazzoli and Rizzo, 1990; Martins et al., 2010). However, the zona pellucida of some species additionally contains carboxylated acid

glycoconjugates, as observed in T. galeatus of this study and in the other Neotropical teleosts studied by Rizzo and Bazzoli (1991) and Gomes et al. (2007). In the follicular cells of T. galeatus, neutral glycoproteins, acid sulphates and carboxylated acid glycoconjugates were detected, which form mucosubstances that can be transferred to the zona pellucida of the egg for adhesiveness (Santos et al., 2013), as also observed in other Neotropical teleosts (Andrade et al., 2001; Martins et al., 2010; Normando et al., 2014). In the present study, the reproductive peak of males and females occurred in the period from November to February when the values of conductivity, dissolved oxygen, and ph of the water were appropriate for the reproduction of T. galeatus. In fact, these physical-chemical factors of water are favorable for reproduction of fish in tropical environments (Lowe-MacConnell, 1987; Boncompagni et al., 2013). During the reproductive peak, high values of the biological indices were observed in T. galeatus females, indicating that reproduction did not interfere with the physiological condition and health of the fish. For males, the variations of these indices were minimal, except for the GSI which showed significantly higher values in stage 3. Changes in the biological indices during reproduction in several Brazilian teleost fish species have also been observed by Ratton et al. (2003), Hojo et al. (2004), Alvarenga et al. (2006), and Carvalho et al. (2009). A long reproductive period and the occurrence of spawning females in almost all bimesters associated with the histological characteristics of spawned ovaries, showing follicles in all stages of development besides post-ovulatory follicles confirm that T. galeatus is a partial spawner. The spawning of the parceled type is the mechanism by which certain species increase the number of

oocytes produced during the reproductive cycle, and is common in Neotropical teleost fish. The various postures during the same reproductive cycle represent a strategy for reducing competition among females of the same population, and to allow asynchronous larval development of larvae occupying different niches for the smaller and larger individuals (Ratton et al., 2003; Normando et al., 2014). Acknowledgements The authors thank the Raquel Loures and Francisco Ricardo de Andrade- Neto of the Peixe Vivo Program of Companhia Energética de Minas Gerais (CEMIG) for logistical support. The authors thank also the partnership of CEMIG and the Brazilian research foundations: CNPq, CAPES and FAPEMIG for financial support.

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Fig. 1 - A: T. galeatus ovaries are sacciform and yellowing. B: testes have the cranial region with longer lobes and whitish colouration (spermatogenic region = asterisks). The caudal region presents a seminal vesicle with and thicker and shorter lobes and pink colour (secretory region = star).

Fig. 2 - Histological sections of T. galeatus ovaries showing follicles in various stages of development, stained with HE A: oogonia nest (*); B: initial perinucleolar follicle (O1) with basophil cytoplasm and nucleus with multiple nucleoli. Advanced perinucleolar follicle (O2) with finely granular basophil cytoplasm and nucleoli next to the nuclear envelope; C: initial pre-vitellogenic follicle (O3i) with cortical alveoli (CA) forming a continuous collar in the peripheral ooplasma, thin zona pellucida (ZP) and cubic follicular cells (FC); D: advanced pre-vitellogenic follicle (O3a) with cortical alveoli (CA) filling almost all ooplasma, thin zona pellucida (ZP) and cubic follicular cells (FC); E: vitellogenic follicle (O4) with ooplasma full of yolk globules (Y); F: Detail of spherical yolk globules and acidophilus (Y), thin zona pellucida (ZP) and highly prismatic follicular cells (FC) in a vitellogenic follicle.

Fig. 3 - Histochemical reactions in T. galeatus vitellogenic follicles A and B: PAS positive reactions in follicular cells, zona pellucida, cortical alveoli, and yolk globules; C and D: positive reaction to AB at ph 2.5 in the follicular cells and zona pellucida; E: positive reaction to AB at ph 0.5 in the follicular cells; F: positive reaction to Sudan black B in yolk globules.

Fig. 4 - Histological sections of T. galeatus ovaries in different maturation stages stained with HE. A: F1- Resting, initial (O1) and advanced perinucleolar (O2) follicles; B: F2- initial maturing, O1, O2 and pre-vitellogenic follicles (O3); C: F3- mature, O3 and vitellogenic follicles (O4); D: F4- spawned, post-ovulatory follicle (POF); E: spermatozeugmata detail (*) in ovarian lamella; F: initial atresia characterised by

hypertrophy follicular cell (FC) and disintegration of the zona pellucida (ZP); G: intermediate atresia with liquefaction of yolk globules and absorption of yolk globules by follicular cells (FC); H: Advanced atresia characterised by the formation of yellow body (YB).

Fig. 5 - Histological sections of T. galeatus testicles in different maturation stages stained with HE. A: M1- resting, containing only spermatogonia (SPG) in the seminiferous tubules; B: M2-initial maturing, small amount of spermatozoa (SPZ) in the lumen of the seminiferous tubules; C: M3- mature, seminiferous tubules filled with sperm (SPZ); D: M4- spent, seminiferous tubules with open lumen containing a small amount of residual spermatozoa (SPZ).

Table 1 Oocyte diameter (FD), thickness of the zona pellucida (ZP), and height of the follicular cells (FC) of T. galeatus females captured from the Irapé reservoir, on the Jequitinhonha River. FEMALES Follicles OD (µm) ZP (µm) FC (µm) O1 91.76 ± 17.75 a - - O2 181.1 ± 31.34 b - - O3 i 281.35 ± 53.62 c 1.57 ± 0.35 a 7.13 ± 1.43 a O3 a 455.12 ± 80.40 d 2.70 ± 0.36 b 14.88 ± 4.04 b O4 1017.53 ± 207.87 e 2.71 ± 0.52 b 45.41 ± 11.88 c O1 = initial perinucleolar, O2 = advanced perinucleolar, O3 i = initial pre-vitellogenic, O3 a = advanced pre-vitellogenic, O4 = vitellogenic follicle. Values followed by the same letter in the same column are statistically similar (Duncan test).

Table 2 Bimonthly absolute frequency of males and females of T. galeatus by gonadal maturation stage, captured from the Irapé reservoir, on the Jequitinhonha River from May 2012 to April 2013. MALES FEMALES Bimester N 1 2 3 4 N 1 2 3 4 M-J/12 42 39 1 1 1 63 54 3 4 2 J-A/12 87 78 7 2 0 88 77 5 4 2 S-O/12 20 16 2 1 1 17 11 6 0 0 N-D/12 57 39 8 7 3 129 43 30 44 12 J-F/13 16 11 1 1 3 30 6 5 15 4 M-A/13 32 27 1 1 3 45 32 7 4 2 Total 254 210 20 13 11 372 223 56 71 22 N = number of fish per bimester. 1 = Resting, 2 = Initial maturation, 3 = mature, and 4 = spent/spawned.

Table 3 Average values of the gonadosomatic (GSI), hepatosomatic (HSI), stomach repletion (SRI), and coelomic fat (CFI) indices as well as the condition factor (K) by gonadal maturation stage (GMS) of males and females T. galeatus, from the Irapé reservoir, on the Jequitinhonha River. MALES GSM N GSI HSI SRI CFI K 1 210 0.26±0.19 a 0.88±0.44 a 2.19±1.97 a 0.15±0.33 a 2.69±0.59 a 2 20 1.84±0.57 b 1.06±0.35 a 3.61±2.42 a 0.39±0.37 ab 2.93±0.38 a 3 13 4.87±1.41 c 1.13±0.49 a 2.71±2.79 a 0.43±0.48 b 3.50±0.54 b 4 11 1.08±0.60 d 1.20±0.45 a 2.32±1.95 a 0.42±0.64 b 2.84±0.70 a Total 254 FEMALES GMS N GSI HSI SRI CFI K 1 223 0.40±0.20 a 0.92±0.34 a 2.28±1.98 a 0.23±0.48 a 2.83±0.64 a 2 56 2.39±1.31 b 1.29±0.39 b 2.99±2.12 a 0.48±0.46 a,b 3.27±0.63 b 3 71 8.48±2.95 c 1.23±0.49 b 2.43±1.33 a 0.40±0.45 a,b 3.31±0.48 b 4 22 1.66±1.58 d 1.53±0.60 c 4.19±3.14 a 0.54±0.54 b 2.94±0.59 a Total 372 N = number of fish per GMS. 1 = Resting, 2 = Initial maturation, 3 = mature, and 4 = spawned/spent. Values followed by the same letter in the same column are statistically similar (Duncan test).

Table 4 Average values of the gonadosomatic (GSI), hepatosomatic (HSI), stomach repletion (SRI), and coelomic fat (CFI) indices as well as the bimonthly condition factor (K) for T. galeatus males and females, captured from the Irapé reservoir, on the Jequitinhonha River, from May 2012 to April 2013. MALES Bimester N GSI HSI SRI CFI K M-J/12 42 0.33±0.81 a 0.88±0.46 a 2.49±2.84 a,c 0.30±0.48 b 2.44±0.44 c J-A/12 87 0.50±0.89 a,b 0.85±0.53 a 1.72±0.93 a 0.07±0.23 a 2.84±0.43 a S-O/12 20 0.54±0.84 a,b 0.95±0.42 a 1.99±2.16 a 0.15±0.22 a,b 2.04±0.31 b N-D/12 57 1.06±1.51 b,c 0.97±0.34 a 3.43±2.73 b,c 0.23±0.33 a,b 3.01±0.51 a J-F/13 16 1.34±2.01 c 1.03±0.37 a 2.51±1.30 a,c 0.48±0.47 c 2.74±0.64 a M-A/13 32 0.57±1.19 a,b 0.93±0.27 a 1.98±1.04 a 0.21±0.45 a,b 2.92±0.92 a Total 254 FEMALES Bimester N GSI HSI SRI CFI K M-J/12 63 0.91±2.06 a 0.92±0.38 a 1.83±1.78 a 0.36±0.71 b 2.54±0.70 c J-A/12 88 0.82±1.42 a 0.88±0.27 a,d 1.97±1.69 a 0.09±0.23 a 3.02±0.56 a S-O/12 17 1.14±1.22 a 1.07±0.27 a,b 2.43±2.56 a,b 0.34±0.44 b 2.16±0.37 b N-D/12 129 3.80±3.83 b 1.18±0.48 b,c 3.09±2.01 b,c,d 0.37±0.44 b 3.21±0.54 a J-F/13 30 5.40±4.99 c 1.35±0.60 c 3.55±2.68 c 0.61±0.56 c 3.17±0.57 a M-A/13 45 1.31±2.41 a 1.14±0.41 b,d 2.40±1.70 a,d 0.36±0.42 b 3.18±0.57 a Total 372 N = number of fish per bimester. Values followed by the same letter in the same column are statistically similar (Duncan test).

Table 5 Physical-chemical parameters of the water from the Irapé reservoir, on the Jequitinhonha River, from October 2012 to February 2013. IRAPÉ RESERVOIR Average ± DP Amplitude Temperature ( C) 26.06 ± 2.67 22.10 29.15 Oxygen (mg L) 7.0 ± 3.53 3.08 7.67 ph 6.85 ± 1.46 5.93 7.21 Conductivity (µs cm) 18.77 ± 10.47 0.4 26.78