The Insulin-Like Androgenic Gland Hormone in Crustaceans: from a Single Gene Silencing to a Wide Array of Sexual Manipulation-Based Biotechnologies

Biotechnology Advances 30 (2012) 1543–1550

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Research review paper The insulin-like androgenic gland hormone in crustaceans: From a single gene silencing to a wide array of sexual manipulation-based biotechnologies

Tomer Ventura, Amir Sagi ⁎

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel article info abstract

Available online 25 April 2012 Due to the over-harvesting and deterioration of wild populations, the ever-growing crustacean market is increasingly reliant on aquaculture, driving the need for better management techniques. Since most cultured Keywords: crustacean species exhibit sexually dimorphic growth patterns, the culture of monosex populations (either Crab all-male or all-female) is a preferred approach for gaining higher yields, with the ecological benefit of reduc- fi Cray sh ing the risk of invasion by the cultured species. Sexual manipulations may also render sustainable solutions to Insulin-like androgenic gland hormone the environmental problems caused by the presence of invasive crustacean species with detrimental impacts Invasive species Lobster ranging from aggressive competition with native species for food and shelter, to affecting aquaculture facilities fi Monosex culture and harvests and causing structural damage to river banks. Recent discoveries of androgenic gland (AG)-speci c Prawn insulin-like peptides (IAGs) in crustaceans and the ability to manipulate them and their encoding transcripts RNA interference (IAGs) have raised the possibility of sexually manipulating crustacean populations. Sexual manipulation is al- Sex reversal ready a part of sustainable solutions in fish aquaculture and in the bio-control of insect pest species, and attempts Shrimp are also being made to implement it with crustaceans. As recently exemplified in a commercially important prawn species, IAG silencing, a temporal, non-genetically modifying and non-transmissible intervention, has enabled the production of non-breeding all-male monosex populations that are the progeny of sexually reversed males ('neo-females'). IAG manipulations-based biotechnologies therefore have the potential to radically transform the entire industry. We review here how this proof of concept could be broadened to meet both aquacultural and environmental needs. We include the major cultured decapod crustacean groups and suggest a sustainable solution for the management of invasive and pest crustacean species. We also review the key considerations for devising a biotechnological approach that specifically tailors the molecular technolog- ical abilities to the management of each target group. © 2012 Elsevier Inc. All rights reserved.

Contents

1. Preface ...... 1544 2. Fromtheandrogenicglandtoabiotechnologicalproofofconcept ...... 1544 2.1. Milestones in the discovery of the androgenic gland and its hormone ...... 1544 2.2. Biotechnological proof of concept: an all-male prawn population – ﬁeld conﬁrmation ...... 1544 3. Tailoringthebiotechnologytoeachofthemajordecapodgroups...... 1545 4. From need to application ...... 1546 4.1. Crustacean aquaculture ...... 1547 4.2. Environmental impact of invasive crustaceans ...... 1547 5. Epilog ...... 1548 Acknowledgments ...... 1548 References ...... 1549

Abbreviations: AG, androgenic gland; Cq-IAG, Cherax quadricarinatus IAG; dsRNA, double-stranded RNA; GMO, genetically modiﬁed organism; IAGs, insulin-like AG-speciﬁcfactors; Mr-IAG, Macrobrachium rosenbergii IAG; RNAi, RNA interference. ⁎ Corresponding author at: Department of Life Sciences and the National Institute for Biotechnology Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel. Tel.: +972 8 6461364; fax: +972 8 6472992. E-mail address: [email protected] (A. Sagi).

1. Preface The chemical nature of the AG hormone remained an enigma for many years. Several ultra-structural studies of the AG suggested that Crustaceans comprise a large (~50,000 species) and diverse sub- the AG hormone is proteinaceous (King, 1964; Okumura and Hara, group of arthropods. Present in almost every habitat, they are, on 2004; Taketomi, 1986). This hypothesis was strengthened by the partial the one hand, economically important as target aquaculture species, purification of an active AG-borne protein extract (Hasegawa et al., but on the other hand, they are also agricultural pests, invasive, and 1987; Juchault et al., 1978; Katakura et al., 1975; Okuno et al., 1997) pathogen carriers. Rampant crustacean over-harvesting and the deterio- and finally, by the full sequence of a glycosylated protein (Martin et ration of wild stocks has left the crustacean trade increasingly reliant on al., 1999) in parallel to its characterization and cDNA cloning, which aquaculture which had an estimated market value of more than US$18 was verified to be AG-specific(Okuno et al., 1999). The encoded protein billion in 2009 (FAO, 2009). The crustacean aquaculture market relies was shown to comprise a linear preprohormone, organized according to primarily on a relatively small number of species (~40 species accounted the insulin-like superfamily of peptides, a signal peptide (cleaved off to for more than 90% of the market value in 2009), most of which are sex- give rise to the prohormone) at the N'-terminus, followed by a B chain, ually dimorphic decapods. In contrast, the estimated number of inva- then a connecting peptide (C peptide), and finally an A chain (Fig. 1A). sive crustacean species is much higher with some studies reporting As is common to all insulin-like peptides, the mature hormone (the B impacts by several hundred species (Galil et al., 2011). In the crayfish and A chains after cleavage of the signal and C peptides) has three disul- sub-group alone, 24 invasive species have been documented, some of fide bridges: two inter-chain bridges, and one intra-chain bridge (within which inflict severe pressure on the populations of the existing natural the A chain; Fig. 1B). These keystone disulfide bridges stabilize the three- crayfish along with a negative impact on aquaculture facilities and har- dimensional structure common to members of the insulin-like super- vests (Gherardi et al., 2011). Solutions for enhancing the profitability of family of peptides (Fig. 1C). The first decapod insulin-like AG-specific crustacean aquaculture and for management of invasive/pest crusta- peptide encoding cDNA (IAG) was discovered almost a decade later cean species may benefit from our better understanding of the sexual (Manor et al., 2007) followed by many others in a relatively short period. differentiation process in crustaceans. With these respects, monosex To date, the IAGs of nine additional decapod species were cloned (six of non breeding population culture was raised as a highly beneficial ap- which are reviewed in detail by Ventura et al. (2011b)), representing proach. Proof of concept was recently shown with the complete and func- the commercially and ecologically most important decapod groups: tional sex reversal through the manipulation of a male differentiating crabs, crayfish, prawns, and shrimps. All of these sequences encode pro- organ and, even more recently, manipulation of the hormone produced teins that share in common linear structure and the identity of the con- by this organ and alteration of the population sex ratio. Here we suggest served cysteine residues, which are the keystone of the insulin-like broadening this approach to include major decapod crustacean groups to super family of peptides (Fig. 1A and B, respectively), and that enable develop biotechnologies that could be tailored specifically to address the three-dimensional folding (Fig. 1C). The multiple sequence alignment each species and the requirements of the industry and/or ecological and of the ten known decapod mature IAGs (B and A chains) are given in environmental issues. Fig. 1D. The recent utilization of RNA interference (RNAi) has revolutionized 2. From the androgenic gland to a biotechnological proof of concept the functional assays of newly discovered genes (Dorsett and Tuschl, 2004) as applied in a number of decapod species (Lugo et al., 2006; While sexual manipulation is currently widely practiced in fish aqua- Shechter et al., 2008). Silencing IAG expression in two decapod species culture, on-going efforts are being made to implement this approach with led to demasculinization accompanied by a shift toward femaleness in crustaceans. In fish, monosex culture is achieved through sex reversal me- one of the species, thus proving the key role of the IAG in male sexual dif- diated by steroid manipulations (Smith et al., 2009). Unlike vertebrates, ferentiation for the first time in decapods (Rosen et al., 2010; Ventura et sexual differentiation in crustaceansisregulateduniquelybyanon- al., 2009). In another recent IAG silencing experiment, full and functional steroidogenic male-specific androgenic gland (AG), which obstructed sex reversal was achieved (Ventura et al., 2012), as detailed below. sporadic attempts made to induce a sexual shift through steroid administration (Ohs et al., 2006). The AG was detected in all studied malacos- 2.2. Biotechnological proof of concept: an all-male prawn population – tracan crustacean species, and its key role in male sexual differentiation field confirmation was exemplified in an amphipod, an isopod, and in several decapod species, marking it as a viable target for sexual manipulation-based bio- In the commercially important freshwater prawn M. rosenbergii, technologies. In recent years, AG-specific insulin-like peptide (IAG)- males grow larger and faster than females (Sagi et al., 1986). A case encoding transcripts (IAGs) were identified in many crustacean species, study in India—a major producer of this species—has shown that hand- of which several are commercially important. IAG silencing in such a segregation of males and the removal of the less-profitable females of decapod species proved useful in obtaining a full and functional sex re- the species increases the grower's income by ~60% (Nair et al., 2006). Al- versal, leading to the production of monosex populations. though AG manipulation dates back half a century (Charniaux-Cotton, 1962), a biotechnological approach for the generation of all-male popula- 2.1. Milestones in the discovery of the androgenic gland and its hormone tions was only recently devised, and it involves the microsurgical removal of the AG from juvenile males. When successful, such removal leads to The crustacean male-specificAGwasfirst observed in the blue swim- the full and functional sex reversal of males into offspring-producing ming crab Callinectes sapidus (Cronin, 1947). Later on, this gland was neo-females (Aflalo et al., 2006). Since M. rosenbergii males are the homo- shown in an amphipod species to be the key regulator of male sexual dif- gametic sex, bearing two homologous sex chromosomes (ZZ) as in sever- ferentiation (Charniaux-Cotton, 1954), and was thus termed the andro- al other studied crayfish and shrimp species, sex-reversed males produce genic gland (Charniaux-Cotton, 1955). Since then, a series of studies 100% male progeny, as first reported by Sagi and Cohen (1990).Fraught have proved that AG implantation masculinize females and its removal with difficulties, however, this biotechnology is hampered by the low feminizes males in an amphipod species (Charniaux-Cotton, 1957, success rate of the microsurgery (~1.3%) and by lengthy (up to ten 1962), an isopod species (Suzuki and Yamasaki, 1991, 1997; Suzuki et months) and labor-intensive progeny testing (Aflalo et al., 2006). These al., 1990), and in several decapod species (Abdu et al., 2002; Aflalo et hurdles led to the exploration for novel technologies to meet market al., 2006; Barki et al., 2003, 2006; Karplus et al., 2003; Khalaila et al., demands. 1999, 2001; Lee et al., 1993; Malecha et al., 1992; Manor et al., 2004; Manipulation of the AG hormone is one such route being explored. Nagamine and Knight, 1987; Nagamine et al., 1980a, b; Sagi and Cohen, IAG (Mr-IAG) silencing in M. rosenbergii arrested spermatogenesis and 1990; Sagi et al., 1990, 1999, 2002; Taketomi and Nishikawa, 1996). temporally inhibited the development of a masculine appendage on a T. Ventura, A. Sagi / Biotechnology Advances 30 (2012) 1543–1550 1545

Fig. 1. AG insulin-like peptides (IAGs): the basic linear structure and a predicted three-dimensional model. All IAGs found thus far (in 10 decapod and 3 isopod species) have characteristics of the insulin-like super family of peptides, including the linear structure of the preprohormone composed of a signal peptide, followed by a B chain, a C peptide, and an A chain (A). The signal and C peptides may be cleaved out (dashed boxes in A) to give rise to the mature hormone, composed of the B chain interlinked by two disulfide bridges to the A chain where another intrachain disulfide bridge occurs (B). These predicted disulfide bridges enable the three-dimensional folding of the predicted mature hormone, as shown here for the predicted folding of Cq-IAG (C; Modified from Manor et al. (2007)). Multiple sequence alignment of ten decapod mature IAGs is given (D; using CLUSTAL W algorithm, manually corrected). The most conserved feature is the predicted backbone consisting of six cysteine residues (highlighted in orange), which suggests disulfide bridges (orange lines). Also, highlighted in orange are two cysteine residues in prawns that may form a third inter-chain disulfide bridge. Each decapod group is represented with a sketch (left, up to bottom): the crab Charybdis feriatus, the red-claw crayfish C. quadricarinatus, the giant freshwater prawn M. rosenbergii, and the white shrimp Litopenaeus vannamei. Species names are represented by the three initial letters of each as follows: Cal. sap., Callinectes sapidus; Por. pel., Portunus pelagicus; Che. des., Cherax destructor; Che. qua., Cherax quadricarinatus; Mac. ros., Macrobrachium rosenbergii; Mac. lar, Macrobrachium lar; Pal. pac., Paleomon pacificus; Pal. pau., Paleomon paucidens; Pen. mon., Penaeus monodon; Mar. jap., Marsopenaeus japonicus. regenerated swimming leg in mature male prawn individuals (Ventura al., 2012), a constraint that required the prior identification of mo- et al., 2009). When performed at early developmental stages, however, lecular sex markers (Ventura et al., 2011a). Since the desired out- prior to the appearance of male sexual characteristics, Mr-IAG silencing come in M. rosenbergii is to obtain all-male populations, and the caused the full and functional sex reversal of males into neo-females. Successfully mated with untreated males, the neo-females produced all-male progenies (Fig. 2)(Ventura et al., 2012). An all-male progeny from such a neo-female was grown in parallel with a normal mixed population, and the growth performances of the two groups were compared. The size distribution of individuals in the mixed population was similar to that of the male individuals in the all-male population, which had twice the number of male individuals (unpublished results). This marked the first field study of a monosex population derived from a single gene silencing-induced sex reversal. Because it does not involve genetic modification of the organism (GMO) under investigation, thereby bypass- ing the regulatory pipeline required of genetically-modified crops (Stein and Rodriguez-Cerezo, 2010), this proof of concept is a boon for monosex biotechnology. As the intervention is temporal, it is not transmissible to next generations. Indeed, this approach may be of tremendous applied merit in the aquaculture industry. Moreover, it could also form part of a sustainable solution for the management of invasive and/or pest crustacean species, where the production of non-reproducing male or female populations is sought.

3. Tailoring the biotechnology to each of the major decapod groups

ThecaseofMr-IAG silencing in M. rosenbergii revealed the constraints that exist and the issues that must be considered when attempting to tailor a specific biotechnology to each of the known decapod groups. fi De ning characteristics of a species' life cycle, reproductive strategy, in- Fig. 2. Mr-IAG-silencing induces full-functional sex reversal of males into all-male tervention timing, mode of inheritance, availability of sex markers and progeny-producing 'neo-females'. A. Ventral view of a Mr-IAG silenced neo-female incu- the desired outcome should all be thoroughly investigated to customize bating eggs in its brood-chamber (arrow, with a few eggs magnified). B. Genomic valida- IAG manipulation-based biotechnology to that species. In the case of tion of the neo-female and broodstock maleness; PCR products generated from intact male and intact female control individuals and from the neo-female and individuals from its M. rosenbergii, Mr-IAG silencing resulted in full and functional sex rever- progeny (upper panel) as templates, as well as a control female and individuals from its sal only when the intervention occurred in the early post-larval stages progeny (bottom panel), separated on an agarose gel. n.c. – negative control, p.c. – positive before the appearance of external sexual characteristics (Ventura et control, f.s. – female-specific marker. Modified from Ventura et al. (2012). 1546 T. Ventura, A. Sagi / Biotechnology Advances 30 (2012) 1543–1550 mode of inheritance involves homogametic males, Mr-IAG silencing crayfish showing that WW females were viable and able to reproduce (to obtain all-male progeny-producing neo-females) is the pre- (Parnes et al., 2003). ferred sexual manipulation. The same set of IAG interventions is also feasible in heterogametic Based on the above, IAG manipulation could drive improved crus- males,asisthecasewithcrabs(Triño et al., 1999)(Fig. 3, right). However, tacean production. Moreover, it could form part of a sustainable solu- since YY males were not shown to be viable, an all-female population tion to the problems associated with invasive and/or pest crustacean production scheme is simpler to achieve than the all-male strategy. species through the production of non breeding monosex cultures. Moreover, since all-female production was shown to be economically This general strategy can be customized according to sex chromosome beneficial in crabs (Triño et al., 1999), the culture of all-female popula- composition and preferred sex to cover all the major groups of industri- tions should be the preferred approach for crabs when addressing aqua- ally grown, invasive, pest, and/or even pathogen carrying crustacean culture management issues (Fig. 3, bottom right). Since little is known species (Fig. 3). IAG expression can either be knocked down in males regarding lobster sex chromosome composition, and lobster IAGs have to obtain neo-females, as was done in M. rosenbergii,oradministered yet to be identified, devising a proper monosex scheme for lobsters will to females to obtain neo-males. Although IAG administration was not be possible only after further study of this commercially valuable group. yet shown to induce full and functional sex-reversal, administration of a recombinant hormone to the isopod Armadilidium vulgare showed 4. From need to application some masculinization (Okuno et al., 2002), and AG implants were shown to induce a full and functional sex reversal in a decapod species Crustaceans comprise a definite number of cultured species with a (Malecha et al., 1992). steady increase in culture volumes in recent years, from more than Four biotechnological routes to achieve monosex populations have 703,000 tons valued at over US$3 billion in 2000 to more than been suggested. In species where the males are homogametic (i.e., 4,127,000 tons with a value exceeding US$18 billion in 2009 (FAO, bear two homologous sex chromosomes, ZZ), and where males are the 2009). This increase dictates the need for better management to support preferred sex, as in crayfish (Parnes et al., 2003) and prawns (Malecha this growing field and enhance yields. Since most cultured crustacean et al., 1992; Sagi and Cohen, 1990), IAG deprivation from males to in- species exhibit sexually dimorphic growth patterns, thus conferring on duce neo-females will enable the production of 100% male populations, monosex population techniques a significant advantage, harnessing solu- as was achieved by Ventura et al. (2012) (Fig. 3, top left). For species tions such as IAG manipulation-based biotechnology has the potential to with homogametic males in which females are the preferred sex, as in radically transform the entire aquaculture industry. Monosex biotechnol- shrimps (Gopal et al., 2010) or prawns under certain management ogies may also contribute to the creation of relatively simple environ- strategies (Malecha, in press), IAG administration to females, designed mental solutions to the problems associated with invasive species, to generate neo-males, will eventually enable the production of 100% ranging from preventing their dissemination to the active eradication of female populations (Fig. 3, bottom left), as supported by findings in those that are already present by releasing sterile monosex individuals.

Fig. 3. Possible production schemes for IAG manipulation-induced monosex populations of the most prominent decapod groups. The IAG can be manipulated in species where males are homogametic (ZZ, left) or heterogametic (XY, right). For each scheme, representative groups of species are denoted. The IAG can be either removed from males to obtain neo-females (top) or administered to females to obtain neo-males (bottom). Sketches represent the giant freshwater prawn M. rosenbergii (top left), the red-claw crayﬁsh C. quadricarinatus (top, second left), the crab Charybdis feriatus (right), and the white shrimp Litopenaeus vannamei (bottom left). T. Ventura, A. Sagi / Biotechnology Advances 30 (2012) 1543–1550 1547

4.1. Crustacean aquaculture that Crustacea is the most successfully introduced phylum in marine systems, accounting for 28% of reports in marine coastal communities As the global crustacean industry becomes increasingly reliant on in North America and 56% of the faunal species found in ballast tanks aquaculture rather than on wild catch (FAO, 2009), new avenues for in Europe (Ashton et al., 2007). Many crustacean species (estimated improving culture techniques must be explored. Table 1 summarizes at hundreds world-wide (Galil et al., 2011)) have been cause for genu- the increase in total yields and incomes of the different cultured decapod ine concern as they are aggressive competitors with native species for groups during the years 2000 and 2009, with the fold-change between food and shelter, they negatively affect aquaculture facilities and har- these years, which emphasizes the immense increase in decapod aqua- vests, they cause structural damage to river banks, and some even culture volumes (data from FAO, 2009). Moreover, the most plausible carry hazardous pathogens. In a recent report that summarized the 27 route for monosex population biotechnology is suggested for each of most threatening alien animal species introduced into Europe for aqua- the above groups based on the desired sex and mode of inheritance culture and related activities (Savini et al., 2010), five decapod species (Fig. 3). In all the above five major farmed crustacean groups, monosex are listed. Table 2 summarizes the number of recorded invasive species population culturing could be a preferred technology. In crabs, either from each of the decapod groups described in Fig. 3, their endemic all-male or all-female populations are favored over mixed populations sources and invaded areas, and the reported impacts. The main vectors (Triño et al., 1999), while in crayfish and prawns, males grow larger of dissemination include man-made canals, live trades, intentional in- and faster and as such, all-male population cultures are desired (Nair et troductions for other purposes, and shipping traffic. Indeed, when im- al., 2006; Sagi et al., 1997). In contrast, female shrimp grow larger and properly managed, aquaculture of commercially important decapod faster, making all-female population cultures preferable (Gopal et al., species is a two edged sword, with its potential to disseminate invasive 2010). Given the economic impact of obtaining single-sex enriched crus- species whose negative impact sometimes eclipses the benefits of its tacean cultures, the idea of generating monosex cultures based on ge- aquaculture. We highlight here several such cases from each group netic manipulation is an attractive one. (described in Fig. 3 and Table 2), which together indicate the need for Monosex crustacean culturing offers several advantages. For in- stringent controls in the relevant aquaculture areas. stance, the fresh marketing season would be prolonged due to faster Among the more than 50 reported alien crab species, the most growth rates as a gender-tailored interface could be implemented (Sagi widely spread is the green shore crab Carcinus maenas. This European et al., 1986). Moreover, the absence of any non-planned reproduction en- crab species was introduced globally through the live bait and sea sures that energy is not diverted from growth and that there are no sib- food industries hand in hand with its substantial contribution to the de- lings competing for resources. Furthermore, selective breeding can mise of the commercial soft-shell clam fishery in the northeastern USA transpire in physically separated surroundings, thereby avoiding com- during the 1940s and 1950s (Grosholz, 2011). This notorious crab spe- promise by parents or siblings drawn from the growout ponds. Monosex cies joins a list of a total of nine crab species that are common predators aquaculture, moreover, is ecologically advantageous: because no repro- of cultured mollusks (Creswell and McNevin, 2008). Another example of duction occurs in the ponds, the environmental risk of introduced aquatic a globally introduced crab is the Chinese mitten crab Eriocheir sinensis, species arising from juveniles invading natural water bodies is reduced. cultured in the Far East as a food delicacy. This species is thought to Even should invasion occur, one gender would not be able to reproduce have been unintentionally introduced to Europe via ballast water dis- and establish an exotic species (Beardmore et al., 2001). charge of larvae in the early 20th century, and later it appeared in the Currently, IAGs are known in representatives of all the major decapod San Francisco Bay area where its burrowing activity causes siltation of groups excluding lobsters. The enhanced yield achievable through mono- the waterways and bank erosion, thus increasing the risk of flooding. sexpopulationculturehasbeenshownincasestudiesinacrabspecies While it requires saline water for breeding, it has been observed as far (Triño et al., 1999), in a crayfish species (Curtis and Jones, 1995; Manor as 1,000 km from the sea, a fact that demonstrates its unfortunate abil- et al., 2002), in a prawn species (Sagi et al., 1986)—where another case ity to move up the river systems, and indeed, this species continues to study reported about a 60% increase in net income to the growers spread throughout Europe and USA (Bentley, 2011). when growing hand segregated all-male populations and discarding the In the crayfish sub-group of over 600 species, 24 invasive species females (Nair et al., 2006)—and in a shrimp species where females were reported worldwide (Gherardi, 2011) and are known to threat- were shown to grow faster and reach higher weights (Gopal et al., 2010). en the mere existence of native crayfish species in some eco-regions (Holdich et al., 2009). Since crayfish are detritivores, they tend to persist 4.2. Environmental impact of invasive crustaceans in water bodies. Like crabs they, too, can travel long distances from their place of dispatch, making them highly successful invaders. Since many Crustacea is among the most abundant groups of species on earth, non-indigenous crayfish species can be easily purchased as ornamental occupying virtually every ecological niche. As such, it is not surprising crustaceans, they can also be easily disseminated (Gherardi et al., 2011).

Table 1 Scale and scope of the increasing aquaculture industry of the sexually dimorphic decapod groups with the potentially advantageous IAG manipulation-based biotechnologies. ⁎ ⁎⁎ ⁎⁎ ⁎ Group Number of Inheritance Preferred monosex Quantity (tons) Quantity (tons) Fold-change USD in USD in Fold-change species mode route in 2000 in 2009 2000 2009

Crabsa 10 XY/XX Neo-males→all-female 328,005 820,770 2.5 1,500,451 4,764,468 3.2 crayﬁshb 7 WZ/ZZ Neo-females→all-male 8,249 526,637 63.8 32,684 2,410,284 73.7 Lobsters 6 ? ? 73 1412 19.3 1,128 14,140 12.5 Prawnsc 4 WZ/ZZ Neo-females→all-male 217,828 438,818 2.0 701,793 2,203,725 3.1 Shrimpsd 8 WZ/ZZ Neo-males→all-female 149,477 2,339,384 15.7 810,020 9,302,973 11.5 Grand totals 35 703,632 4,127,021 5.9 3,046,076 18,695,590 6.1

⁎ ⁎⁎ -3 Fold-change – represents the value in 2009 divided by the value in 2000. USD – US dollars (values are represented as 1×10 of the amount). a – A commercial evaluation of mud crab monosex culture indicated all-male culture is more profitable; however, all-female culture is easier to achieve and also more profitable than mixed populations (Triño et al., 1999). b – A case study of the red claw crayfish Cherax quadricarinatus showed substantial increase in marketable yields of all-male populations compared with all-female and mixed populations (Curtis and Jones, 1995; Manor et al., 2002). c – All-male populations of the giant freshwater prawn Macrobrachium rosenbergii were shown to obtain up to twice the yield compared with mixed populations (Sagi et al., 1986) and an increase in the net income of farmers by ~60% when practiced via manual discarding non-profitable females in India (Nair et al., 2006). d – A case study in Penaeus monodon showed females grow larger than males (Gopal et al., 2010). 1548 T. Ventura, A. Sagi / Biotechnology Advances 30 (2012) 1543–1550

Table 2 The most prominent invasive/pest decapod crustacean species.

Group Number of Endemic sources Invaded regions Reported impacts Bibliographic invasive species source

Crabs >50 Asia; Atlantic Europe; Australia; Caribbean; Antarctica; Australia; Black Sea; Central America; Aquaculture impairment; River (Grosholz, China, Yellow sea; Eastern United States; lorida; Hawaii; Japan; Korea; Mediterranean; bank erosion. 2011) Indo-Pacific; Mediterranean; Mexico; New Zealand; North East United States; North New Zealand; North West America; West America; Scandinavia; Scandinavia, Barents South Central America; Tropical Atlantic. sea; South Africa; South America; South Eastern United States; West Europe; West United States. crayfish 24 Asia; Europe; North America; Oceania. Africa; Asia; Central America; Europe; North Aquaculture impairment; (Gherardi, America; Oceania; South America. Extinction pressure on native 2011) crayfish populations; Pathogen carrier; River bank erosion. ⁎ Shrimps 2 Northwest United States. Northwest United States. Aquaculture impairment. (Dumbauld et al., 2006) Total >76

⁎ The two reported pest shrimp species are not invasive but endemic to the Northwest United States.

Marketing of monosex cultured populations could thus prevent this line Efforts to minimize the impacts of invasive and/or pest crustacean of dissemination, with the added value of enhanced yields. The most ex- species may benefit from sexual manipulations, as has been success- tensively studied case of an invasive crayfish species is that of the red fully realized with other arthropods such as pest insects, where the swamp crayfish Procambarus clarkii. This species, native to north- controlled distribution of monosex sterile specimens led to infertile eastern Mexico and south-central USA, was legally introduced into copulations which eventually suppressed the natural population (Nolan southern Spain in 1973 from Louisiana (USA) and later illegally intro- et al., 2011; Vilcinskas et al., 2011). As was recently suggested for the duced throughout Spain, France, Italy, and other countries from eradication of invasive fish species, stocking with high proportions of 1970–1990. It is known to affect native European crayfish (family YY 'neo-females' (Trojan Y chromosome) may lead to a disproportionate- Astacidae) because it out-competes them and acts as a vector for the ly high percentage of males in the next generations (Gutierrez and Teem, transmission of the crayfish fungus plague, Aphanomyces astaci.Italso 2006). This strategy could be applicable to invasive crab species where reduces the value of invaded freshwater habitats by consuming inverte- males are heterogametic. brates and macrophytes and by degrading riverbanks through its Clearly, the vast behavioral, morphological, anatomical, and phys- burrowing activity. It accumulates heavy metals and toxins produced iological changes accompanying sex reversal could not be attributed by Cyanobacteria, such as Microcystis aeruginosa, and can transfer solely to the action of a single component. Improved molecular tech- them to its consumers, including humans. It is an intermediate host of nologies may be of great importance in putting all the pieces of the trematodes of the genus Paragonimus, which are potential pathogens of puzzle of crustacean sexual differentiation cascades into place. The humans if undercooked crayfish are consumed. If present in irrigation discovery of these novel molecular components, combined with the structures, such as reservoirs, channels, or rice fields, it may cause signif- already available knowledge will be applicable for many sexual inter- icant economic losses. This is due to both its burrowing activity, which ventions. For instance, as the silencing of IAG enabled a full and func- alters soil hydrology and causes water leakage, and its feeding habit, tional sex reversal, silencing a key component of gonad maturation which causes damage to rice plants (Souty-Grossetetal.,2006). All of to be identified and characterized will enable sterility induction. Com- these impacts make P. clarkii one of the 100 worst invasive species in bined, the two approaches could enable the production of monosex- Europe and among the worst 27 invasive animal species (Savini et al., sterile populations, which could provide sustainable solutions for 2010). aquaculture and for the improved management of invasive and/or In the case of shrimp species, the Pacific white shrimp Litopenaeus pest crustacean species. This approach may prove beneficial for the vannamei is the world's most widely cultured alien crustacean species, aquaculture industry, since monosex-sterile populations might exhibit and as such, it poses a dire threat through its competition with indige- better growth rates due to energy allocation toward growth rather than nous shrimp species and as a vector of viral diseases (Liao and Chien, to gonad ripening (primarily in all-female populations) and could also 2011). While there is little evidence of a genuine impact from this wide- defend commercial lines from theft. A precise intervention aimed at the ly cultured, introduced species, the impact of two indigenous burrow- expression of a single molecule could constitute a solution preferable to ing shrimp species Neotrypaea californiensis and Upogebia pugettensis the polyploidy approaches currently practiced (Sellars et al., 2010). In was devastating to the Pacific Northwest USA oyster aquaculture indus- case of invasive species management, a combined approach of the pro- try. These species reduce the stability of the bottom substrate where duction of monosex-sterile populations, as was done in insects, could oysters are raised and cause them to be covered with sediment and prove useful by spreading sterile male individuals to suppress pest popu- die (Dumbauld et al., 2006). Forty years of using a pesticide to eradicate lations (Nolan et al., 2011). these fouling species is due to phase-out by 2012 (Creswell and As IAGs were already discovered in ten different decapod species over McNevin, 2008), raising the need for a new, viable solution. such a short period (2007–2012), it is logical to assume that with advances in technology and the already existing high commercial incentive, 5. Epilog additional IAG cascade components instrumental for sexual manipulations will soon be discovered. Decapod crustaceans comprise a definite number of aquaculture important species that is on the rise, hand in hand with the augmenta- tion of the detrimental effects of some of these species and many others. Acknowledgments Through a single gene silencing, a full and functional sex-reversal has been achieved recently in a decapod (Ventura et al., 2012), enabling We thank Dr. Shmuel Parnes of the ornamental aquaculture company the development of species-specific biotechnologies for monosex popu- “Colors” for contributing his sketches for Figs. 1 and 3.Weacknowledge lation culture and the design of solutions to manage invasive and pest grants from Tiran Ltd. through BGN and from the National Institute for decapod species. Biotechnology in the Negev (NIBN). T. Ventura, A. Sagi / Biotechnology Advances 30 (2012) 1543–1550 1549

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