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Indirect evidence indicates that penaeids may have a W/Z sex heritability mechanism with the androgenic gland (AG) mediating masculine differentiation. The present study evaluated the immune response against grafts and the expression of primary and secondary male sexual characters in Litopenaeus vannamei females implanted with terminal ampoules and associated AGs. Experiments included grafting females at PL 34–48 . Grafting at PL 37 generated few cases of melanization exhibiting dark coloration covering parts or the whole implant while most implants showed white coloration, without any trace of melanization. Histology of melanized implants showed necrotic tissue, encapsulated by black depositions. These melanized capsules were externally surrounded by normal haemocytes and flat haemocytes in the middle. An experiment evaluating possible absorption of the grafts based on recovery, gross coloration, size of implants, and histology showed that Complete terminal ampoule-AG implants, Control-vas deferens implants and Abdominal muscle implants, recovered at day 7 and 15, exhibited gradual and statistically significant decrease in size, which was more evident for muscle implants. Grafts with normal appearance from this experiment remained viable during 7 and 15 days post-implantation; these tissues showed a normal cellular structure with neither signs of melanization nor haemocyte infiltration. Appendices masculinae did not develop in any implanted females or controls. Concerning petasma development, two females implanted with AG showed a male phenotype in their first endopodites, characterized by a straight shape at the distal region, a middle protuberance and absence of setae along the middle curved edge of the structure. Regenerated and intact endopodites of Control-proximal terminal ampoule females showed the typical female phenotype with a slender shape with a curved distal edge, without a middle protuberance, and with setae along the middle curved edge of endopodites. Implanted females showed no evidence of masculinization or abnormal development of oocytes. In the light of the present study the effects of AG transplantation on sex in penaeids should be further evaluated in younger PL stages. Statement of relevance This contribution presents new information on sex reversal technology on Litopenaeus vannamei based on androgenic gland implants . The findings are novel for the family Penaeidae concerning the immunological response to tissue grafting and the plasticity for sex reversal in this world aquaculture species. [ABSTRACT FROM AUTHOR]

The recent introduction into aquaculture of RNA interference (RNAi) for producing the preferred all-male monosex cultures, which give improved yields, has raised awareness of the need to investigate the consequences of such novel biotechnological manipulations. Here, we present meta-analysis style study on data from observations of three consecutive cultured all-male (ZZ) generations of the giant freshwater prawn Macrobrachium rosenbergii (De Man). Each consecutive generation comprised the progeny of RNAi-manipulated sex-reversed males. The manipulation was achieved through the administration of dsRNA encoding the insulin-like androgenic hormone into males (which transformed them into ‘neofemales,’ ZZ) of the previous all-male generation. Each generation was cultured in a separate earthen pond for a short (~ 4–5 months) growout period. At harvest of each of the generations, the typical M. rosenbergii population structure comprising three male morphotypes was obtained. An anatomical examination of the male reproductive system of a representative specimen of the third all-male generation showed normal reproductive outputs, even though the prawns had been grown without the presence of females (WZ) for three generations. At the molecular level, expression of vital male-specific genes in the third generation of all-male M. rosenbergii culture was demonstrated. Thus, the present study showing the lack of any overtly apparent long-term consequences of the RNAi-based biotechnology provides support for the responsible use of temporal RNAi in aquaculture. Finally, the absence of the W chromosome for three generations raises questions with regard to its role and content with respect to crustacean sexual determination and differentiation. [ABSTRACT FROM AUTHOR]

Quantitative analysis of hypertrophy and hyperactivity in tissue of the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei was made during the molt cycle by measuring the area of androgenic gland cells located in the subterminal region of the terminal ampoule and measuring the expression of the insulin-like androgenic gland hormone precursor gene ( Lv-IAG ) in the distal vas deferens and terminal ampoule, respectively. Data were compared to intact shrimp at the same molt stages. In eyestalk-ablated shrimp, the lack of negative control from the X-organ–sinus gland complex produced hypertrophy in androgenic gland cells, because the area of the cells folded approximately 1.5 times up to the intermolt stage, when compared to the intact group. It also produced hyperactivity because the relative expression of Lv-IAG was significantly higher than the intact group at all molt stages. After gathering data from the vas deferens and terminal ampoule, the relative expression of Lv-IAG was maintained at higher levels, without a significant difference during the molt stages. However, after each tissue was analyzed independently, a bell-shape curve pattern occurred, according to the molt cycle, as also occurred in intact shrimp. This suggests that in the absence of eyestalks, the brain and nervous system are operating secondarily to synchronize Lv-IAG expression into the molt cycle. The relative expression of Lv-IAG was significantly greater in the terminal ampoule than in the distal vas deferens. In summary, these findings support a reproductive endocrine axis in L . vannamei , as an eyestalk–androgenic gland linkage concomitant with the molt cycle. [ABSTRACT FROM AUTHOR]

Abstract: Sex reversal technology, realized through androgenic gland (AG) manipulation, was recently introduced as a process for production of all-male producing broodstock. This technology exploits however, a relatively small number of sex-reversed broodstock. Thus, both genetic improvement via a breeding program and prevention of inbreeding are needed to ensure the sustainability of such technology. Three wild strains of prawns originating from geographically (though not necessarily genetically) isolated locations in India [Gujarat (G), Kerala (K) and West Bengal (WB)] were assessed for their suitability as breeders for all-male production. In addition, their potential for a selective breeding program was evaluated. A comparative evaluation of early sex segregation, sex reversal, growth performance, and population structure in the three selected strains was performed. Among the purebred strains, after eight months of grow out in earthen ponds, growth performance of the WB strain was the best (59.39±1.08g), while that of G was the poorest (26.50±0.94g). Strain-additive genetic effects for body weight at harvest were highest for the WB strain (+45.9%) and lowest for the G strain (−28.3%). Body masses of WB×K and WB×G crosses were 14.2% and 8.8% above the mean mass of the purebred strains, respectively, while that of the K×G cross was 23% below this value. In most crosses, males reached heavier mean body weights than did females with higher frequencies of the large male morphotypes being seen in the WB purebred strain and its respective crosses. Reciprocal effects for body mass ranged from 4% to 14.9% below the mean of the purebred strains. These negative signs mean that in the two crosses involving the WB strain, growth performance is higher when this stain was used as the sire strain. Similarly, the growth performance of the K×G cross was higher when the former was used as the sire strain. Average heterosis effect for body weight was minor (−0.51±0.73) and did not differ significantly from zero. The high correlation between strain additive effects (the major source of variation in growth) and total performance for body weight (r =0.927) indicate the existence of valuable genetic variation that could be exploited in a selective breeding program. For all-male production, males from the three strains were segregated at early post-larval stages and microsurgical AG removal was performed. In all the strains, similar low levels of complete sex reversal into functional neo-females (genetic males) were realized (0.17% −0.34%). These produced relatively small numbers of neo-female to be crossed with normal males to produce the desired all-male population, but raise the possibility that such a process could result in a genetic bottleneck. Thus, a genetic improvement scheme for each strain integrated with periodical crosses of the resulting neo-females from one strain with males from another strain is suggested to avoid inbreeding. [Copyright &y& Elsevier]

Abstract: Sexual differentiation in male crustaceans is known to be controlled by the androgenic gland (AG), possibly through a peptide hormone. Recently, three freshwater and one marine crustacean decapod genes encoding AG-specific insulin-like peptides were characterized. We report here the molecular cloning of the complete sequence encoding an AG-specific insulin-like peptide (Pm-IAG) in the commercially important marine Penaeid prawn, Penaeus monodon. The deduced precursor sequence consists of a signal peptide, B chain, C peptide and an A chain. It exhibits the same structural organization as that of previously identified crustacean insulin-like androgenic gland specific peptides (IAGs). The positions of cysteine residues of the putative A and B chains, which govern the folding of the mature peptide via the formation of disulfide bridges, are highly conserved among the prawn and other crustaceans, while the rest of the amino acids show low sequence similarity. Gene expression analysis of Pm-IAG in several tissues, including the closely juxtaposed sperm duct and muscle, confirmed that it is specifically expressed in the AG. The findings suggest that with an appropriate intervention, sexual differentiation could be manipulated and thus might be instrumental for the establishment of monosex culture in this bimodally growing shrimp. [Copyright &y& Elsevier]

Abstract: Timing of organogenesis and subsequent development of genital organs were studied in female and male Pacific white shrimp Penaeus (Litopenaeus) vannamei postlarvae. This was linked to the timing of differentiation of external structures that differentiate the genders. Anatomy of the gonad appears to be unique for penaeid species. The genital organ was fully recognized from postlarve day-16 (PL16) as a bilateral lobe located in the anterior region of the midgut gland (first anterior lobes) that connects to an anterior perpendicular collector tube that extends dorsally towards the posterior region of the midgut gland and forms an inverted U-shape collector. Eight bilateral lobes in females (second to ninth) and the bilateral oviduct between the seventh and eighth lateral lobes and seven bilateral lobes in the male (second to eighth) are connected along the inverted U-shape collector tube. These lobes extend over the surface of the midgut gland beneath the pericardium. Shortly after organogenesis of the female gonad, the tenth bilateral lobe emerges from the distal region of the collector tube and continues dorsally along the intestine, and the fourth bilateral lobe did not develop and regressed until apparently absorbed. In males, the posterior bilateral vas deferens emerges from the same region. Around PL50 (0.5–0.6 g; 45–50 mm), external gender differentiation was recognized in the form of the thelycum in females and the gonopores in males. Additionally, the male androgenic gland appears at the posterior-external wall of each anterior vas deferens, surrounded by connective tissue that attaches to the anterior vas deferens and the eighth testicular lobe. Gonad differentiation occurred from PL68 (1.8–2.2 g; 70–74 mm), where it was possible to differentiate the female ovary from the male testes. Timing of sex reversal studies in penaeids is discussed. [Copyright &y& Elsevier]

Abstract: Males of the giant freshwater prawn Macrobrachium rosenbergii grow faster and reach higher weights at harvest than females a fact which makes the culture of all-male populations desirable. All-male populations were produced by mating sex-reversed males, i.e., neofemales, with normal males. Neofemales capable of mating and spawning were produced by removal of the androgenic gland (AG) from immature M. rosenbergii males. The main obstacle to developing a technology based on this type of manipulation is the difficulty of identifying males at a sufficiently early stage of development. To overcome this problem, we developed a novel two-step scheme for large-scale microsurgical andrectomy. Phase I post larvae were andrectomized at ages 25–60days after metamorphosis (PL25–60). A low success rate of functional sex reversal, resulting with all male progeny was obtained (1.3%). In the light of the low success rate and a number of cases of abnormal reproductive development, a second phase was introduced in which the progeny (presumed males) of neofemales from phase I were andrectomized at earlier ages (PL20–30). This two-phase protocol enabled a large quantity of juvenile males to be andrectomized. In phase II there was a significant increase in the number of sex-reversed animals (developed ovaries) and a significant shortening of the time to maturation. Comparison of the all-male progeny with a normal mixed population showed higher growth performance of the all-male population. This advantage together with the shorter maturation time in phase II opens the possibility to scale-up the system to field conditions. Since sexual dimorphic growth patterns are common among decapod crustaceans, it is obvious that the results of this study will have applied significance for many aquacultured species. [Copyright &y& Elsevier]