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In crustacean aquaculture, size dimorphism between males and females is the main key factor determining the advantage of monosex aquaculture over that of mixed populations. This factor is particularly relevant for the freshwater prawn, Macrobrachium rosenbergii, for which intensification of cultures is complicated by a complex social structure in which large dominant males are territorial and inhibit the growth performance of smaller males and females. It has therefore been suggested that all-female mono-culture could be the practice of choice, since females are less aggressive and less territorial and are believed to exhibit a relatively homogenous growth pattern. Here we report the first large-scale comparative field study of all-female and mixed populations under extensive and intensive stocking conditions in earthen ponds. The study was facilitated by application of our novel biotechnology based on a single injection of suspended hypertrophied androgenic gland cells. Under both our intensive and extensive conditions, the all-female cultures showed better performance than the mixed cultures in most key aquaculture parameters including survival rate and yield per hectare. Also, the intensively stocked all-female ponds showed better feed conversion ratio than mixed ponds. Furthermore, while the mean size of the animals did not differ significantly between the two treatments, the all-female populations exhibited significantly higher size uniformity. Our study suggests that for M. rosenbergii, female monosex aquaculture is a sustainable method to yield a homogenous crop.

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.

An unresolved problem in aquaculture ponds is the susceptibility of freshwater fish to parasitic diseases, such as those caused by Centrocestus trematodes, which are transmitted by snails of the Thiaridae family. Two species of this family are common in Tilapia aquaculture ponds in Israel, the endemic Melanoides tuberculata and the invasive Thiara scabra, both being hosts of various disease-causing parasitic trematodes. Promising biocontrol agents are freshwater prawns of the genus Macrobrachium, which are known to be voracious predators of freshwater snails. Prawns in contrast to fish, occupy a benthic niche in the aquaculture pond and may thus be expected to prey on disease-carrying snails, which burrow into the pond bottoms. Prawns also bring the added advantage of constituting a high-value side-product to the pond-produced biomass without the need for additional feed. Following recent biotechnological advances in monosex prawn production, non-reproducing monosex prawn populations are now commercially available for testing as ecologically safe biocontrol agents in aquaculture. Laboratory predation experiments examining the ability of monosex Macrobrachium rosenbergii prawns to prey on Melanoides tuberculata and T. scabra snails showed that even a single prawn weighing >4 g is capable of exterminating dozens of snail hatchlings per day and hence of preventing hatchling recruitment in both snail species. Large prawns exhibited significantly superior predation abilities with respect to large snails of both species, with Melanoides snails being more susceptible to predation than Thiara snails. This study is the first to integrate laboratory studies with field observations on the utility of monosex all-female Macrobrachium rosenbergii prawns as biocontrol agents of damaging snails in fish ponds. These prawns were found to be effective biocontrol agents in Tilapia growout aquaculture ponds in terms of reduction in both snail abundance and rates of fish infection with the snail-borne parasites. The promising results of this biocontrol method call for further study aimed at optimizing the biocontrol power and profit of monosex prawn populations in polyculture with fish as part of wider and more comprehensive ecological risk assessment studies.

The recent introduction of RNA interference (RNAi) based biotechnology in aquaculture has aroused public awareness regarding safety aspects related to the use of such temporal transcript manipulation. In the present study, we demonstrate use of this biotechnology in large-scale production of all-male freshwater prawn Macrobrachium rosenbergii, relying on a two-phased approach. First, the insulin-like androgenic gland hormone (Mr-IAG) was silenced by dsRNA injections, causing sex-reversal of males into ‘neo-females’ (genetic males with female phenotype, with 86% success in this study). The next step involved mating the neo-females with normal males to produce all-male progeny. The administered exogenous dsRNA was fully cleared from the prawn's tissues after 7 days. Gene silencing through dsMr-IAG appeared to be temporary, with decay seen over time and nearly full expression of Mr-IAG (~ 82%) being restored after 28 days. Assessment of reproduction and fecundity in normal females versus neo-females revealed that in both groups, ~ 80% of the prawns were in a reproductive state and no significant difference was found in their reproductive output, either in terms of brood mass or number. Population structure, as indicated by the normal occurrence of male morphotypes, in both mixed-sex and all-male populations was examined in terms of weight and morphotype frequency distribution. The final harvest and marketing of the all-male prawn offspring occur about 19 months, i.e., one generation after the actual RNAi procedure. Thus, on top of the aquaculture yield advantage, the use of such RNAi procedure in crustacean aquaculture seems to be safe due to its temporary nature.

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.

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 sexreversed 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.

Rearing crayfish in individual compartments could be a means of overcoming social-dependent density limitations in order to increase yields per unit area in intensive culture. Two experiments were conducted to evaluate the impact of neighbors on the growth of redclaw crayfish (Cherax quadricarinatus) males in three-dimensional units, consisting of 144 individual compartments, designed for intensive battery culture. The first experiment revealed significantly lower growth rates in crayfish surrounded by neighbors on all sides than in those reared with no neighbors in adjacent compartments, or with neighbors only in compartments on the same level but not above or below them. The second experiment revealed significantly lower growth rates in relatively small crayfish surrounded by large crayfish in a mixed-size unit than in those surrounded by similar-sized small crayfish in a uniform-size unit. No such effect was found for relatively large crayfish surrounded by small crayfish. The results clearly indicated interactions between crayfish in our system, despite them being housed in separate compartments. Growth was adversely affected by the presence of neighbors in adjacent compartments, especially vertically adjacent ones, and the presence of larger individuals in adjacent compartments. This social effect was attributed to partial tactile contact between neighbors in our system. The consequences of these findings for battery culture of crayfish are discussed.