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Abstract: Sex reversal technology, realized through androgenic gland (AG) manipulation, was recently introduced as a process for production of all-male producing broodstock. This technology exploits however, a relatively small number of sex-reversed broodstock. Thus, both genetic improvement via a breeding program and prevention of inbreeding are needed to ensure the sustainability of such technology. Three wild strains of prawns originating from geographically (though not necessarily genetically) isolated locations in India [Gujarat (G), Kerala (K) and West Bengal (WB)] were assessed for their suitability as breeders for all-male production. In addition, their potential for a selective breeding program was evaluated. A comparative evaluation of early sex segregation, sex reversal, growth performance, and population structure in the three selected strains was performed. Among the purebred strains, after eight months of grow out in earthen ponds, growth performance of the WB strain was the best (59.39±1.08g), while that of G was the poorest (26.50±0.94g). Strain-additive genetic effects for body weight at harvest were highest for the WB strain (+45.9%) and lowest for the G strain (−28.3%). Body masses of WB×K and WB×G crosses were 14.2% and 8.8% above the mean mass of the purebred strains, respectively, while that of the K×G cross was 23% below this value. In most crosses, males reached heavier mean body weights than did females with higher frequencies of the large male morphotypes being seen in the WB purebred strain and its respective crosses. Reciprocal effects for body mass ranged from 4% to 14.9% below the mean of the purebred strains. These negative signs mean that in the two crosses involving the WB strain, growth performance is higher when this stain was used as the sire strain. Similarly, the growth performance of the K×G cross was higher when the former was used as the sire strain. Average heterosis effect for body weight was minor (−0.51±0.73) and did not differ significantly from zero. The high correlation between strain additive effects (the major source of variation in growth) and total performance for body weight (r =0.927) indicate the existence of valuable genetic variation that could be exploited in a selective breeding program. For all-male production, males from the three strains were segregated at early post-larval stages and microsurgical AG removal was performed. In all the strains, similar low levels of complete sex reversal into functional neo-females (genetic males) were realized (0.17% −0.34%). These produced relatively small numbers of neo-female to be crossed with normal males to produce the desired all-male population, but raise the possibility that such a process could result in a genetic bottleneck. Thus, a genetic improvement scheme for each strain integrated with periodical crosses of the resulting neo-females from one strain with males from another strain is suggested to avoid inbreeding. [Copyright &y& Elsevier]

Abstract: This work investigates the effects of androgenic gland (AG) ablation on the structure of the reproductive system, development of secondary sexual characters and somatic growth in Cherax quadricarinatus males. The AG ablation, which was performed at an early developmental stage (initial weight: 1.85±0.03g), had no effect on the somatic growth parameters (specific growth rate and growth increment), but it prevented the re-formation of male gonopores and appendices masculinae. However, the red patch differentiation and chelae size were similar to those in control males. All the ablated animals developed a male reproductive system. Testis structure was macroscopically and histologically normal. The distal portion of the vas deferens (DVD) was enlarged in some animals, with histological alterations of the epithelium and the structure of the spermatophore. Results suggest that the higher growth in males than in females may be due to an indirect effect of the AG on energy investment in reproduction rather than to a direct effect of an androgen. This is the first report of a potential action of the AG on the secretory activity of the distal VD and the structural organization of the spermatophore. Although the AG may play a role in the development of male copulatory organs, its association with the red patch development deserves further research. The results obtained in the present study support and complement those from intersexes of the same species. [Copyright &y& Elsevier]

Abstract: Over the past six decades, a unique crustacean endocrine organ, the androgenic gland (AG), has occupied the minds of groups researching Crustacea the world over. Unlike male sexual differentiation and maintenance of sexual characteristics in other arthropods, in crustaceans these processes are regulated by the unique male AG. Crustaceans present a particular case in which the gametogenic organ (testis) is clearly separated from the organ regulating sex differentiation (the AG), enabling endocrine manipulations. The AG was first discovered in a decapod species and later investigated in detail not only in decapods but also in amphipods and isopods. The key role of the AG in regulating sex differentiation was subsequently validated in a number of representative species of a wide array of Malacostraca. It was in an isopod species that the AG hormone was first discovered. Later, orthologous genes were found in isopods and decapods, with all these genes sharing the key features of the insulin-like superfamily of peptides. This review unfolds the story of the AG and AG-specific insulin-like factors (IAGs) from a historical perspective, highlighting the main achievements in the field and giving a glimpse of future challenges to be addressed. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4μm in diameter), stain strongly with hematoxylin–eosin (H&E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6μm in diameter), stain lightly with H&E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction. [Copyright &y& Elsevier]

Abstract: The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5–7μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13–15μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease. [ABSTRACT FROM AUTHOR]

This study investigated the histology of the androgenic gland in Eriphia verrucosa (Forskål, 1775). The results obtained with light microscopy show that the androgenic gland in this species consists of 2 cell types. Type 1 cells are small and do not contain a secretory product. Type 2 cells are considerably larger and may produce a proteinaceous secretion. This study identified the location and structure of the androgenic gland in Eriphia verrucosa, which is an economically important brachyuran crab, and revealed the chemical nature of the secretions produced by its cells. [ABSTRACT FROM AUTHOR]

Abstract: Timing of organogenesis and subsequent development of genital organs were studied in female and male Pacific white shrimp Penaeus (Litopenaeus) vannamei postlarvae. This was linked to the timing of differentiation of external structures that differentiate the genders. Anatomy of the gonad appears to be unique for penaeid species. The genital organ was fully recognized from postlarve day-16 (PL16) as a bilateral lobe located in the anterior region of the midgut gland (first anterior lobes) that connects to an anterior perpendicular collector tube that extends dorsally towards the posterior region of the midgut gland and forms an inverted U-shape collector. Eight bilateral lobes in females (second to ninth) and the bilateral oviduct between the seventh and eighth lateral lobes and seven bilateral lobes in the male (second to eighth) are connected along the inverted U-shape collector tube. These lobes extend over the surface of the midgut gland beneath the pericardium. Shortly after organogenesis of the female gonad, the tenth bilateral lobe emerges from the distal region of the collector tube and continues dorsally along the intestine, and the fourth bilateral lobe did not develop and regressed until apparently absorbed. In males, the posterior bilateral vas deferens emerges from the same region. Around PL50 (0.5–0.6 g; 45–50 mm), external gender differentiation was recognized in the form of the thelycum in females and the gonopores in males. Additionally, the male androgenic gland appears at the posterior-external wall of each anterior vas deferens, surrounded by connective tissue that attaches to the anterior vas deferens and the eighth testicular lobe. Gonad differentiation occurred from PL68 (1.8–2.2 g; 70–74 mm), where it was possible to differentiate the female ovary from the male testes. Timing of sex reversal studies in penaeids is discussed. [Copyright &y& Elsevier]

Abstract: The ability of anthropogenic chemicals to cause reproductive disorders has been the focus of toxicologists for many years. Whilst the focus of endocrine disrupting chemicals has mainly been associated with vertebrate groups, there have been continued calls for more research on the invertebrates. Surprisingly, within the Crustacea, many studies have focussed on female or growth/moulting related endpoints despite many of the vertebrate studies highlighting male related effects such as abnormal male reproductive development. Furthermore, a large number of the invertebrate studies have focussed on vertebrate estrogens or their mimics. Considering the biology of the crustacean endocrine systems, this paper shall argue that unlike the vertebrates, it is a lot more difficult to feminise a crustacean than it is to de-masculinise one. Consequently, crustacean toxicologists, by following the tact of vertebrate biologists, may have been trying to address the right questions, but in the wrong way. Studies have shown that intersexuality in crustaceans may arise through the masculinisation of heterogametic (WZ) females or the de-masculinisation of males through aberrations in male androgenic gland activity. It is recommended that the focus be put on understanding the mechanisms of sex determination in Crustacea, and the expression of male secondary sexual characteristics at the molecular, biochemical and physiological level are fully explored so that appropriate assessments can be made as to whether sexual endocrine disruption is occurring in this ecologically important group. [Copyright &y& Elsevier]

Abstract: Members of the insulin family of hormones are generally not regarded as gender-specific, although there is sporadic evidence for the possible involvement of insulin pathways in sexual differentiation. In crustaceans, sexual differentiation is controlled by the androgenic gland (AG), an organ unique to males. To date, attempts to identify active AG factors in decapods through either classical purification methods or sequence similarity with isopod AG hormones have proven unsuccessful. In the present study, the first subtractive cDNA library from a decapod AG was constructed from the red-claw crayfish Cherax quadricarinatus. During library screening, an AG-specific gene, expressed exclusively in males even at early stages of maturation and termed Cq-IAG (C. quadricarinatus insulin-like AG factor), was discovered. In situ hybridization of Cq-IAG confirmed the exclusive localization of its expression to the AG. Following cloning and complete sequencing of the gene, its cDNA was found to contain 1445 nucleotides encoding a deduced translation product of 176 amino acids. The proposed protein sequence encompasses Cys residue and putative cleaved peptide patterns whose linear and 3D organization are similar to those of members of the insulin/insulin-like growth factor/relaxin family and their receptor recognition surface. The identification of Cq-IAG is the first report of a pro-insulin-like gene expressed in a decapod crustacean in a gender-specific manner. Its expression in a male-specific endocrine gland controlling sex differentiation supports the notion that insulin may have evolved in the context of regulating sexual differentiation. [Copyright &y& Elsevier]

Environmental effects on sex determination in Litopenaeus vannamei were studied by rearing day 1 postlarvae at three temperatures, under three photoperiods, at high density and by starving. None of the environmental conditions affected sex determination or differential development of gender in this species. From day 50, the development of the endopodite of the first pair of pleopods revealed the first external differentiation, showing a triangular structure with three setae in females, whereas a tubular structure remained in males. Juvenile shrimp sex differentiation took place from days 50–90, independent of size, only if postlarvae reached a development threshold of 150 mg of body weight and 20 mm of body length previously. Histology and scanning electron microscopy of the vas deferens revealed that the androgenic gland (AG) is a single 2-mm cord attached in the subterminal ejaculatory region, just before the distal vas deferens narrows. The AG is composed of large oval cells containing vacuolated cytoplasm, and each cell has a prominent rounded nucleus, similar to all descriptions of the AG in Malacostracans, so we assume that it should have the same function in sex differentiation. [ABSTRACT FROM AUTHOR]

Abstract: In crustaceans, male differentiation and primary and secondary characteristics are regulated by the androgenic gland (AG). In gonochoristic crustaceans, the AG is also linked to intersexuality. Whereas the co-occurrence of various male and female characteristics has been demonstrated in intersex crustaceans, little is known regarding sexually dimorphic behavior patterns in such individuals. In the present study, we used an intersex crayfish model to investigate – for the first time in crustaceans – the agonistic and mating behavior of intersex individuals, and to explore the effects of AG ablation on behavior, morphology and physiology. As was the case for their morphological and physiological reproductive traits, intersex individuals – despite being genotypically females – generally resembled males in terms of behavior: they engaged in fighting with males and copulated with receptive females. However, fighting durations of intersex animals were intermediate between those of males and females, and the durations of the copulations were remarkably short. Adult intersex individuals that had been AG ablated at the juvenile stage were unlikely to engage in fighting with males (similar behavior to females) and did not exhibit any mating behavior with receptive females. AG ablation resulted in feminine morphological and physiological shifts in the treated intersex individuals and enabled vitellogenin gene transcription and the onset of secondary vitellogenesis. It thus appears that an as-yet-unknown AG-secreted factor(s) regulating maleness also seems to regulate the organization of male behavior in crustaceans. [Copyright &y& Elsevier]

Abstract: Males of the giant freshwater prawn Macrobrachium rosenbergii grow faster and reach higher weights at harvest than females a fact which makes the culture of all-male populations desirable. All-male populations were produced by mating sex-reversed males, i.e., neofemales, with normal males. Neofemales capable of mating and spawning were produced by removal of the androgenic gland (AG) from immature M. rosenbergii males. The main obstacle to developing a technology based on this type of manipulation is the difficulty of identifying males at a sufficiently early stage of development. To overcome this problem, we developed a novel two-step scheme for large-scale microsurgical andrectomy. Phase I post larvae were andrectomized at ages 25–60days after metamorphosis (PL25–60). A low success rate of functional sex reversal, resulting with all male progeny was obtained (1.3%). In the light of the low success rate and a number of cases of abnormal reproductive development, a second phase was introduced in which the progeny (presumed males) of neofemales from phase I were andrectomized at earlier ages (PL20–30). This two-phase protocol enabled a large quantity of juvenile males to be andrectomized. In phase II there was a significant increase in the number of sex-reversed animals (developed ovaries) and a significant shortening of the time to maturation. Comparison of the all-male progeny with a normal mixed population showed higher growth performance of the all-male population. This advantage together with the shorter maturation time in phase II opens the possibility to scale-up the system to field conditions. Since sexual dimorphic growth patterns are common among decapod crustaceans, it is obvious that the results of this study will have applied significance for many aquacultured species. [Copyright &y& Elsevier]

Abstract: Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab. [Copyright &y& Elsevier]

Males of the freshwater prawnMacrobrachium rosenbergii(De Man) grow faster and reach a larger size at harvest than females of the species. It is thus obvious that culture of monosex all-male populations would be economically advantageous. Sexual differentiation in crustaceans is regulated by the androgenic gland (AG), which plays a pivotal role in the regulation of male differentiation and in the inhibition of female differentiation. InM. rosenbergii, AG removal from immature males resulted in sex reversal, with complete female differentiation. Similarly, AG implantations into immature females lead to the development of the male reproductive system. Sex-reversedM. rosenbergiianimals were capable of mating with normal specimens to produce offspring. Early attempts in Israel and more recently, attempts in other countries to establish all-male populations through manual segregation showed that for the production of monosex prawn populations to be economically feasible, intervention via the AG is probably required. However, a suitable biotechnology is still to be developed, and an androgenic hormone has yet to be identified in decapods. Three lines of aquacultural and biotechnological research and development are proposed for the future: (1) Establishment of monosex cultures through manual segregation, together with the application of selective harvesting and claw ablation, as well as examination of different monosex culture strategies under a variety of economic conditions. (2) Microsurgical intervention in the AG, leading to the development of functional neo-females, which would subsequently be mated with normal males to produce all-male progeny. (3) Elucidation of AG bioactive products to enable biochemical or molecular manipulation of sex differentiation. [ABSTRACT FROM AUTHOR]

Among all of the decapods that have been studied, only one kind of gland, the androgenic gland, has been found attached to the subterminal ejaculatory region of the vas deferens. In the present study of the red swamp crayfish, it was found that two kinds of gland-like tissues were attached to the subterminal ejaculatory region of the vas deferens. One of the tissues, which is referred to as TIB, is located inside the body cavity, whereas the other tissue, which is referred to as TIC, is inside the coxa. The characteristics of TIB resemble those of the androgenic gland of other malacostracans. The TIC cells, however, are smaller and more basophilic due to the small size and high density of their cytoplasm and nuclei. Moreover, black fibers, similar to the nerve fibers of the ganglion, were detected among the TIC cells after Bodian's stain. The conclusion is that TIC may be a newly found male sexual characteristic and further research is needed to confirm its functions. [ABSTRACT FROM AUTHOR]

This study reports the results of examination of the gonadal morphology and ultrastructural features of the androgenic hormone (AH)-producing androgenic gland cells of laboratory stocks of functional male woodlice, Armadillidium vulgare, with female genital apertures (♂fga), with and without experimentally induced infections of the sex-ratio-distorting endobacterial parasite, Wolbachia. Males (♂fga) have been reported in wild populations containing individuals infected with this maternally transmitted sex-ratio-distorting parasite. We report a reduction of testicular segment (utricle) number, androgenic gland cell hypertrophy, and electron-dense ultrastructural cytological features in ♂fga males. The presence of the Wolbachia parasite had no effect on the features we examined. These results suggest that ♂fga males are produced as the result of a delayed expression/action of the male sex-determining AH which causes a “lag-phase” delay in male differentiation in genetic males and is not due to the presence, in genetic females, of a hypothetical, epigenetic “M” gene as suggested by Rigaud and Juchault [Genetics 133 (1993) 247]. Our results favor the interpretation of males as true genetic (ZZ) males in which the delayed AH action appears to involve cellular AH trafficking pathways which may be controlled by an impaired autosomal gene responsible for AH action. [Copyright &y& Elsevier]

Intersexuality has been found in both males and females of the marine/estuarine amphipod, Echinogammarus marinus, at polluted and reference sites in East Scotland. Polluted sites had significantly more intersex specimens than reference sites, however the cause of intersexuality is unclear. Discriminant analysis of morphometric data showed that normal male specimens from the most polluted site resembled pooled intersex males, suggesting that subtle endocrine disruption (ED) maybe occurring in these otherwise apparently normal males. The main discriminating character was gnathopod size, recognised to be under androgenic gland control. The association of distinctive morphometry with intersexuality may provide a new approach to biomarkers of ED in crustaceans. The opportunities for other novel biomarkers, for example biochemical or behavioural markers, may also be explored through study of intersex animals. [Copyright &y& Elsevier]

The effect of the androgenic gland (AG) on female agonistic behavior was studied in the Australian freshwater crayfish Cherax quadricarinatus. Interactions between large males and smaller individuals of three types - sham-operated females, females implanted with the AG and intact males - were compared. Half-hour-long pair encounters were staged in large aquaria and monitored with a video camera. Interactions between crayfish of the same or opposite gender(s) were of a clearly agonistic character, without any courtship or reproductive elements. The large males were the winners of all fights. Small males fought more often and for longer periods of time, and grasped their male opponents for longer periods of time, than sham operated females. AG-implanted females occupied an intermediate position between the two genders in their fighting behavior. In addition, AG-implanted females performed 'crawling over' (a tolerance-indicative act which is more pronounced towards the other sex) over the large males less frequently than the sham operated females did, and were also treated by these males more like males than females, with regard to this behavior. The behavioral changes in AG-implanted females followed the general trend of masculinization also evident in the morphological and physiological changes in their gonads and the appearance of male secondary sexual characters. [ABSTRACT FROM AUTHOR]

Oocyte maturation in decapod crustaceans is a two step process. Primary vitellogenesis is followed by a variable hiatus that lasts up to the onset of secondary vitellogenesis, which is marked by the rapid accumulation of yolk proteins in the oocytes. We have cloned a complete Cherax quadricarinatus vitellogenin cDNA. The sequenced cDNA contains a 2584 aa open reading frame which shows sequence similarity to vitellogenins from other crustaceans. The mRNA encodes at least two of the previously identified vitellin components, indicating that the primary translation product is subject to post-translational modification, including proteolytic cleavage. The region close to the 3′ end of the mRNA encodes a previously characterized negatively charged protein (provisionally designated P106). We show here that the negative charge of P106 could be due to its ability to bind calcium. Northern blot data show that this gene is expressed as a single 8000 nt transcript and is present in the hepatopancreas of secondary-vitellogenic females. Primary vitellogenic and other tissues examined in male and female animals were negative. In sexually plastic intersex animals, removal of the androgenic gland results in vitellogenin transcription, indicating that the gene is negatively regulated by the androgenic gland. [Copyright &y& Elsevier]

In decapod crustaceans, a number of neurohormones regulating a variety of physiological processes, including reproduction, are to be found in the X-organ–sinus gland complex of the eyestalk. Bilateral eyestalk ablation was thus performed in mature males of the Australian red claw crayfish Cherax quadricarinatus with the aim of studying the role of eyestalk-borne hormones on spermatogenic activity in the testis and on the androgenic gland (AG). The latter gland controls the differentiation and functioning of male sexual characteristics in crustaceans. Eyestalk ablation caused hypertrophy of the AG, as indicated by an increase in gland weight (3.9±0.44 mg vs <0.1 mg in intact males) and by overexpression of AG polypeptides. In the testes of eyestalk-ablated males, empty spermatogenic lobules were common, while lobules containing primary spermatocytes were infrequent. These findings were reflected in decreased amounts of DNA in these testes and a consequent increase in the relative weights of the sperm ducts. Since it was found that eyestalk ablation affected both the AG and the reproductive system, in vitro experiments were conducted to study the direct effects of the sinus gland on the AG and testes and of the AG on the testes. Sinus gland extracts inhibited by 30% the incorporation of radiolabeled amino acids into AG polypeptides and almost totally inhibited the secretion of radiolabeled AG polypeptides into the culture medium. However, sinus gland extracts had no significant effects on testicular tissue. On the other hand, AG extracts affected the in vitro phosphorylation of a testicular polypeptide (of 28 kDa), in a time- and dose-dependent manner, suggesting a direct effect of AG-borne hormones on the testes. The above findings, together with the evidence for direct inhibition by the sinus gland on the AG, suggest an endocrine axis-like relationship between the sinus gland, the AG, and the male reproductive system in decapod crustaceans. [Copyright &y& Elsevier]

Although the mechanism of sex-differentiation in crustaceans has yet to be defined, the androgenic gland (AG) is thought to be the exclusive organ that produces the androgenic hormone (AH) which induces male sexual development. This paper presents results of light and transmission electron microscopy and total protein analysis of androgenic glands from three male morphotypes (orange-claw, orange-blue-claw and blue-claw) of the freshwater prawn, Macrobrachium rosenbergii. Highest protein content (76 µg//AG) was found in the blue-claw morphotype as compared to the orange-blue-claw (45 µg/AG) and the orange-claw morphotype (19 µg/AG). Sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-page) analysis of the cell free extract of the AG from the three morphotypes revealed four polypeptides (∼16, ∼18, ∼23 and ∼26 Kd) which quantitatively increase from the sexually immature orange-claw to the sexually mature blue-claw morphotype. The 16 and 18 Kd polypeptides could be the AHs. [ABSTRACT FROM AUTHOR]

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The androgenic gland of M. lamerrii is situated near the seminal vesicle and along the vas deferens of the male prawns. Histologically it is made up of cords of cells which are arranged loosely. Histochemical analysis of the androgenic gland cells showed the presence of cystine/cysteins, protein bound amino acid groups. Androgenic gland of M. lamerrii shows signs of increased secretory activity during the sexually active phase of the male. In adult prawns, eyestalk ablation results in the hypertrophy of androgenic gland. Brain and thoracic ganglion extracts also showed enhanced secretory activity of androgenic gland and corresponding gonadal activity in the male prawns. [ABSTRACT FROM AUTHOR]

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The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata , by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens , a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place. Image 1 • Male estuarine crabs were exposed to 1 mg/L of glyphosate for one month. • A significant decrease in sperm count was observed. • Abnormal spermatophores were also seen. • A lower sperm count was also observed in vitro , in gonads exposed to glyphosate. • An apparent interference of glyposate with the androgenic gland was seen in vitro. [ABSTRACT FROM AUTHOR]

Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts ( Sp-CFSH1 and Sp-CFSH2 ) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain . Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females ( P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain , the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro . It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10 -6 M ( P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.