Eliahu D. Aflalo

Temporal Silencing of an Androgenic Gland-Specific Insulin-Like Gene Affecting Phenotypical Gender Differences and Spermatogenesis

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis.

A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.

Reciprocal Changes in Calcification of the Gastrolith and Cuticle During the Molt Cycle of the Red Claw Crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l−1). On the other hand, pH varied greatly, from 8.7 ± 0.15 in the gastrolith cavity through 7.6 ± 0.2 in muscle to 6.9 ± 0.5 in the stomach.

A sexual shift induced by silencing of a single insulin-like gene in crayfish: ovarian upregulation and testicular degeneration.

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.

Androgenic gland implantation promotes growth and inhibits vitellogenesis in Cherax quadricarinatus females held in individual compartments

Summary Androgenic glands (AGs) were implanted into young female red claw crayfish, Cherax quadricarinatus, with the aim of investigating the role played by the AG in the balance between growth and reproduction under conditions of minimal social interaction (individual compartments). The growth rate of the females with AG implants was significantly higher than that of the control non-implanted females (0.11 ± 0.03 g/day vs. 0.08 ± 0.02 g/day). This difference was attributed to the larger molt increments and slightly shorter molt intervals of the females with implants vs. the control females. At the end of the experiment (538 days), the mean weight of the implanted females was significantly higher than that of the control females (64.58 ± 18.24 g vs. 51.07 ± 12.71 g, respectively), a lead of 26.4% for the implanted females that started 91 days after implantation and became significant at 153 days after implantation. By that time, 55.5% of the implanted females had developed typical male secondary characters, such as the red patch on the propodus. The shift of energy from female reproduction to growth was further demonstrated by the level of expression of the vitellogenin gene in the hepatopancreas: gene expression was high in control females but lower by several orders of magnitude in the AG-implanted females, as shown by real time RT-PCR relative quantification. Confirmation of these findings was provided by an ELISA test, which showed that the level of vitellogenic cross-reactive protein in the hemolymph of AG-implanted females was significantly lower than that in intact females. The significant growth promotion in AG-implanted females was clearly not due to social interaction. It may be attributed to a direct growth factor—like the effect of androgens in vertebrates—in combination with an indirect effect, through the shift of energetic investment from reproduction and vitellogenesis to growth. Since the AG implant had a more marked effect on molt increment than on molt interval, it seems likely that the AG acts as a growth promoter rather than as a molt enhancer.

Timing Sexual Differentiation: Full Functional Sex Reversal Achieved Through Silencing of a Single Insulin-Like Gene in the Prawn, Macrobrachium rosenbergii

ABSTRACT In Crustacea, an early evolutionary group (∼50 000 species) inhabiting most ecological niches, sex differentiation is regulated by a male-specific androgenic gland (AG). The identification of AG-specific insulin-like factors (IAGs) and genomic sex markers offers an opportunity for a deeper understanding of the sexual differentiation mechanism in crustaceans and other arthropods. Here, we report, to our knowledge, the first full and functional sex reversal of male freshwater prawns (Macrobrachium rosenbergii) through the silencing of a single IAG-encoding gene. These “neofemales” produced all-male progeny, as proven by sex-specific genomic markers. This finding offers an insight regarding the biology and evolution of sex differentiation regulation, with a novel perspective for the evolution of insulin-like peptides. Our results demonstrate how temporal intervention with a key regulating gene induces a determinative, extreme phenotypic shift. Our results also carry tremendous ecological and commercial implications. Invasive and pest crustacean species represent genuine concerns worldwide without an apparent solution. Such efforts might, therefore, benefit from sexual manipulations, as has been successfully realized with other arthropods. Commercially, such manipulation would be significant in sexually dimorphic cultured species, allowing the use of nonbreeding, monosex populations while dramatically increasing yield and possibly minimizing the invasion of exotic cultured species into the environment.

Expression of the Reproductive Female-Specific Vitellogenin Gene in Endocrinologically Induced Male and Intersex Cherax quadricarinatus Crayfish

Abstract In oviparous females, the synthesis of the yolk precursor vitellogenin is an important step in ovarian maturation and oocyte development. In decapod Crustacea, including the red-claw crayfish (Cherax quadricarinatus), this reproductive process is regulated by inhibitory neurohormones secreted by the endocrine X-organ-sinus gland (XO-SG) complex. In males, the C. quadricarinatus vitellogenin gene (CqVg), although present, is not expressed under normal conditions. We show here that endocrine manipulation by removal of the XO-SG complex from male animals induced CqVg transcription. The CqVg gene was expressed differentially during the molt cycle in these induced males: no expression was seen in the intermolt stages, but expression was occasionally detected in the premolt stages and always detected in the early postmolt stages. Relative quantitation with a real-time reverse transcriptase-polymerase chain reaction showed that expression of CqVg in induced early postmolt males was an order of magnitude lower than that in reproductive females, a finding that was consistent with RNA in situ hybridization results. The SDS-PAGE of high-density lipoproteins from the hemolymph of endocrinologically induced early postmolt males did not show the typical vitellogenin-related polypeptide profile found in reproductive females. On the other hand, removal of the XO-SG complex from intersex individuals, which are chromosomally female but functionally male and possess an arrested female reproductive system, induced the expression, translation, and release of CqVg products into the hemolymph, as was the case for vitellogenic females. The expression of CqVg in endocrinologically manipulated molting males and intersex animals provides an inducible model for the investigation and understanding of the endocrine regulation of CqVg expression and translation in Crustacea as well as the relationship between the endocrine axes regulating molt and reproduction.

A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix

Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

Solubility and Bioavailability of Stabilized Amorphous Calcium Carbonate

Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC‐C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC‐C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole‐body retention over a 34‐hour period. Calcium serum analysis revealed that calcium absorption from ACC and ACC‐C preparations was up to 40% higher than from CCC, whereas retention of ACC and ACC‐C was up to 26.5% higher than CCC. Absorbed calcium in the femurs of ACC‐administrated rats was 30% higher than in CCC‐treated animals, whereas 15% more calcium was absorbed following ACC‐C treatment than following CCC treatment. This study demonstrates the enhanced solubility and bioavailability of ACC over CCC. The use of stable ACC as a highly bioavailable dietary source for calcium is proposed based on the findings of this study.

Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

The crustacean male-specific androgenic gland (AG) regulates sexual differentiation. In the prawn Macrobrachium rosenbergii, silencing an AG-specific insulin-like encoding transcript (Mr-IAG) inhibited the development of male sexual characters, suggesting that Mr-IAG is a key androgenic hormone. We used recombinant pro-Mr-IAG peptide to generate antibodies that recognized the peptide in AG cells and extracts, as verified by mass spectrometry. We revealed the temporal expression pattern of Mr-IAG and studied its relevance to the timetable of sex differentiation processes in juveniles and after puberty. Mr-IAG was expressed from as early as 20 days after metamorphosis, prior to the appearance of external male sexual characters. Mr-IAG expression was lower in the less reproductively active orange-clawed males than in both the dominant blue-clawed males and the actively sneak mating small males. These results suggest a role for Mr-IAG both in the timing of male sexual differentiation and in regulating reproductive strategies.