Aquaculture and Fisheries 5 (2020) 283–288

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Aquaculture and Fisheries

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aquaculture-and-fisheries

Sex reversal and the androgenic gland (AG) in Macrobrachium rosenbergii: A

review
Kianann Tana, Huigong Jiangb, Donghuo Jiangb, Weimin Wanga,∗
a
College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of
Agriculture, Huazhong Agricultural University, Wuhan, 430070, China
b
College of Natural and Applied Sciences, University of Guam, Mangilao, Guam, 96913, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The male giant freshwater prawn (Macrobrachium rosenbergii) generally exhibit faster growth rates compared to
Macrobrachium rosenbergii females of similar age. Consequently, the practice of culturing monosex (all-male) prawn can increase both the
Androgenic gland (AG) production yield and income. Manually selecting males for culture during the grow-out period has long been
Sex reversal practiced in commercial prawn farming, but is labor-intensive and generally unsuccessful in producing a male
Monosex culture
monoculture. The androgenic gland (AG) is essential for sexual differentiation in prawn and development of
RNAi (dsRNA)
Andrectomization
male primary and secondary sexual characteristics. Microsurgical removal of the AG (andrectomy) from male
prawn at an early development stage will transform the male into a fully-functioning neo-female. The M. ro-
senbergii androgenic gland-specific insulin-like hormone (Mr-IAG) plays critical roles in male spermatogenesis
and male sexual development. RNA interference (RNAi) is a highly specific post-transcriptional interference or
silencing of gene expression in cells. Silencing Mr-IAG had induced full and functional sex reversal of male to
neo-female (genetic male with a female phenotype). Microsurgical removal of AG and RNAi are two different
approaches to producing neo-females and both methods target the manipulation of AG for efficient sex-reversal.
Neo-females can mate with normal males to produce all-male prawn progeny, which can be used for commercial
prawn farming and to improve production efficiency and profitability. This article reviewed recent research
related to the development and functions of AG and the testis and their interactions with morphological dif-
ferentiation at various stages for male M. rosenbergii, aiming to increase understanding on this topic and facilitate
discovery of new techniques for advancing M. rosenbergii aquaculture.

1. Introduction depression from repeated use of wild M. rosenbergii broodstocks con-

The giant freshwater prawn (Macrobrachium rosenbergii) is com-
monly found in rivers and estuaries of tropical and subtropical regions
around the world. It has become an economically significant crustacean
species cultured in many countries including: China, India, Bangladesh,
Thailand, Vietnam, and Malaysia in Asia; and Brazil and Ecuador in
South America. Global production of farmed M. rosenbergii was 194,000
tons in 2004, 99% of which was produced from Asia, and the market
value was above US$ 810 million (FAO, 2009). The global production
of farmed M. rosenbergii reached a peak of 226,800 tons in 2007 and has
fluctuated since that time (FAO, 2009). In recent years, there have been
serious production issues for M. rosenbergii farming, such as disease
outbreaks and deteriorating seed quality, producing a considerable
decline in farmed M. rosenbergii production since 2008. Inbreeding
tributed to decreases of overall production in Taiwan and Thailand
(Mather, 2003; Thanh et al., 2010).
Genetic improvement through selective breeding has previously
been applied to improve production performance in fish aquaculture.
Successful examples include Salmo salar (Quinton et al., 2005), Or-
eochromis niloticus (Eknath et al., 2007; Ponzoni, Hamzah, Tan, &
Kamaruzzaman, 2005), and Oncorhynchus kisutch (Neira et al., 2006),
where 10% – 20% genetic improvement per generation has been
achieved. Selective breeding programs were also initiated for a few
crustacean species of commercial importance (Jerry, Purvis, Piper, &
Dennis, 2005; Jones, McPhee, & Ruscoe, 2000) and several marine
penaeid species (Argue, Arce, Lotz, & Moss, 2002; De Donato,
Manrique, Ramirez, Mayer, & Howell, 2005; Preston, Crocos, Keys,
Coman, & Koenig, 2004). The information on phenotypic variation and

∗
Corresponding author. College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China.
E-mail addresses: kianann1987@webmail.hzau.edu.cn (K. Tan), hgong@triton.uog.edu (H. Jiang), dhjiang101@outlook.com (D. Jiang),
wangwm@mail.hzau.edu.cn (W. Wang).

https://doi.org/10.1016/j.aaf.2019.11.004
Received 26 December 2018; Received in revised form 18 November 2019; Accepted 19 November 2019
Available online 27 November 2019
2468-550X/ © 2019 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
K. Tan, et al.
both non-additive and additive genetic variation of each species is es-
sential to the successful development of any breeding project (Gjerde,
1986). Selective breeding over the generations can achieve significant
long-term economic benefits, and alternatively, monosex culture is also
a promising approach for increasing production yield. For crustaceans,
monosex culture has been used when there are large differences in
behavioral patterns and growth rates between males and females
(Curtis & Jones, 1995; Sagi, Ra’anan, Cohen, & Wax, 1986). All-male
cultures of M. rosenbergii have been carried out by manual segregation
(Sagi et al., 1986) and the results showed a significant increase of
harvest yield and an income increase over 60% (Mohanakumaran,
Salin, Raju, & Sebastian, 2006). Aflalo et al. (2006) developed a new
technique to produce all-male populations through sex reversal of males
in which a two-phase biotechnological approach was employed. The
two phases included removing AG of male prawn during its sexual
differentiation process to develop neo-females, which are genetically
male but with a female phenotype, and, secondly, utilizing the neo-
female to mate with regular males to produce monosex (all-male) off-
spring. Studies showed that the removal of AG plays a vital role in
manipulating the sex reversal in M. rosenbergii, yet the functions of AG
and testis in the development of the male reproductive system are still
not well understood.
2. Androgenic gland and testis
The AG of crustaceans was first characterized in the amphipod
(Charniaux-Cotton, 1954) and is situated on the subterminal portion of
the sperm duct (Kleinholz, 1979). For mature male M. rosenbergii
prawn, the endocrine and gametogenic functions are divided into two
distinct organs: the AG and the testis. The AG of M. rosenbergii consists
of cell bundles in a pyramidal cellular cluster and is closely linked with
the posterior section of the ejaculatory duct (Veith, 1983). The male
reproductive system of M. rosenbergii, situated in the cephalothorax, can
be morphologically divided into three parts: testis, vas deferens, and
terminal ampullae (Lynn and Clark, 1983). The testes are responsible
for spermatogenesis. The epithelium of vas deferens secrets products for
the formation of a sperm mass and spermatophoric wall, functions of
which are to maintain a proper environment for the sperm and to
transport sperm down the ducts. The mature spermatophores are stored
in the terminal ampullae. The paired testes of M. rosenbergii are pitch-
fork-shaped and joined anteriorly (Nagamine, Knight, Maggenti, &
Paxman, 1980a, b). Under histological observations, testes form an
outer tunica layer and plenty of branching spermatogenic lobules. In-
side the testis, spermatogenic lobules are synchronized with sperma-
togenesis phase, but seasonal patterns of spermatogenesis were not
observed. Spermatogonia could be found along the walls of the sper-
matogenic lobule. The lumen of the lobule consists of spermatogonia,
spermatids, and spermatocytes, or a combination of these components,
depending on the stage of spermatogenesis. There was considerable
evidence of interactions among AG, testes, and vasa deferentia. Pre-
vious histological studies indicated that if a male prawn underwent an
andrectomy (a microsurgical procedure to remove the AG), the vasa
deferentia and testes of the prawn would be severely atrophied and the
regeneration of vasa deferentia would also be inhibited (Nagamine
et al., 1980a, b). The reduction of spermatogenic lobules and sperma-
togenesis were observed in an andrectomized male prawn (Nagamine
et al., 1980a, b). Primary spermatocytes and spermatogonia could be
commonly observed, but the spermatids and secondary spermatocytes
could only be seen occasionally. The spermatogenesis in the testes of an
andrectomized M. rosenbergii was less developed than that in the normal
male prawn (Sagi, Cohen, & Yoram, 1990). The AG clearly altered and
affected the development of testis and reproductive system (Nagamine
et al., 1980a, b). The AG plays a more crucial function than testes in sex
reversal; therefore, it is essential to learn more about AG development
of M. rosenbergii.
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3. Development of androgenic glands
Rungsin, Swatdipong, and Na-Nakorn (2012) studied the develop-
ment of AG in juvenile M. rosenbergii and discovered that AG develop-
ment coincided with the emergence of vas deferens in Stage I, similar to
that of Orconectes nais (Carpenter, 1970). The AG in Stage I was not able
to function properly, as nucleoli were absent under histological ob-
servations, because nucleoli play an essential role in RNA synthesis
within the cells of the AG. In the second development stage (Stage II),
the nucleoli appeared in the cells of the AG and the AG started to
function (Thampy, 1973). Under histological observations using hae-
matoxylin and eosin counterstain, the cells appeared polygon-shaped
and were strongly stained. The nucleoli could not be seen during the
inactive stage, but could be easily observed during the active stage.
These cells were similar to the type I cells of the AG found in mature M.
rosenbergii (Okumura & Hara, 2004; Phoungpetchara et al., 2011). At
Stage II, the appearance of the gonopore complex was super-positioned
with the nucleoli of AG cells.
Immunohistochemistry studies showed that the M. rosenbergii in-
sulin-like androgenic gland factor (Mr-IAG) was weakly expressed in
the type I cells that appeared in the AG of mature M. rosenbergii
(Phoungpetchara et al., 2011). As shown in the recent studies at the
genetic- (Tomer Ventura et al., 2009) and protein-levels (Ventura,
Aflalo, Weil, Kashkush, & Sagi, 2011a, b), Mr-IAG is an important
functional gene in AG hormone and Mr-IAG gene expression starts to
occur 20 days after post-larvae metamorphosis (PL20), when the go-
nopore complex appears. The results indicated that the AG initiated the
growth of the gonopore complexes and that Mr-IAG in the AG led to the
enlargement of the gonads size during stage II development.
During Stage III, the nucleoli disappeared from the nuclei of the AG
cells, likely due to the rapid increase in cell numbers in the AG. At full
development, Stage IV, nucleoli appeared again and the AG resumed its
function and was involved in the morphological differentiation of testes
(Rungsin et al., 2012). Based on these observations, the AG may be
periodically turned on or off during the reproductive cycle, similar to
what has been observed in O. nais (Carpenter, 1970) and Scylla para-
mamosian (Liu, Cheung, & Chu, 2008). Even at the juvenile stage, the
AG was clearly developed because it is essential in the sexual differ-
entiation. The absence or presence of the AG is the key to determining if
individual male prawn could develop into males or neo-females.
4. Morphology differentiation
Male M. rosenbergii can be differentiated by the appearance of ap-
pendixes masculine, gonopores, and associated gonopore flaps (gono-
pore complexes). Appendixes masculine and gonopore complexes are
situated on the mature chelipeds and second pair of pleopods. The
carapace of male M. rosenbergii are sturdier and longer compared to
those of females. The male M. rosenbergii can be classified into three
developmental phases since sexually-dimorphic structures develop at
different times. During Phase I, the gonopore complex only appears
when the carapace length is 6 mm to 10 mm. At Phase II, when the
carapace length is between 10 mm and 28 mm, both the gonopore
complexes and appendices masculine appear. In Phase III, when the
carapace length is greater than 28 mm, the appendixes masculine, go-
nopore complexes, and mature chelipeds are all fully developed
(Nagamine et al., 1980a, b).
Three types of dominance hierarchies in male M. rosenbergii have
been reported (Kuris, Ra’anan, Sagi, & Cohen, 1987). These three adult
morphotypes were described as: small males (having shorter claws,
semi-transparent in color), orange-claw males (having long claws but
shorter than blue-claw males, orange in color), and blue-claw males
(having very long claws, deep blue in color) (Ra’Anan, 1985). Previous
studies showed that all three adult morphotypes could coexist in a M.
rosenbergii population with difference sizes and growth rates (Kuris
et al., 1987), with each morphological type representing a distinct
K. Tan, et al.
Fig. 1. Orange-claw male morphology of giant freshwater prawn (M. ro-
senbergii). (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
Fig. 2. Blue-claw male morphology of giant freshwater prawn (M. rosenbergii).
(For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
reproductive strategy. The small males utilized a lurking-copulation
strategy and could further transform into males with orange claws
(Telecky, 1984). The orange-claw males with (Fig. 1) showed faster
physical growth compared to small males and blue-claw males. These
orange-claw males did not perform any courtship and mating behavior
and some of them could further evolve into a superior male with blue-
claws (Fig. 2). Blue-claw males performed courtship behavior and
mated with female prawn (Cohen, Raanan, & Brody, 1981; Ra’Anan &
Sagi, 1985), but their somatic growth eventually stopped. The small
males evolved into orange-claw morphological phenotypes gradually,
where orange-claw was an intermediate form, and strong orange-claw
males would further transform into the dominant blue-claw males
(Kuris et al., 1987). All male M. rosenbergii were capable of evolving
into the next morphological stage, with the associated changes in: claw
coloration and shape (Kuris et al., 1987), somatic growth rate, carapace
length, reproductive behavior (Ra’Anan & Sagi, 1985), and the anatomy
of the reproductive organs and midgut glands (Sagi, Yoram, & Cohen,
1988; Sagi & Ra'anan, 1988). It is evident that the androgenic gland
alters the sexual differentiation and that morphological changes are
closely related to the emergence of the AG (Sagi et al., 1990).
The presence of the AG is essential for the natural regeneration and
differentiation of male secondary sexual characteristics in M. rosenbergii
(Nagamine & Knight, 1980). To further confirm the function of the AG
in the morphological differentiation of M. rosenbergii, bilateral surgical
ablations of the AG (Sagi et al., 1990) were performed on small males
and orange-claw males. Previous research revealed that when the AG
were removed from small males, 80% of the andrectomized males
evolved into orange-claw males, but none of them further developed
into the dominant blue-claw morphological phenotype (Sagi & Cohen,
1990). When the AG was removed from orange-claw males, 76% of
them successfully transformed into the dominant blue-claw males. In
the control group, almost all of the small males and orange-claw males
eventually evolved into the blue-claw morphological types (Sagi et al.,
1990). The genital papillae, a secondary sexual organ, was not found in
any of the andrectomized small males and 95% of the orange-claw
males. C onsequently, the andrectomized prawns developed a female-
like appearance in the genital region. The removal of AG not only
changed the external morphological appearance, but also the anato-
mical framework of the reproductive system. After AG removal, the
testes and sperm ducts were malformed and ampullae were missing.
Compared with non-andrectomized males’ prawns, the andrectomized
prawn had shorter sperm ducts, the malformed testes resulted in a
lower gonadosomatic index, and the ampullae could not regenerate
(Sagi et al., 1990). When the AG of juvenile males were removed, no
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further differentiation of claws occurred, in terms of shape and col-
oration, and some of the reproductive tract and genital papillae were
feminized.
The conversion from the strong orange-claw to blue-claw morpho-
logical type would carry on even after the AG was removed. The an-
dretomized males, regardless of their morphological stage, could evolve
into the next stage, but further transformation was inhibited (Sagi et al.,
1990). This suggested that the AG is essential for morphological
change, while the atrophy of sperm ducts and testes inhibits the mor-
phological process. Sperm could be found in the testis of the andreto-
mized males, indicating that the andrectomy does not inhibit sperma-
togenesis. However, the quality of sperm and the rate of sperm
production were affected (Sagi et al., 1990). Based on the under-
standing of changes in sex after alteration of the AG, we can utilize
innovated biotechnologies to produce the monosex (all-male) progeny.
5. Sex reversal
The first sex reversal in a decapod crustacean was produced with M.
rosenbergii. The AG tissues were implanted into immature females M.
rosenbergii to achieve neo-males (Nagamine et al., 1980a, b). All female
offspring were obtained by crossing the neo-males with normal females.
In the female sex reversal process, a tiny hole was created at the right
ventral side of the abdomen, near the cephalothorax. The AG tissues
were injected into the abdominal musculature through this hole. The
molting of the injected female prawns was observed to identify the
emergence of a male gonopore complex. Researchers concluded that the
appearance of the male gonopore complex is a more trustworthy in-
dicator compared to the appendix masculinum (Nagamine et al., 1980a,
b). As a result, there were 41 neo-males identified via the appearance of
a male gonopore complex and 13 of these were further certified to be
normal males. When the neo-males became larger in size, they were
easier to identify because neo-males not only have male gonopore
complex, but they also possess a female gonopore complex, though with
abnormal setae. Yet, neo-males, created after AG tissues transplantation
and AG cells injection, eventually developed into phenotypically-
normal males (Malecha et al., 1992). In the end, neo-males differ-
entiated into the three adult morphotypes (small males, orange-claw
males, and blue-claw males) (Kuris et al., 1987). N eo-males with blue-
claws were mated with normal females to obtain an all-female progeny.
The removal of the AG in male M. rosenbergii post-larvae (PL) led to
successful transformation into neo-females (andrectomized prawn) with
either ovotestes (Nagamine et al., 1980a, b) or functional ovaries
(Aflalo et al., 2006; Sagi & Cohen, 1990). The neo-females were capable
of producing all-male progeny when mating with normal males, how-
ever, the success rates of the neo-female production via andrectomy
technology were low and variable, which was one of the limiting factors
for mass producing neo-females at a commercial scale for all-male
prawn PL production. The stage of the PL during andrectomy was the
key to the success of generating neo-females. Previous studies showed
that the most suitable stage to increase the success rates of feminization
using an antrectomy is at PL stage when the appendix masculina was
absence (Aflalo et al., 2014). Aflalo et al. (2006) showed that early
stages (i.e. PL20-30) could achieve high success rate in feminization.
Other studies showed that the success rate of feminization can be im-
proved by using a late stage of PL, such as PL85-105 (Aflalo et al., 2006).
Regardless of age or stage, feminization could be successful with the
removal or interfering with the AG before the appendix masculina de-
velops (Nagamine et al., 1980a, b).
Another method utilizes RNA interference technology (RNAi) to
produce neo-females. RNAi works on the cellular level, which is dif-
ferent from external microsurgical removal of the AG. These two
methods both aim to interfere with AG function and produce sexual
differentiation.
K. Tan, et al.
5.1. Gene silencing by RNA interference (RNAi) related to sexual
differentiation
RNA interference (RNAi) is a highly specific post-transcriptional
interfering or silencing of gene expression in cells. The active compo-
nent is small interfering RNA (siRNA) that can be synthesized en-
dogenously to silence or interfere with gene activities. There are three
types of RNAi that can be synthesized in vitro: small interfering RNA
(siRNA), short hairpin-loop RNA (shRNA), and long double strand RNA
(dsRNA). siRNA can be produced externally through in vitro tran-
scription. After synthesis, the siRNA, shRNA, or dsRNA can be admitted
into cells through microinjection or transfection. At the cellular level,
the shRNA and dsRNA are cleaved into a small fragment of siRNA by an
enzyme, Dicer. These small fragments of siRNA work like a sequence-
specific locator for the target RNA and become part of the RNA-induced
silencing complex (RISC) (Agrawal et al., 2003; Qi and Hannon, 2005;
Watson, Fusaro, Wang, & Waterhouse, 2005). RNAi is capable of tem-
porarily silencing or interfering with the specific gene expression
without genetically modifying the target organism. The microinjection
of RNAi has been successfully demonstrated in the laboratory and
proven to be the most efficient and cost-effective method for RNAi in-
duction in crustaceans for silencing gene expression. In addition to the
rapid and mass-scale RNAi production, other innovative protocols have
to be developed. Kamath and Julie (2003) conducted an experiment
with a mass-scale administration method by feeding the plathyhelminth
Caenorhabditis elegans with a bacterium containing dsRNA and the re-
sults showed the effectiveness of such an approach (Kamath & Julie,
2003). In crustaceans, interfering with or silencing a gene using RNAi
technology enables the interference functions of the gene in the em-
bryonic or organ growth and development, cellular metabolism, and
reproduction mechanisms (Kato, Kobayashi, Watanabe, & Iguchi,
2011). Kato et al. (2011) demonstrated that silencing and interfering
with Doublesex1, Dsx1 via microinjecting dsRNA into male embryos led
to the development of female secondary characteristics. The male-spe-
cific androgenic gland is an important gland regulating the sexual dif-
ferentiation in malacostracan crustaceans. For the red-claw crayfish
Cherax quadricarinatus, an androgenic gland specific insulin-like gene
was identified and known as C. quadricarinatus insulin-like androgenic
gland specific factor (Cq-IAG). Cq-IAG was confirmed affecting the
male-specific sexual differentiation by silencing the Cq-IAG with RNAi
(Rosen et al., 2010). In C. quadricarinatus, the co-occurrence of an ac-
tive male reproductive system along with male secondary sex char-
acters commonly appear in intersex individuals and could permanently
inhibit ovary development. Silencing Cq-IAG in intersex individuals of
C. quadricarinatus resulted in sex modifications including: male femin-
ization, reduced sperm production and low-quality sperm, compre-
hensive testicular degeneration, and build-up of yolk protein in the
developing oocytes. In a series of experiments silencing of Mr-IAG in
juvenile M. rosenbergii males, Ventura et al. (2009) demonstrated the
importance of the M. rosenbergii androgenic gland-specific, insulin-like
hormone (Mr-IAG) in spermatogenesis and the development of male
primary and secondary sexual characteristics (Ventura et al., 2009).
Since the RNAi technology is mature enough for inducing sexual di-
morphism, it can be applied to produce neo-females regularly and en-
hance the mass production of all-male prawn.
5.2. Sexual dimorphism and monosex culture of prawns via RNAi
technology
Fast development of gene silencing technology in decapod crusta-
ceans has generated tremendous opportunities for its applications. The
first commercialized dsRNA-based technology was realized in com-
mercial prawn aquaculture industry (Ventura et al., 2009). Under
aquaculture conditions, reproductive and sexual maturity affects the
growth rates of crustaceans (Aiken, 1992; Hartnoll, 2001). Monosex
culture of all-male prawn has a superior application in commercial
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Aquaculture and Fisheries 5 (2020) 283–288
farming. The manual segregation of male prawn for culture was carried
out in a small-scale, intensive culture system and the results showed
higher growth rates (Sagi et al., 1986). In India, the culture of all-male
M. rosenbergii demonstrated a 60% increase in income
(Mohanakumaran et al., 2006). However, the early practice of manually
segregating males and females during the culture period was not an
efficient way to implement a monosex culture because of the labor and
time demanded. Following the discovery of RNAi, specific dsRNA was
used to interfere and silence the insulin-like androgenic gland hormone
(Mr-IAG) in the M. rosenbergii (Ventura et al., 2009). A two-step method
was used to synthesize the dsRNA. First, a forward and reverse oligo
was designed based on the Mr-IAG mRNA sequence. Then, the forward
and reverse oligo were annealed by a polymerase chain reaction (PCR)
using Mr-IAG as template DNA. PCR products were purified and tran-
scribed into dsRNA. The 2 μg/g dose of dsRNA was microinjected into
the sinus of the fifth set of walking legs at an early developmental stage.
The dsRNA was able to synthesize in vitro efficiently and silenced Mr-
IAG, which led to a sex reversal from males into neo-females. The neo-
females were identified via molecular sex markers (Ventura et al.,
2011a, b). After three months, the neo-females mated with normal
males to produce all-male prawn offspring (Ventura & Sagi, 2012). The
success in producing all male progeny via RNAi silencing biotechnology
was the first commercialized method of sex reversal in the crustacean.
The prawns produced by this technology are considered to be safer to
consume because of the absence of hormones, chemicals, and genetic
modifications. Moreover, this dsRNA-based silencing technology is
temporary and only affects the current generation and is not trans-
mitted vertically to the next generation. Therefore, the all-male prawn
can be consumed safely and free from the regulatory barriers that
prohibit a genetically-modified organism (Stein and Rodriguez-Cerezo,
2010).
5.3. Sex reversal via microsurgery of AG ablation
Sexual maturity, reproductive activity, and gender are the most
common factors affecting the growth of animals in aquaculture systems
(Aiken, 1992). Males and females were cultured separately in earlier
management strategies, shifting the energy used in reproduction ac-
tivities to the growth of an animal. Monosex culture has long been
practiced in fish aquaculture (Beardmore et al., 2001; Gomelsky, 2003).
For crustacean culture, it was implemented in prawn and crab farming
because males have superior growth rates to females (Curtis & Jones,
1995; Lawrence, 2004).
For crustacean species, the sexual differentiation and development
in male primary and secondary characters is genetically regulated by
the AG (Sagi et al., 1990). Sexual differentiation of M. rosenbergii could
be manipulated via the ablation of the AG without destroying the go-
nads, which was critical for producing monosex (all-male) prawn. After
the AG of the male prawn was removed (andrectomized) at an earlier
developmental stage, oogenesis and development of oviducts and fe-
male gonopores, was observed. The process was called feminization and
the andrectomized males further matured into a neo-female phenotype
with functioning female organs (Nagamine et al., 1980a, b). Such neo-
females can breed with normal males to yield an all-male progeny
(Katakura, 1989; Sagi, Snir, & Khalaila, 1997).
The microsurgical ablation of an AG was performed by excision of
the AG located at the fifth pair of walking legs. Both the AG and part of
the sperm ducts had to be taken out to ensure the full removal of the
AG. Full removal of the AG is vital for a successful sex reversal into neo-
females (Aflalo et al., 2006). Three months after an andrectomy, DNA of
prawn with female phenotypic characters was extracted to check
against molecular sex markers to identify neo-females (Ventura et al.,
2011a, b). Neo-females were sorted out and reared separately and go-
nadal development was observed every two weeks. When neo-females
showed clear ovarian development, where the orange-colored ovary
was easily observed via the transparent cuticle from a dorsal view, they
K. Tan, et al.
were ready to mate with the normal males. Mated neo-females then
started to berry eggs, the eggs would mature gradually, and egg color
would change from orange to gray (Aflalo et al., 2006). Such a process
would produce all-male progeny. Microsurgical removal of the AG re-
quires skillful personnel to perform but can be practically implemented
under farm conditions without sophisticated instruments. Typical suc-
cess rates were 3–5%. With this technology, a large number of neo-
females can be produced and the mass production of all-male prawn PL
can then be achieved commercially.
6. Conclusions
The existence of the AG is critical for the development of morpho-
logical phenotypes and sexual differentiation in M. rosenbergii. The
excision of the AG causes atrophy in the testis and reproduction system
and also inhibits the male primary and secondary sexual development,
consequently leading to the feminization in which oogenesis and ovary
development occur. The feminization of male prawn generates neo-fe-
males. Microsurgical removal of an AG and RNAi (dsRNA) silencing are
two different approaches that produce neo-females. Microsurgical re-
moval of an AG requires skillful personnel, but is more accessible and
more likely to be implemented at farms without complex laboratory
conditions. RNAi technology requires dsRNA produced in specific la-
boratory conditions and microinjection demanding well-trained tech-
nical personnel and sophisticated precision equipment. Both technolo-
gies can produce sufficient numbers of neo-females broodstocks to yield
all-male prawn PL at commercial scale. Monosex (all-male) culture of
M. rosenbergii can reach marketable size in a shorter time, yield higher
production biomass along with larger harvest size, which enables
farmers generate higher income and greater profit than mixed culture.
For crustacean species that demonstrate sexual dimorphism, such as
prawn, shrimp and crab, manipulation of androgenic gland-utilizing
modern biotechnologies can facilitate the expansion of monosex culture
to increase aquaculture production efficiency and profitability.
Declaration of competing interest
The authors declare no conflict of interest.
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