Identification and Characterization of an Insulin-Like Receptor Involved in Crustacean Reproduction.

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.

High-density lipoprotein associated with secondary vitellogenesis in the hemolymph of the crayfish Cherax quadricarinatus.

The high-density lipoproteins LPI and LPII were isolated from the hemolymph of the crayfish Cherax quadricarinatus by gradient ultracentrifugation and high-performance liquid chromatography (HPLC). Both lipoproteins contained a carotenoid moiety. LPI is comprised of a single polypeptide with an approximate molecular mass of 96 kDa. LPII was composed of two similar native components, LPIIa and LPIIb, both having polypeptides of 80 and 177 kDa. Both under natural conditions and after endocrine manipulations, LPI was present in males and in females, regardless of the female reproductive stage. LPII was present only in secondary-vitellogenic females, but not during the winter reproductive arrest period. LPII was also absent from young females that had received androgenic gland implants. LPII also appeared in the hemolymph of intersex individuals from which the androgenic gland had been removed. It is therefore suggested that LPII serves as a marker indicating the onset of secondary vitellogenesis in C. quad'iariicarintus females.

Sex determination in crayfish: are intersex Cherax quadricarinatus (Decapoda, Parastacidae) genetically females?

In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1:1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1:3 (male:female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of intersex individuals.

Changes in protein kinase C during vitellogenesis in the crayfish Cherax quadricarinatus--possible activation by methyl farnesoate.

During ovarian maturation in the crayfish Cherax quadricarinatus, changes in ovarian protein kinase C (PKC) isoenzymes take place in parallel to yolk accumulation (as shown by immunoblot analysis). Significant changes were recorded in the amounts of specific isoenzymes and in their distribution between the cytosol and the membranes. Ovarian maturation was accompanied by the appearance of high- and low-molecular-weight immunoreactive PKC isoenzyme species. Among the isoenzymes tested, PKC alpha was the most clearly activated during ovarian maturation, as shown by significant translocation from the cytosol to the particulate fraction and the appearance of high-molecular-weight species. Moreover, a similar picture was obtained in the ovaries of intersex individuals upon induction of secondary vitellogenesis by androgenic gland ablation. Immunohistological staining showed PKC alpha to be localized mainly in the cytosol of premature oocytes, whereas in later maturation stages, it was concentrated around the nucleus in a vesicular structure and in the oocyte membrane. In secondary vitellogenic stages, PKC was localized in the plasma membrane and apparently in follicular cells. In addition, its activity was demonstrated by in vitro phosphorylation assays of a crayfish ovarian homogenate. Activation of total PKC phosphorylation of histone, an external substrate, was induced by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate (TPA) or methyl farnesoate. Both TPA and methyl farnesoate stimulated activation of PKC alpha in organ culture, causing its translocation from the cytosol to the membranes and inducing autophosphorylation of threonine residues. The changes in PKC isoenzymes during ovarian maturation in the crayfish suggest their involvement in this process as well as a possible regulatory role for methyl farnesoate through a direct effect on some PKC isoenzymes.

Intersex Red Claw Crayfish, Cherax quadricarinatus (von Martens): Functional Males with Pre-vitellogenic Ovaries.

Intersex individuals, possessing both male and female genital openings, were assessed in two groups-7 and 19 months old-of Australian red claw crayfish (Cherax quadricarinatus). All intersex individuals investigated were functional males, as suggested by their malelike morphology and the presence of testes, sperm ducts, androgenic glands, and viable spermatozoa. When an ovary was present in an intersex individual from either group, the gonadosomatic index, the diameter of the oocytes, and the ovarian cytosolic polypeptide profile were similar to those of immature, pre-vitellogenic females. We conclude that intersexuality in C. quadricarinatus does not indicate a case of protandric sequential hermaphroditism, as previously suggested. The case of intersexuality described here presents a unique model for the study of the role of the androgenic gland in the regulation of sex differentiation in crustaceans.

A newly established ELISA showing the effect of the androgenic gland on secondary-vitellogenic-specific protein in the hemolymph of the crayfish Cherax quadricarinatus.

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS-PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r = 0. 953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 microg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8-35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 microg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.

Effects of implantation of hypertrophied androgenic glands on sexual characters and physiology of the reproductive system in the female red claw crayfish, Cherax quadricarinatus.

The role of the androgenic gland (AG), an organ unique to male Crustacea, in the development of sex characters and physiology of the reproductive system has not been fully documented in the red claw crayfish, Cherax quadricarinatus. To investigate the role of the AG in this species, the effect of implanting hypertrophied AGs into immature female animals was followed. Of the female animals with AG implants, 91.6% developed male-like propodi, including the red patch characteristic of males of this species. The development of female secondary sex characteristics such as a wider abdomen, a wider endopod, and simple setation was inhibited. At the end of the experiment, the ovaries of the AG-implanted females contained mostly lipid-stage oocytes, with a small number of oocytes at the early yolk stage. The gonadosomatic index of the AG-implanted females was significantly lower than that of the control (sperm duct-implanted or sham-operated) females, which had mature oocytes with a well-defined perinuclear zone and yolk globules. An immunohistochemical test using an antibody developed against a 106-kDa secondary vitellogenic polypeptide showed only slight immunoreactivity in the oocytes of AG-implanted females compared with abundant immunoreactivity in control ovaries. In the polypeptide profile of the high-density lipoprotein (HDL) from the hemolymph of AG-implanted females, the 206- and 79-kDa secondary vitellogenesis-specific polypeptides were not found, whereas they were present in the profile of control females. In contrast, the female-specific 177-kDa polypeptide was present in the polypeptide profile of hemolymph HDL of both AG-implanted females and control females. It seems therefore that while secondary sex characters were masculinized under the influence of the implanted AG, the process of vitellogenesis was suppressed but not fully eliminated in the AG-implanted females.