Instituto Nacional de Investigación y Desarrollo Pesquero. Paseo Victoria Ocampo N°1, Escollera Norte. 7600 Mar del Plata Argentina, and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Rivadavia 1917, 1033 Buenos Aires Argentina
The Mariculture Experimental Station of the National Institute of Research and Fishery Development (INIDEP, Mar del Plata City, Buenos Aires Province) has been developing culture techniques for marine finfish aquaculture since 1994. Among the species studied, red porgy, Pagrus pagrus, and flounder, Paralichthys orbignyanus, are considered the more promising. The euryhalinity of P.orbignyanus enables its culture under experimental conditions (Wasielesky and Miranda, 1995; Sampaio et al., 2007) demonstrating the potential of this species as an aquaculture candidate.
It is known that wild adult female as well as cultured juvenile female P.orbignyanus grow faster and larger than males (López Cazorla 2005; López et al. 2006). Hence, methods to control sex with the final purpose of culturing monosex populations of only faster growing females have become an important issue for the optimization of future commercial production. Studies on the reproductive biology of P.orbignyanus are scarce, either on wild or lab-reared specimens. Mellito Da Silveira et al. (1995) described gonadal morphology of P.orbignyanus adult fish caught on the coasts of Río Grande do Sul in Brazil (31°S). López Cazorla (2005) studied the age, growth, and macroscopic gonadal maturity of P.orbignyanus caught in Bahía Blanca estuary. Assessment of the stage of gonad development is essential in studies of fish reproductive biology (West 1990). Histological methods have been important for the description of early stages of sexual differentiation in juveniles of other flounder species such as southern flounder, P.lethostigna (Luckenbach et al. 2003) and Japanese flounder, P.olivaceus (Tanaka 1987; Yamamoto 1999).
As nothing is known about the timing of sex differentiation in flounder, P.orbignyanus, the present study was conducted to define the schedule of sex determination in lab-reared juvenile based on sex-specific histological criteria.
Materials and methods
Juveniles used for this study were produced during the 2004 and 2005 reproductive seasons at the Mariculture Experimental Station. Different egg batches were obtained by natural tank spawning from the same broodstock with no control of individual spawning pairs (N = 39; sex ratio 1 female : 2.5 male). Spawning technique, larval culture, and grow-out protocols used for juvenile mass production were described by Bambill et al. (2003, 2006), Radonic et al. (2005, 2007) and Müller et al. (2006a, b). A total of 61 fish were histologically sampled at approximately 10-mm total length (TL) size intervals following metamorphosis. Tissues were preserved in Bouin’s fixative for at least 48 h and transferred to 70% ethanol until histological processing. Samples were dehydrated in ethanol, embedded in paraffin, and serially sectioned at 7-μm thickness. Sections were stained with hematoxylin and counter-stained with eosin. Structural and cellular sex distinguishing markers were identified through optic microscopy to describe the stages of oogenesis in females and spermatogenesis in males. Gonadal sections were staged based on the most advanced phase of oogenesis or spermatogenesis observed according to Luckenbach et al. (2003).
Results and discussion
Undifferentiated gonads of flounder P.orbignyanus were located at the posterior end of the abdominal cavity, beneath the ventral surface of the renal region. They appeared as paired structures on each side of the mesentery mainly composed by epithelial and gonial cells surrounding the lumen (Fig. 1).
In presumptive testes, formation of seminal lobules separated by fibrous connective tissue were observed (Fig. 2). Advanced stages of testicular development included the presence of primary and secondary spermatocytes and spermatids. Active spermatogenesis was distinguished including proliferation, growth, and differentiation until presence of spermatozoa in maturing fish.
Female sex differentiation was first characterized by the formation of an ovarian cavity, surrounded by germinal cells and a thin albuginea tunica (Fig. 3A and B). Later, septa develop from the tunica and extend into the ovarian cavity, supporting ovigerous folds (Fig. 3C). This germ cells develop by mitosis into oogonia characterized by a central nucleus and a prominent nucleolus (Fig. 3D). During the immature stage, oocytes in first growth phase (chromatin nucleolar and perinucleolus stage) can be seen. These oocytes have a basophilic cytoplasm with a central round nucleus with several peripheral nucleoli.
The timing of ovarian and testicular differentiation relative to fish size (mm TL) is illustrated in Figure 4. According to Nakamura et al. (1998) and Komatsu et al. (2006), the ovarian cavity formation is a useful criterion for female sex differentiation in teleost fishes. Therefore, in P.orbignyanus juveniles, early gonad differentiation occurs between 41- and 75-mm TL, corresponding to 3.5 and 5.6 mo of age, respectively. Likewise, in P.lethostigma sex differentiation was observed in fish ranging from 75 to 120-mm TL (Luckenbach et al. 2003), while in P.olivaceus this event occurred between the sizes of 30- and 100-mm TL (Tanaka 1987). Nevertheless, Yamamoto (1999) recently reported that sex is determined between 27- and 37-mm TL for the latter. In the present study, males of 1-yr old and minimum size of 134-mm TL were ripe, indicating early maturation under controlled conditions. According to Mellito Da Silveira et al. (1995), sperm was detected in wild P.orbignyanus males over 269-mm TL, whereas immature females with early perinucleolus stage oocytes ranged between 259- and 450-mm TL. Moreover, females in final maturation and spawning stages were over 500-mm TL. López Cazorla (2005) observed mature females (505-mm mean TL) and spermiating males (410-mm mean TL) only during the warmer months (November to January). Tanaka (1987) recorded spermatogenesis in lab-reared males of P.olivaceus under 200-mm TL (179 d after hatching), whereas Luckenbach et al. (2003) observed spermatozoa for the first time in 112-mm TL P.lethostigma specimens. In salmonid species, males often show signs of early maturation before reaching the market size (Bromage 1996). This situation has also been reported for commercial marine finfish species such as European sea bass, Dicentrarchus labrax (Zanuy et al. 2001). Precocious maturation reduces growth rate and food conversion efficiency, and thus constitutes a major problem for commercial aquaculture. A useful strategy to delay early maturation in captivity is the use of appropriate photoperiod regimes, as studied for salmonids and sea bass (Bromage 1996; Zanuy et al. 2001).
The present findings provide a detailed understanding of sexual differentiation and its timing in P.orbignyanus juveniles, that would help develop sex control strategies for the optimization of faster growing culture in commercial operations.
We thank H. E. Christiansen (CONICET PIP 5009) for his assistance with all aspects of fish histology procedures and analysis. INIDEP Contribution No. 1448.