Short CommunicationCloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: Implications for eyestalk regulation of IAG expression
Highlights
► Isolation and characterization of cDNA encoding an insulin-like androgenic gland factor (Cs-IAG) from the androgenic gland (AG) of the male blue crab, Callinectes sapidus. ► Eyestalk removal caused in the increase of Cs-IAG in the AG. ► Implication of the regulation of an eyestalk neuropeptide (s) on IAG expression. ► Male hepatopancreas contained a small expression of IAG.
Introduction
Most crustacean species are gonochoristic and exhibit sex dimorphism whose expression may be hormonally controlled (unlike insects, in which genetic input alone is believed to determine sex differentiation [27]). In malacostracan crustaceans, sex differentiation and secondary sex characteristics are thought to be controlled largely by a hormone secreted by the androgenic gland (AG) [5], an organ unique to this family of animals. The presence of the AG, a male-specific endocrine gland, was first described some 60 years ago in the crab Callinectes sapidus [11]. Thereafter, it was also discovered in other crustacean species, including several isopod and decapod species [5], [38].
Studies showing the AG to be the key regulator of male sex differentiation were based largely on AG manipulations, including AG implantation and injection of AG extracts into females and AG removal from males. AG implantation and injection of AG extracts in female isopod and amphipod species caused masculinization [1], [6], [17], [20], [40] and in decapod females led to the inhibition of vitellogenesis, the development of secondary male sex characteristics and the regression of ovarian development [12], [23], [26], [31], [32]. In contrast, AG removal led to feminization in amphipods and decapods [6], [32], [37]. The data from the above studies suggested the presence of a putative AG factor and gonad plasticity, even in mature crustaceans. Although the AG factor was determined to be proteinaceous in nature [18], [25], studies conducted over several decades with the aim of isolating and characterizing this elusive factor have, to date, revealed very little information, as is reviewed briefly below.
The primary amino acid sequence with three disulfide bridges and the full-length cDNA of the first AG hormone (AGH) to be described were characterized from the isopod Armadillidium vulgae [30], [36]. This AGH was shown to be a heterodimeric glycoprotein, linked by three disulfide bridges – two between the B and A chains and one in the A chain – which shared structural similarity with vertebrate insulin [30], [35]. A few years later, using suppression subtractive hybridization (SSH), our laboratory identified two AG-specifically expressed genes, which were found to encode insulin-like androgenic gland factors (IAGs), one in Cherax quadricarinatus [29] and the other in Macrobrachium rosenbergii [42], designated Cq-IAG and Mr-IAG, respectively. Thereafter, several other genes that encode IAGs in decapods were identified in Portunus pelagicus (Pp-IAG; [39]), Penaeus monodon (Pem-IAG; GU208677), and Cherax destructor (Cd-IAG; EU718788).
As is the case of other peripheral glands in crustaceans, the AG is thought to be negatively regulated by the X-organ sinus gland (XO-SG) complex residing in the eyestalk. It is believed that an endocrine interaction between the eyestalk ganglia and the AG controls male reproduction [19], [24]. The activity of the AG is generally known to be down-regulated by a substance secreted from the eyestalk ganglia, as eyestalk ablation caused the hypertrophy of the AG and stimulation of spermatogenesis [24]. However, the hormonal status of IAG or AGH, i.e., its concentration in hemolymph in relation to male sex maturity, has not yet been confirmed in any crustacean species.
The blue crab Callinectes sapidus displays clear sexual dimorphism: the abdomen is semi-circular in adult females and T-shaped in males [43], and the chelae are orange–red in females and blueish in males [10]. Despite extensive progress in the understanding of endocrine regulation of vitellogensis in female crustaceans, including Callinectes sapidus [46], [47], [48], much less is known about the reproductive physiology of male Callinectes sapidus, despite the fact that the AG was first discovered in this species six decades ago.
The objectives of this study were thus to isolate the full-length cDNA of Cas-IAG from adult male Callinectes sapidus by using homology-based cloning and to examine a putative endocrine axis between the eyestalk ganglia and the AG. First, we isolated the full-length cDNA of the insulin-like androgenic gland factor of Callinectes sapidus, designated Cas-IAG, by using molecular cloning in combination with degenerate PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE). We also employed a qRT-PCR assay to determine the regulatory role of the eyestalk ganglia on the AG. To this end, we measured the regulatory effect of the eyestalk on the expression levels of Cas-IAG.
Section snippets
Animals
Juvenile males were obtained from the blue crab hatchery [Institute of Marine and Environmental Technology (IMET), Maryland] and reared to adulthood under the same conditions as those described previously [21]. Adult male Callinectes sapidus at intermolt (carapace width 120–140 mm) were bilaterally eyestalk-ablated three and seven days prior to the AG collection. On days 3 and 7, intact and ablated animals were anesthetized on ice for 10 min before being dissected.
cDNA synthesis for 5′ and 3′ rapid amplification of cDNA ends (RACE)
Total RNA from different tissues
Molecular cloning of the full-length cDNA encoding the putative Callinectes sapidus Cas-IAG, multiple sequence alignment of IAGs of several crustacean species, and phylogenic analysis
The full-length cDNA (1126 bp) of Cas-IAG (GenBank HM594946) was isolated from an AG by using PCR with degenerate primers, followed by 5′ and 3′ RACE. It was found that Cas-IAG consists of a putative coding region (462 bp) is flanked by a short 5′ UTR (128 bp) and a long 3′ UTR (536 bp) containing a putative polyadenylation site (AATAAA, underlined in Fig. 1).
The open reading frame (ORF) of Cas-IAG cDNA was predicted by the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The signal peptide
Discussion
In the present study, we characterized an insulin-like gene – Cas-IAG – from the androgenic gland of the blue crab Callinectes sapidus. The expression of Cas-IAG found in the AG and hepatopancreas was up-regulated after eyestalk ablation. This finding is in accordance with those of previous studies in decapod crustaceans, which suggested that the eyestalk AG is part of an endocrine axis [24].
Cas-IAG cDNA (1126 bp) is shorter than Cq-IAG (1444 bp), [29] and Mr-IAG (1824 bp) [42], but longer than
Acknowledgments
The authors are grateful to the staff of the blue crab hatchery and Aquaculture Research Center (ARC, Institute of Marine and Environmental Technology, Baltimore, MD) for supplying young juvenile crabs and maintaining re-circulated, closed artificial seawater system. We wish to thank Tomer Ventura and Ohad Rosen for their valuable comments on the manuscript. The research was supported by Research Grant MB-8714-08 from the United States-Israel Binational Agricultural Research and Development
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