In vertebrate reproduction, metabolism, growth and development, essential roles are played by glycoprotein hormones, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), all of which are heterodimers consisting of two subunits, a structurally identical alpha subunit, and a variable beta subunit, which provides specificity. A 'new' glycoprotein hormone heterodimer identified in both vertebrates and invertebrates, including decapod crustaceans, was shown to be composed of the glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5) subunits. The putative receptor for GPA2/GPB5 in invertebrates is the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1). In this study in the giant freshwater prawn, Macrobrachium rosenbergii, we identified and characterized the GPA2 (MrGPA2), GPB5 (MrGPB5) and LGR1 (MrLGR1) encoding genes and revealed their spatial expression patterns in female animals. Loss-of-function RNA interference (RNAi) experiments in M. rosenbergii females demonstrated a negative correlation between MrGPA2/MrGPB5 silencing and MrLGR1 transcript levels, suggesting a possible ligand-receptor interaction. The relative transcript levels of M. rosenbergii vitellogenin (MrVg) in the hepatopancreas were significantly reduced following MrGPA2/MrGPB5 knockdown. MrLGR1 loss-of-function induced MrVg receptor (MrVgR) transcript levels in the ovary and resulted in significantly larger oocytes in the silenced group compared to the control group. Our results provide insight into the possible role of GPA2/GPB5-LGR1 in female reproduction, as shown by its effect on MrVg and MrVgR expression and on the oocyte development. Here, we suggest that the GPA2/GPB5 heterodimer act as a gonad inhibiting factor in the eyestalk-hepatopancreas-ovary endocrine axis in M. rosenbergii.

Various material-strengthening strategies have evolved in the cuticle and the feeding tools of arthropods. Of particular interest is the crustacean mandible, which is frequently reinforced with calcium phosphate, giving a minerology similar to that of human bones and teeth. We report here a biological strengthening method of apatite by Zn substitution, found in the incisor teeth of the freshwater prawn Macrobrachium rosenbergii. Nanoindentation measurements show a clear positive correlation between the Zn/Ca ratio and the stiffness and hardness of the composite. In the incisor, Zn-substituted apatite forms an internal vertical axis, extending from the sharp outer edges of the tooth to its basal segment. The substitution level in this zone (up to 40%) is very high compared with the levels achieved in synthetic ceramics (<20%). Finite element simulation suggests that the high-Zn axis acts as a unique internal load transfer element, directing stress from the biting cusps to the more compliant underlying layers. In light of the considerable research being invested in developing synthetic calcium phosphate derivatives for human bone and tooth grafts, the innovative mineralogy of the M. rosenbergii incisor may inspire beneficial biomimetic applications. STATEMENT OF SIGNIFICANCE: The controlled incorporation of impurities into biominerals is a widespread phenomenon in biomineralization that may pave the way to new classes of biomimetic materials. The present study reveals a biogenic mineral of zinc-substituted apatite found in the incisor teeth of a prawn. A clear correlation between zinc substitution level and stiffness and hardness, suggests that zinc substitution serves to mechanically reinforce the bioapatite. The spatial arrangement of the high-zinc apatite unveils a material-level adaptation strategy for tooth fortification, in which the rigid high-Zn structure servs as an internal load-transfer element that transmits the stress directly from the tooth's sharp cusps to the more compliant underlying layers.

Sexual differentiation and maintenance of masculinity in crustaceans has been suggested as being regulated by a single androgenic gland (AG) insulin-like peptide (IAG). However, downstream elements involved in the signaling cascade remain unknown. Here we identified and characterized a gene encoding an insulin-like receptor in the prawn Macrobrachium rosenbergii (Mr-IR), the first such gene detected in a decapod crustacean. In mining for IRs and other insulin signaling-related genes, we constructed a comprehensive M. rosenbergii transcriptomic library from multiple sources. In parallel we sequenced the complete Mr-IR cDNA, confirmed in the wide transcriptomic library. Mr-IR expression was detected in most tissues in both males and females, including the AG and gonads. To study Mr-IR function, we performed long-term RNA interference (RNAi) silencing in young male prawns. Although having no effect on growth, Mr-IR silencing advanced the appearance of a male-specific secondary trait. The most noted effects of Mr-IR silencing were hypertrophy of the AG and the associated increased production of Mr-IAG, with an unusual abundance of immature sperm cells being seen in the distal sperm duct. A ligand blot assay using de novo recombinant Mr-IAG confirmed the existence of a ligand-receptor interaction. Whereas these results suggest a role for Mr-IR in the regulation of the AG, we did not see any sexual shift after silencing of Mr-IR, as occurred when the ligand-encoding Mr-IAG gene was silenced. This suggests that sexual differentiation in crustaceans involve more than a single Mr-IAG receptor, emphasizing the complexity of sexual differentiation and maintenance.

Background: In Crustacea, a male-specific insulin-like androgenic hormone governs sexual differentiation. Results: A crayfish insulin-like growth factor-binding protein (Cq-IGFBP) was expressed and found to bind the androgenic hormone. Conclusion: Cq-IGFBP is part of the scheme modulating the crustacean androgenic hormone. Significance: Cq-IGFBP is the first IGFBP found that interacts with an insulin-like gender-specific ligand linking early evolution of insulin pathways with gender. Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (∼3 kb long) revealed the presence of tandem and inverted repeats. The ∼3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ∼3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.

In malacostracan crustaceans, sex differentiation is uniquely regulated by a hormone secreted by the male-specific androgenic gland (AG). An isopod AG hormone was the first to be structurally elucidated and was found to belong to the insulin superfamily of proteins. Recently, it has been found that the AGs of several decapod crustaceans express insulin-like androgenic gland factors (IAGs), whose function is believed to be similar to that of the isopod AG hormone. Here we report the isolation from the blue crab Callinectes sapidus of the full-length cDNA encoding a candidate insulin-like AG hormone, termed Cas-IAG. The predicted protein Cas-IAG was encoded as a precursor consisting of a signal peptide, the B chain, the C peptide, and the A chain in that order. While the AG was the main source of Cas-IAG expression, as found in other decapod species, the hepatopancreas of male Callinectes sapidus crabs displayed minor Cas-IAG expression. Eyestalk ablation confirmed the presence of a possible endocrine axis between the eyestalk ganglia and the AG, implying that Cas-IAG expression is negatively regulated by (a) substance(s) present in the eyestalk ganglia.

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.