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by Amir Sagi

In many crustacean species a sexualbimodal growth pattern is exhibited wherefemales grow larger than males of the

species or vice versa. In two of the mosteconomically important penaeid shrimps,Litopenaeus vannamei and Penaeusmonodon, females grow larger than males.On the contrary, in several other culturedspecies such as the Australian red-claw

Monosex culture of prawns throughandrogenic gene silencing

crayfish Cherax quadricarinatus, males growfaster and reach heavier weights thanfemales. This is also the case for the giantfreshwater prawn Macrobrachiumrosenbergii, as males reach market size fasterthan the females. Thus, an all-male monosexpopulation culture of the species is desirable.

Commercial production of freshwaterprawn Macrobrachium rosenbergii has been

the subject ofresearch andcommercialenterprise inmany countries in Asia for several decades.This species is native to the tropical Indo-Pacific region of the world and is aneconomically important crop in China, India,Vietnam and many other Asian countries. It

Male prawns at harvest.

Amir Sagi

A newly developed technology of producing all-male populations offreshwater prawns by gene silencing in the androgenic gland through

RNA interference methods results in a sustainable technology to increaseyields for farmers.

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AQUACULTURE

has high demand as a food item and as anexport product. Its relatively limited wild catchhas resulted in a gradual increase intraditional culture of the prawn. The globalmarket for prawns was growing annually till2009. China, India and Vietnam togetherproduced more than 200 000 mt of prawn perannum (with an annual value of US$ 2.4billion). Bangladesh, Thailand and othercountries produce smaller amounts.

Monosex culture

Culture of monosex populations is acommon procedure in animal husbandry.Differences in growth rate, alimentary needsand behavioural patterns between males andfemales of the same cultured species dictatethe need to establish managementprocedures specifically adjusted to one sex orthe other. Moreover, since a monosex culturepopulation is inherently non-breeding, energy

Monosex culture of prawns through androgenic gene silencing

Reader enquiry number 5

One large male and several reproductive female Macrobrachium resenbergii prawns from thesame batch.

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Monosex culture of prawns through androgenic gene silencing

is focused on growth and unwanted breedingis prevented. Reproduction can be carriedout in such systems under separate,controlled conditions. The monosex culturestrategy has become a common practice infish culture and attempts have been made toapply it to crustacean culture. A small scaleexperiment conducted as early as 1986 inIsrael, by hand-segregating M rosenbergiimonosex populations in happa nets, resultedin significantly higher yields when all-malepopulations were cultured. More recently, aneconomic analysis of all-male populationculture in India showed an income increase ofabout 60% over mixed and all-femalepopulations, taking into account expensescaused by labour-intensive hand segregationand related losses.

Sex reversal and themicrosurgerybiotechnology

In M rosenbergii a fully functional sexreversal could be achieved by microsurgicalmanipulation of the androgenic gland (AG) inearly post-larval males. Although AGmanipulation dates back more than half a Reader enquiry number 6

Professor Amir Sagi is former dean ofthe Faculty of Natural Sciences, and a

member of the Department of LifeSciences and the National Institute forBiotechnology, Ben Gurion University

in the Negev, Israel. He is currentlyholding the Lily and Sidney OelbaumChair in Applied Biochemistry at the

University and is President of theInternational Society for Invertebrate

Reproduction and Development.

century, a biotechnological approach for thegeneration of all-male populations was onlyrecently devised, and it involves themicrosurgical removal of the AG from juvenilemales. AG removal from immature malesresults in sex reversal. We found that sex-reversed animals (termed “neo-females”) arecapable of mating with normal males toproduce an all-male offspring population.When successful, such removal leads to thefull and functional sex reversal of males intoneo-females (phenotypic females with malegenotype) that develop feminised gonads.Since M rosenbergii males are thehomogametic sex, bearing two homologoussex chromosomes (ZZ) as in several otherstudied crayfish and shrimp species, sex-reversed males produce 100% maleprogeny. The microsurgery biotechnology forall-male monosex culture of prawns has beenused in India, Thailand and Vietnam.

Biotechnologies basedon gene silencing andthe BGU line

The gene silencing based biotechnologywas developed in our laboratory at the

National Institute of Biotechnology, Ben GurionUniversity (BGU) of the Negev, Israel. It isbased our recent finding of a new geneencoding an insulin-like androgenic glandhormone from M rosenbergii (termed Mr-IAGand fully patented). The patent is licensed toTiran Shipping Ltd (1997) with exclusive,worldwide rights to utilise the intellectualproperty (IP) and to commercialise the licensedproducts. It was found that silencing of this genethrough RNAi (RNA interference) at an earlypostlarval stage could cause complete sexreversal of a male into a functional neo-female.The technology includes the application ofgene silencing in males of the fast growingBGU line of M rosenbergii prawns. Throughthe use of specific molecular sex markers, theidentified males are transformed through atemporal single gene silencing into neo-females. The neo-females are grown and bredto produce all-male populations.

The technology does not use hormones orchemicals and it does not produce geneticallymodified prawns (a completely non-GMOtechnology). Because it does not involvegenetic modification of the organism, therebyby-passing the regulatory pipeline required ofgenetically-modified crops, this is a boon formonosex biotechnology. As the intervention istemporal with the silencing agent beingnaturally occurring RNA which degrades in afew days, it is not transmissible to futuregenerations. Indeed, this approach may be oftremendous applied merit in the aquacultureindustry. Moreover, it could also form part of asustainable solution for the management ofinvasive and/or pest crustacean species,where the production of non-reproducing maleor female populations is sought.

How Mr-IAG silencing results in sex reversal