The reproductive conditions of male shrimps, genus Penaeus, sub-genus Litopenaeus (open thelyca penaeoid shrimps): A review

Aquaculture 300 (2010) 1–9

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Review article

The reproductive conditions of male shrimps, genus Penaeus, sub-genus Litopenaeus (open thelyca penaeoid shrimps): A review Jorge Alfaro-Montoya ⁎ Estación de Biología Marina, Escuela de Ciencias Biológicas, Universidad Nacional, Puntarenas, Costa Rica

a r t i c l e

i n f o

Article history: Received 6 August 2009 Received in revised form 7 December 2009 Accepted 8 December 2009 Keywords: Penaeidae Penaeoid Shrimp Male reproduction Spermatophores Sperm quality

a b s t r a c t Male reproductive performance in penaeoid aquaculture is a major issue. This review evaluates the current knowledge on male reproduction of open thelyca penaeoid shrimps. This group of shrimp belongs to the genus Penaeus, sub-genus Litopenaeus, and presents a unique reproductive model, characterized by complex spermatophores and thelyca without seminal receptacles; however, sperm seem to reach maturation and capacitation on the open thelyca. Males of this group adapt differently to captivity, being P. (Litopenaeus) vannamei the best adapted species. Nevertheless, three problematic conditions develop in confined environments in one or more species: male reproductive tract degenerative syndrome (MRTDS), male reproductive system melanization (MRSM), and spermatophore deterioration (SD). © 2009 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . 2. Functional anatomy of the reproductive system 3. Endocrine control of male reproduction . . . . 4. Male sexual maturation . . . . . . . . . . . 5. Male sexual problems . . . . . . . . . . . . 6. Spermatophore renovation . . . . . . . . . . 7. Nutrition in male reproduction . . . . . . . . 8. Acrosome reaction and sperm maturation . . . 9. Spermatophore preservation . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Introduction World aquaculture has grown at an average annual rate of 8.8% from 1950 to 2004. Latin America and the Caribbean region have the highest average annual growth of 21.3%, mainly with salmonids and shrimps (FAO, 2006, 2009). The production of introduced Penaeus (Litopenaeus) vannamei in Asia and the Pacific region was 1.1 million MT; whereas in Latin America and the Caribbean it was 266,000 MT (FAO, 2006). In 2004, Asia produced more P. vannamei (52%) than the

⁎ Tel.: + 506 277 3324; fax: + 506 237 6427. E-mail address: [email protected]. 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.12.008

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native species, mainly P. monodon (48%); China cultured P. vannamei at 71% of the 600,000 MT produced (FAO, 2005). Penaeoid male reproductive performance is currently regarded as one of the major drawbacks in the fragile shrimp aquaculture industry (Parnes et al., 2004). Open thelyca penaeoid shrimps present a complex male reproductive system, which has received less attention than the female reproductive system, morphologically, endocrinologically and functionally (Browdy, 1992; Browdy, 1998; PerezVelazquez et al., 2003). This review will focus on developments related with the functioning of the male reproductive system of four American species belonging to the genus Penaeus, sub-genus Litopenaeus: P. (Litopenaeus) vannamei, P. (Litopenaeus) stylirostris, and P. (Litopenaeus) occidentalis

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from the eastern Pacific, and P. (Litopenaeus) setiferus from the western Atlantic. Few data were available on the fifth species: P. (Litopenaeus) schmitti from the western Atlantic. These species of shrimp are unique among the 12 original genera of the family Penaeidae and the 6 original sub-genera of Penaeus (Flegel, 2007), in the sense that their reproduction is based on an open thelyca model, with complex spermatophores and no seminal receptacles in females (Perez Farfante, 1975; Bauer and Cash, 1991). Cross-fertilization between these species is limited by prezygotic and postzygotic barriers. Misamore and Browdy (1997) demonstrated that mating behaviors are different in P. setiferus and P. vannamei, inducing no interspecific matings. Interspecific artificial insemination and in vitro fertilization between these species generated no hybrids. Successful hybridization in Litopenaeus has been reported only for P. setiferus × P. stylirostris (Lawrence et al., 1984) and P. setiferus × P. schmitti (Bray et al., 1990) at very low fertilization rates (b1%). Sperm–egg incompatibility seems to be an important prezygotic barrier in penaeoid shrimps (Alfaro et al., 2003). However, the primary binding technique developed by Rojas and Alfaro (2007) indicates that P. stylirostris and P. vannamei male sperm are capable of binding to P. occidentalis eggs, but interspecific acrosome reaction induction has not been demonstrated yet. To our knowledge, a review of this nature has not been published ever and the accumulated research has been partially considered in the process of understanding male reproduction. For the nomenclature of penaeoid shrimps, I have adopted the binomials according to Holthuis (1980). 2. Functional anatomy of the reproductive system The anatomy of the male reproductive system of P. setiferus was described by King (1948) and Talbot et al. (1989), with posterior definitions of the functions of the vas deferens and the terminal ampoule (Fig. 1). An impregnated P. occidentalis female is presented in Fig. 3; the complex structure of spermatophores is similar among

the five species of Litopenaeus and was described by Perez Farfante (1975). Spermatogenesis has been studied mainly in closed thelycum species (Nagabhushanam and Kulkarni, 1981; Shigekawa and Clark, 1986; Kang et al., 2008). In the sub-genus Litopenaeus, Alfaro (1994) made some ultrastructural observations on P. stylirostris, including the definition of the place for spike elongation, which occurs gradually in the descending medial vas deferens. Recently, final sperm maturation and capacitation in the Litopenaeus thelyca was reported by Alfaro et al. (2007). The proximal vas deferens transports spikeless spermatids from the testes into the ascending medial vas deferens. The primary and secondary layers of spermatophores are synthesized in the ascending and descending vas deferens, respectively (Ro et al., 1990). The main body and sperm sac of spermatophores are formed in one of the two channels of vas deferens (Bauer and Cash, 1991). The terminal ampoule is histologically complex, with separated chambers for the assembling of additional spermatophore sub-units: dorsal plate, wings, glutinous mass and adhesive (Talbot et al., 1989). Sperm of penaeoid shrimp are unistellate, have a non-motile spike, and undergo acrosome reaction during sperm–egg interaction (Clark et al., 1981; Griffin et al., 1988). The ultrastructure of P. vannamei sperm from spermatophores was described by Dougherty and Dougherty (1989), who reported on the pathology of melanized spermatophores of pond-cultured P. vannamei. Sperm cells consist of a spike, a hemispherical cap, a nucleus, a filamentous meshwork between the nucleus and hemispherical cap, and a hemispherical rim of cytoplasmic particles. Sperm of P. vannamei and P. occidentalis have a similar length (body diameter = 3–4 μm); whereas P. stylirostris is bigger (6.5 μm; Alfaro et al., 2007). 3. Endocrine control of male reproduction Okumura (2004) summarized our current knowledge of the regulatory mechanism on male shrimp reproduction, based on three

Fig. 1. Male reproductive system of Penaeus (Litopenaeus). A: Anatomical location (modified from Shigueno, 1975). B: Isolated reproductive system (modified from King, 1948).

J. Alfaro-Montoya / Aquaculture 300 (2010) 1–9

levels: central nervous system/ x-organ-sinus gland level, androgenic gland (AG)/mandibular organ (MO) level, and primary/secondary sexual characteristics level. However, Nagaraju (2007) proposed an endocrine model with the mandibular organ at the highest regulatory level. The protocerebrum of male natantian crustaceans produces a substance or substances that regulate the integrity of the genital system (Kleinholz and Keller, 1979, for review; Fingerman, 1987, for review). The X-organ-sinus gland complex in the eyestalk of decapod crustaceans is the major neuroendocrine control center (Fingerman, 1987, for review). The effect of the sinus gland neurohormones on decapod male reproduction is less well known than that in the female (Khalaila et al., 2002). Unilateral eyestalk ablation was demonstrated to improve the quality of P. vannamei spermatophores (Leung-Trujillo and Lawrence, 1985; Salvador et al., 1988; Alfaro and Lozano, 1993). Testicular maturation in decapod crustaceans is controlled from the central nervous system by the stimulatory action of 5-hydroxytriptamine (5-HT), which stimulates the release of a putative gonad stimulating hormone (GSH) that activates the synthesis and release of the AGH by the AG. AGH triggers testicular maturation and spermatogenesis (Fingerman, 1997). Complementary, dopamine (DA) inhibits testicular maturation by blocking GSH release and or stimulating the release of gonad inhibiting hormone (GIH; Fingerman, 1997). However, recent findings in female endocrinology are improving this basic approach. Serotonin and its receptors have been identified in the ovary of P. monodon, suggesting a direct role of serotonin in ovaries (Ongvarrasopone et al., 2006; Wongprasert et al., 2006). Gonadotropin-releasing hormone (GnRH) was identified in the protocerebrum of P. monodon, and this molecule may be involved in the regulation of serotonin, as well as a possible GSH (Ngernsoungnern et al., 2008). Methyl farnesoate (MF) from the MO of crustaceans acts as a gonadotropin and a morphogen in both males and females (Laufer et al., 1988; Homola et al., 1991; Rotllant et al., 2000; Laufer and Biggers, 2001). MF treatment has demonstrated to have a direct stimulatory effect on the testicular development, because it was taken by MF receptors present in the testes (Nagaraju, 2007, for review). This molecule regulates the development of different male morphotypes in the spider crab, Libinia emarginata (Huberman, 2000, for review), and it has been identified in penaeoid shrimps, including P. vannamei and P. stylirostris (Laufer and Biggers, 2001). Recently, Alfaro et al. (2008) reported a significant improvement in sperm counts and sperm abnormalities in P. vannamei after five injections of MF at 120 ng g− 1 b.w; however, MF at 1200 ng g− 1 b.w. had no effect. The MO is under the inhibitory action of the MO-inhibiting hormone from the sinus gland (Huberman, 2000, for review; Nagaraju, 2007, for review), supporting the pathway proposed by Okumura (2004). The AG of crustaceans is the endocrine organ that controls the differentiation and functioning of the male reproductive system, and the development of secondary sexual characteristics (CharniauxCotton, 1954; Charniaux-Cotton and Payen, 1985). However, it has been recently proposed that the AG of penaeoid shrimps is not involved in sex determination or sex differentiation because the gender differentiates earlier, soon after transformation of larvae (Garza-Torres et al., 2009). A proteinaceous androgenic gland hormone (AGH) has been identified from an isopod (Hasegawa et al., 1987; Sagi and D Aflalo, 2005, for review), but in decapod crustaceans only lipidic molecules like farnesylacetone and steroids have been identified and they may play complementary roles to AGH (Sagi, 1988; Sagi and D Aflalo, 2005, for review). The injection of 17-alpha-methyl testosterone at 0.01 or 0.1 µg g− 1 body weight (b.w.) induced a significant improvement of P. vannamei spermatophores (Alfaro, 1996). A direct inhibition by the sinus gland on the AG secretion has been demonstrated (Khalaila et al., 2002). The AG of penaeoid shrimps has been studied in few species: P. (Melicertus) kerathurus (Charniaux-Cotton and Payen, 1985),

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P. japonicus (Nakamura et al., 1992), P. (Fenneropenaeus) chinensis (Li and Li, 1993). The first electron microscopy observations for Litopenaeus were provided by Alfaro (1994) and recently CamposRamos et al. (2006), made histological observations for P. vannamei. These studies indicate that the AG of P. vannamei and P. stylirostris is a cord-like cellular mass attached to the proximal part of the ampoule, the distal vas deferens, and the distal part of the descending medial vas deferens. Crustaceans have not demonstrated any endocrine function from the testes (Fingerman, 1987; Fingerman, 1997). However, the male reproductive system could also be a source for ovarian-maturationinducing pheromones (Huberman, 2000, for review) and prostaglandins (Harrison, 1990, for review), but these topics require more attention.

4. Male sexual maturation Quality of spermatophores has been measured by a series of parameters which include the following variables: spermatophore weight, sperm count, live sperm percentage, and abnormal sperm percentage (Leung-Trujillo and Lawrence, 1987; Parnes et al., 2004). Sperm viability has been analyzed by vital dyes and recently by flow cytometry (Lezcano et al., 2004). We proposed a model for male sexual maturation based on three independent levels: testis maturation, vas deferens maturation and spermatophore synthesis (Fig. 2; Alfaro, 1993). The model explains that spermatogenesis produces spikeless spermatids in young males, which vasa deferentia are not fully prepared for spermatid maturation as indicated by the low proportion of sperm with spike elongation. Complementary, spermatophores are synthesized and their normal appearance is no indication of the quantity and quality of the contained sperm cells. This model was further supported by Ceballos-Vázquez et al. (2003), who used it to explain their findings on spermatophore quality of P. vannamei, cultured in ponds. To this model, we added our recent findings on final sperm maturation and capacitation on the female thelycum (Alfaro et al., 2007).

Fig. 2. Proposed model for male sexual maturation and sperm capacitation of Penaeus (Litopenaeus) based on Alfaro and Lozano (1993) and Alfaro et al. (2007).

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Table 1 presents spermatophore quality data for wild and captive species of the sub-genus Litopenaeus. In general, wild adult animals show low and high sperm counts, fluctuating from 11 to 50 million; sperm abnormalities are normally low, below 24% in large size males. Males in captivity respond differently depending on species; P. setiferus show very low sperm counts, P. occidentalis and P. vannamei generate spermatophores of adequate quality as compared to wild animals, and P. stylirostris show low sperm counts. P. vannamei males grown in tidal ponds develop an age-dependent improvement in spermatophore quality, which is superior in 12-monthold animals (38 g b. w; sperm count = 5 million; abnormality = 17%) than in younger males (Ceballos-Vázquez et al., 2003). A similar response was reported for 9–10-month-old P. schmitti (Ramos et al., 1995). P. vannamei and P. stylirostris young males cultured in earthen ponds show low spermatophore quality (sperm count = 2 million; abnormalities = 52–86%; Alfaro, 1993; Alfaro and Lozano, 1993). A low water temperature (26 °C) has a positive effect on the quality of the reproductive system, including spermatophores, but high temperatures (29 °C–32 °C) are detrimental in P. setiferus and P. vannamei (Pascual et al. 1998; Perez-Velazquez et al., 2001). A stable captive environment such as maturation facilities has a beneficial effect on male sexual performance as compared to pond culture in P. vannamei and P. stylirostris (Alfaro, 1993; Alfaro and Lozano, 1993). Wild males of P. occidentalis and P. stylirostris seem to adapt well to captivity, improving their spermatophore quality (unpublished data). However, in P. occidentalis the use of artificial ejaculation always induces the expulsion of ampoules (Alfaro et al.,

Table 1 Sperm counts—abnormalities per compound spermatophore and reproductive conditions for wild and captive species of the sub-genus Litopenaeus. Species

Wilda

Captivityb

Conditions in captivity

P. setiferus

40 g: 45 m.—23% (1) 39.5 g: 39.7 m.— 4.0% (8)

MRTDS MRSM

P. occidentalis

44 g: 11 m.—24% (5)

40 g: RT: 0 m. (1) 39.5 g: RT: 7.46 m.—89% (8) 39 g: RT (26 °C): 13.2 m.—5.4% (13) 34 g: RT (30 °C): 6.5 m.—16.1% (13) 44 g: RT: 39 m.—32% (5)

P. vannamei

46 g: 50 m.—22% (4) 36 g: 12 m.— 38% (10)

SD-MRSM

P. stylirostris

N.A.

48 g: RT: 18.6 m.— 36.7% (11) 38 g: MP (tidal): 4.6 m.—17.4% (6) 38 g: MP (earthen): 0.7 m.—26.8% (6) 30–35 g: RT: 15.5–22.6 m.— 52.8–74% (7) 29.1 g: RT: 8.7 m.— 25.5% (12) 25 g: BP: 10 m.—18% (9) 24 g: RT: 20 m.—33% (3) 21 g: MP: 8 m.—13% (10) 50 g: RT: 8 m.—15% (2) 35 g: MP: 5 m.—50% (2) 25 g: MP: 2 m.—75% (2)

N.A.

SD?-MRSM

N.A.: data not available. References: 1) Alfaro, 1990; 2) Alfaro, 1993; 3) Alfaro and Lozano, 1993; 4) Alfaro et al., 1993b; 5) Alfaro unpublished data; 6) Ceballos-Vázquez et al., 2003; 7) Heitzmann et al., 1993; 8) Leung-Trujillo and Lawrence, 1987; 9) Parnes et al., 2004; 10) Rendón et al., 2007; 11) Perez-Velazquez et al., 2001); 12) Perez-Velazquez et al., 2003; 13) Pascual et al., 1998. a Animal body weight in grams: sperm count in millions—sperm abnormality in percentage. b Culture systems: RT = reproduction tank, MP = marine water pond, BP = brackish water pond.

1993b), maybe as a consequence of their heavy spermatophores (0.30–0.13 g) in comparison to P. setiferus (0.14 g), P. vannamei (0.06 g) and P. stylirostris (0.06 g). The specific factors that induce the sexual improvement have not been studied. 5. Male sexual problems Male Reproductive System Melanization (MRSM; Figs. 3 and 4) is a condition that affects open thelyca shrimps: P. vannamei, P. stylirostris and P. setiferus (Chamberlain et al., 1983) and Macrobrachium rosenbergii (Harris and Sandifer, 1986). The signs were clearly described by Chamberlain (1988) and are summarized in Fig. 5. Pioneering research dealing with the cause of this condition revealed the presence of Vibrio (Brown et al., 1979) and Pseudomonas (Chamberlain et al., 1983), but infectivity was not accomplished. Then, MRSM was proven to be a pathological condition in P. setiferus caused by at least three chitinolytic bacterial species, based on bacteria isolation and infectivity assays (Alfaro et al., 1993a). The signs of MRSM were induced by challenging with 50-fold-diluted 24-h bacteria cultures injected directly into the gonopores. Complementary studies with the other species of Litopenaeus have not been published yet. A particular condition occurs in P. setiferus, named by Talbot et al. (1989) as Male Reproductive Tract Degenerative Syndrome (MRTDS; Fig. 3). Alfaro (1990) proposed that this degenerative syndrome of the male reproductive tract was a different condition than MRSM of P. setiferus. Posterior findings by other authors have supported these different etiologies (Sánchez et al., 2001; Pascual et al., 2003; Goimier et al., 2006). The MRTDS is a condition that generates a dramatic decrease in sperm count and increase in percentage of abnormal sperm from spermatophores. At day 35 from capture, spermatophores show no living sperm, but no melanization of sperm ducts is observed (Leung-Trujillo and Lawrence, 1987; Talbot et al., 1989). PerezVelazquez et al. (2001) stated that MRTDS also occurs in P. stylirostris and P. vannamei, but our contributions (Alfaro, 1993; Alfaro and Lozano, 1993), clearly show that these species do not develop the signs of MRTDS of P. setiferus. Based on our previous contributions, it was established that males in captivity, from other species of the sub-genus Litopenaeus, may develop two types of alterations: 1) MRSM and 2) spermatophore deterioration (SD; Figs. 3 and 4). These conditions have also been considered as a same problem, but their signs are completely different, as indicated in Fig. 5. The major difficulty dealing with MRSM has been the definition of the specific condition in relation with SD, since the first gross diagnostics is made by looking at the pigmentation of spermatophores. However, MRSM is a condition that stars in the ampoules and progresses towards the vas deferens, melanizing spermatophores and sperm ducts, causing irreversible sterility and death. On the contrary, SD is a condition associated only with spermatophores, causing sperm renovation (Alfaro and Lozano, 1993). Sánchez et al. (2001) and Pascual et al. (2003) have proposed an explanation for the occurrence of MRSM and MRTDS observed in P. setiferus. In captivity, nutritional and environmental stress reduces the immunological capacity, including the regulation of the phenoloxidase system, which increases melanin production. An increase of melanin is then present in sperm cells producing cell degeneration and finally sterilization of the males. Stress makes the animal more susceptible to microbial infections and disease, reducing its capacity to resist bacteria normally present in sea water. We suggest that this model could also explain the occurrence of SD. 6. Spermatophore renovation Our current understanding of spermatophore renovation in P. vannamei is summarized in Fig. 6. Heitzmann et al. (1993) and Parnes et al. (2006) demonstrated the existence of a molt-dependent

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Fig. 3. Male reproductive conditions of three species of Litopenaeus. Photographs: 1–2) Penaeus (Litopenaeus) setiferus with MRSM and MRTDS, 3) P. (Litopenaeus) occidentalis with an impregnated normal compound spermatophore (I.S.), 4) P. (Litopenaeus) stylirostris with a normal system (N) and SD.

spermatophore renovation mechanism, which empties the ampoules before molting and produces new spermatophores after molting. Since this mechanism is associated with the molt cycle, the renovation occurs cyclically every 2 weeks, depending on the size of males. The molt cycle durations of adult P. stylirostris (52 g b.w.) and P. setiferus (43 g b.w.) are 11–12 days and 13–14 days, respectively (Robertson et al., 1987). This mechanism of renovation seems to play a key role in the wild, but in captivity a complementary mechanism is also activated: spermatophore deterioration (SD). This mechanism was first studied in P. vannamei by Alfaro and Lozano (1993), who reported that SD is a condition for the degradation and the renovation of spermatophores. This condition generates new normal spermatophores after the complete deterioration of the old units (Fig. 3). Recently, Parnes et al. (2006) also confirmed the existence of this mechanism for spermatophore renovation in P. vannamei. It seems that in captivity, males renovate spermatophores by mating or molting, as in the wild, but also by SD. Molting also occurs cyclically in captivity, but a fraction of the male population develops SD in different culture environments such as brackish water ponds (Parnes et al., 2004), and reproduction tanks (Alfaro and Lozano, 1993). Therefore, the molt-dependent renovation of spermatophores is not operational in these males, for unknown reasons, and the SD is activated as an alternative mechanism. Fig. 7 shows the individual response in spermatophore condition of P. vannamei males during eight weeks (Alfaro and Lozano, 1993); some males produced normal spermatophores during the experimental time, suggesting a moltdependent renovation of spermatophores, according to Parnes et al. (2006). Other males activated the deterioration process, that turned spermatophores into partially brown units (stage II), which followed a complete deterioration until their degradation. This process involves

various molting cycles without renovation by mating or molting (Heitzmann et al., 1993), and the artificial ejaculation of deteriorated spermatophores accelerates the renovation of normal white spermatophores (Alfaro and Lozano, 1993; Alfaro, 1996; Parnes et al., 2006). The study by Diamond et al. (2008) analyzed males with SD since no male developed MRSM, only spermatophores presented light tan to dark brown color. The authors did not prove an auto-immune mechanism for melanized spermatophores, and supported the noninfectious melanization for spermatophore renovation as suggested by Parnes et al. (2006). However, the authors found bacteria associated with normal and melanized spermatophores. The fact that no male developed MRSM indicates that these bacteria were not pathogenic or the immune system controlled their propagation. It was proposed that SD is an adaptation to captivity since no melanization cases have been reported from the wild (Parnes et al., 2006), and the incidence of SD is higher than MRSM in captivity. Therefore, it is possible that under some stressful culture conditions, males with SD may develop MRSM. Based on the present understanding, the following model for the renovation mechanism is proposed. According to Parnes et al. (2006), the molt-dependent spermatophore disappearance is an endogenous, cyclic phenomenon that occurs in all mature P. vannamei males at all times. This phenomenon involves the internal degradation of the spermatophores during the 12 h before molt, possibly by acellularmatrix degradation processes and phagocytosis of the spermatozoa. Our observations of the SD phenomenon (Alfaro and Lozano, 1993) also indicate that spermatophores degrade internally showing melanization and size reduction until complete disappearance after some weeks. Therefore, it seems that the fast renovating mechanism for white spermatophores is deeply delayed in males that activate a melanization mechanism. Still, the precise cellular mechanism that eliminates spermatophores has to be elucidated. However, the model

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Fig. 4. Male reproductive conditions of Penaeus (Litopenaeus) vannamei. Photographs: 5) male with normal spermatophores stage I, 6) male with SD stage II, 7) male with MRSM stage III, 8) isolated sections of a normal (N) reproductive system and MRSM stage III.

proposed by Sánchez et al. (2001) and Pascual et al. (2003) for P. setiferus syndromes, seems a logical hypothesis to explain the noninfectious melanization of spermatophores. The degradation of spermatophores has proven not to be an autoimmune response. A pioneering effort to this hypothesis was presented by Alfaro (1990), who followed an intraspecific transplantation approach. The implantation of spermatophore sections (2 × 2 mm) into female P. setiferus did not induce graft melanization as compared to 31% induction in control cuticle sections after 8 days from surgery. A different approach was followed by Diamond et al. (2008), using haemocyte–sperm interaction assays. Their findings indicate that normal sperm do not trigger a phagocytic response; only damaged sperm and associated debris are apparently recognized as foreign leading to its engulfment by phagocytic haemocytes. Consecutive spermatophore renovations by artificial ejaculation have demonstrated to be a practical procedure for improving sperm quality in P. vannamei (Alfaro and Lozano, 1993; Ceballos-Vázquez et al., 2004) and P. stylirostris (Alfaro, 1993). This simple technique substitutes the molt-dependent spermatophore renovation mechanism and avoids the activation of SD. 7. Nutrition in male reproduction The nutritional needs of male brood shrimp have received little attention as compared to female reproduction and general nutrient requirements of penaeoid shrimps (Harrison, 1990, for review; Browdy, 1992, 1998, for review; Shiau, 1998, for review; Wouters et al., 2001, for review; Perez-Velazquez et al., 2003). Reduced sperm quality has been associated with vitamin E (Chamberlain, 1988) and ascorbic acid deficiencies (Leung-Trujillo and Lawrence, 1988), and diets over-saturated with vitamin E and/or

astaxanthin affected negatively spermatophore regeneration in P. vannamei (Wouters et al., 1999). A supplement of soy lecithin has a beneficial effect on sperm counts of P. stylirostris (Bray et al., 1989). Perez-Velazquez et al. (2003) found that the common fresh-food diet used in maturation laboratories for P. vannamei provides a deficiency of vitamins and/or minerals, affecting sperm quality. Goimier et al. (2006) reported on the effect of dietary protein level over sperm quality of P. setiferus and suggested that a 45% level is the optimum. They indicate that an immune reaction can occur in response to excess dietary protein affecting several physiological functions included sperm synthesis and sperm quality. 8. Acrosome reaction and sperm maturation It has been stated that induction of the acrosome reaction is the only definitive criterion for determining sperm viability in penaeoid shrimp since the sperm is non-motile (Griffin and Clark, 1987). The egg water (EW) technique for in vitro induction of the acrosome reaction in a shrimp (Sicyonia ingentis) was first described by Griffin et al. (1987), generating 75% reactive sperm by 5 min exposure. The technique uses the jelly precursor released by eggs during spawning in artificial seawater, contained in beakers. The technique was used for sperm analysis in P. vannamei (Wang et al., 1995), reporting 37.4% reactive sperm for pond grown males. Sperm from wild P. occidentalis males reacted against conspecific EW, but at a low rate (4–17%), suggesting that further maturation may be required in the external surface of the thelycum (Alfaro et al., 2003). Recently, Alfaro et al. (2007) demonstrated that P. vannamei sperm initiate the synthesis of the filamentous meshwork (FM) in the male reproductive system and that more material accumulates after mating. On the contrary, the male reproductive system of P. stylirostris

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Fig. 7. Individual spermatophore evaluation over time for cultured Penaeus (Litopenaeus) vannamei based on Alfaro and Lozano (1993). N: normal white spermatophore, F: spermatophore in formation, EA: empty ampoule, PB: partially brown spermatophore, CB: completely brown spermatophore.

does not appear to activate the synthesis of the FM. It is proposed that the FM region is an essential part of the acrosome that continues its formation after mating in some species of Litopenaeus. After mating, sperm cells from P. occidentalis are subjected to physiological changes, which improve their capacity to react against conspecific EW. 9. Spermatophore preservation

Fig. 5. Male sexual problems of Penaeus (Litopenaeus) in captivity, according to Chamberlain (1988), Talbot et al. (1989) and Alfaro and Lozano (1993).

Goguenheim et al. (1999) and Le Moullac et al. (2003) described a protocol for chilled storage (4 °C, 1 week) of P. stylirostris spermatophores, using a preservation medium composed of 0.22 µm filtered seawater adjusted to pH 7 using Tris–HCl containing penicillin and streptomycin. Nimrat et al. (2006) developed a protocol for chilled storage (1 month) of P. vannamei spermatophores at 2–4 °C, using mineral oil with 0.1% penicillin–streptomycin to prevent bacterial proliferation; apparent sperm viability was measured using eosin– nigrosin staining. Cryopreservation efforts for penaeoid spermatophores have been published for P. vannamei (Lezcano et al., 2004) and P. monodon (Vuthiphandchai et al., 2007). For P. vannamei, the cryopreservation of spermatic mass had the highest percentage of cell viability postthawing, measured by flow cytometry, as compared to spermatic suspension and complete spermatophore, using slow cooling rate in the presence of methanol (61.6%). Successful cryopreservation of P. monodon spermatophores was achieved using Ca-free saline containing 5% DMSO and one-step cooling rate of −2 °C min− 1 between 25 and −80 °C before storing in liquid nitrogen; sperm viability was measured by eosin–nigrosin staining and artificial insemination. 10. Conclusions

Fig. 6. Spermatophore renovation in Penaeus (Litopenaeus) vannamei based on Heitzmann et al. (1993), Alfaro and Lozano (1993) and Parnes et al. (2006).

The male reproductive system of the open thelyca penaeoid shrimps is a complex physiological unit, for the hormonal control of male sexual differentiation, the production of sperm, and possibly for pheromone release. Sperm production has received more attention for its immediate practical application on the controlled reproduction of shrimps. Manipulation of the AG in the family Penaeidae has not been developed. Basic and applied research in this field will improve our knowledge on the sex-determining mechanism (Campos-Ramos et al., 2006). From a practical approach, monosex culture based on bimodal growth patterns as developed for M. rosenbergii (Aflalo et al., 2006) could also be applied for penaeoid shrimps since females exhibit superior growth to males. P. vannamei males grow slower than females after reaching maturity around 20 g. b.w. (Parnes et al., 2004); however, these animals are normally harvested below that

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weight. Pheromones are another interesting research subject for the near future. The quality of Litopenaeus spermatophores in culture facilities is different among species, being P. vannamei the species that better adapt to captivity; however, recent data from P. occidentalis also show a positive male sexual adaptation (Alfaro, unpublished data). P. setiferus is the most sensitive species since massive nauplii production by means of natural mating has not been reported (Pascual et al., 2003). Few data available from P. stylirostris suggest that these males exhibit low sperm counts and good percentage of abnormalities in reproduction facilities. Three major problems have been discovered associated with the male reproductive system of Litopenaeus. Similar conditions have not been reported from other decapod crustaceans, except for MRSM in M. rosenbergii. The present understanding of these problems allow us to manage them by applying appropriate protocols. SD is a noninfectious mechanism for spermatophore renovation, and manual ejaculation can be used to accelerate the process. MRSM is a bacteriainduced pathology of low incidence, which cannot be treated but it can be prevented by applying good water sanitation and adequate male handling. MRTDS of P. setiferus seems to be a stress-induced physiological condition that will require a nutritional and or genetic approach to be solved. In vitro fertilization of Litopenaeus has been difficult to accomplish (Alfaro et al., 1993b; Misamore and Browdy, 1997). Recent findings indicate that sperm of open thelyca penaeoid shrimps also complete maturation and capacitation on the thelycum (Alfaro et al., 2007); therefore, a completely different approach is needed to achieve this biotechnological tool. Acknowledgements This review was supported by Ley de Pesca from the Government of Costa Rica. Special thanks to unknown referees for their valuable comments. References Aflalo, E.D., Hoang, T.T.T., Nguyen, V.H., Lam, Q., Nguyen, D.M., Trinh, Q.S., Raviv, S., Sagi, A., 2006. A novel two-step procedure for mass production of all-male populations of the giant freshwater prawn Macrobrachium rosenbergii. Aquaculture 256, 468–478. Alfaro, J., 1990. A contribution to the understanding and control of the male reproductive system melanization disease of broodstock Penaeus setiferus. Master's Thesis, Department of Wildlife and Fisheries Sciences, Texas A & M University, USA. Alfaro, J., 1993. Reproductive quality evaluation of male Penaeus stylirostris from a grow-out pond. J. World Aquac. Soc. 24, 6–11. Alfaro, J., 1994. Ultraestructura de la glándula androgénica, espermatogénesis y oogénesis de camarones marinos (Decapoda: Penaeidae). Rev. Biol. Trop. 42, 121–129. Alfaro, J., 1996. Effect of 17-alpha-methyltestosterone and 17-alpha-hydroxyprogesterone on the quality of white shrimp, Penaeus vannamei spermatophores. J. World Aquac. Soc. 27 (4), 487–492. Alfaro, J., Lozano, X., 1993. Production and deterioration of spermatophores in pondreared Penaeus vannamei. J. World Aquac. Soc. 24, 522–529. Alfaro, J., Lawrence, A.L., Lewis, D., 1993a. Interaction of bacteria and male reproductive system blackening disease of captive Penaeus setiferus. Aquaculture 117, 1–8. Alfaro, J., Palacios, J.A., Aldave, T.M., Angulo, R.A., 1993b. Reproducción del camarón Penaeus occidentalis (Decapoda: Penaeidae) en el Golfo de Nicoya, Costa Rica. Rev. Biol. Trop. 41, 563–572. Alfaro, J., Muñoz, N., Vargas, M., Komen, J., 2003. Induction of sperm activation in open and closed thelycum shrimps. Aquaculture 216, 371–381. Alfaro, J., Ulate, K., Vargas, M., 2007. Sperm maturation and capacitation in the open thelycum shrimp Litopenaeus (Crustacea: Decapoda: Penaeoidea). Aquaculture 270, 436–442. Alfaro, J., Zúñiga, G., García, A., Rojas, E., 2008. Preliminary evaluation of the effect of juvenile hormone III and methyl farnesoate on spermatophore quality of the white shrimp, Litopenaeus vannamei Boone, 1931 (Decapoda: Penaeidae). Rev. Biol. Mar. Oceanogr. 43, 167–171. Bauer, R.T., Cash, C.E., 1991. Spermatophore structure and anatomy of the ejaculatory duct in Penaeus setiferus, P. duorarum, and P. aztecus (Crustacea: Decapoda): homologies and functional significance. Trans. Am. Microsc. Soc. 110 (2), 144–162. Bray, W.A., Lawrence, A.L., Leung-Trujillo, J.R., 1989. Reproductive performance of ablated Penaeus stylirostris fed a soy lecithin supplement. J. World Aquac. Soc. 20 (1), 19A (Abstr.).

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