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During their life, crustaceans undergo several molts, which if theoretically compared to the human body would be equivalent to replacing all bones at a single event. Such a dramatic repetitive event is coupled to unique molecular mechanisms of mineralization so far mostly unknown. Unlike human bone mineralized with calcium phosphate, the crustacean exoskeleton is mineralized mainly by calcium carbonate. Crustacean growth thus necessitates well-timed mobilization of bicarbonate to specific extracellular sites of biomineralization at distinct molt cycle stages. Here, by looking at the crayfish Cherax quadricarinatus at different molting stages, we suggest that the mechanisms of bicarbonate ion transport for mineralization in crustaceans involve the SLC4 family of transporters and that these proteins play a key role in the tight coupling between molt cycle events and mineral deposition. This discovery of putative bicarbonate transporters in a pancrustacean with functional genomic evidence from genes encoding the SLC4 family—mostly known for their role in pH control—is discussed in the context of the evolution of calcium carbonate biomineralization.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

Spectacular colors and visual phenomena in animals are produced by light interference from highly reflective guanine crystals. Little is known about how organisms regulate crystal morphology to tune the optics of these systems. By following guanine crystal formation in developing spiders, a crystallization mechanism is elucidated. Guanine crystallization is a "non-classical," multistep process involving a progressive ordering of states. Crystallization begins with nucleation of partially ordered nanogranules from a disordered precursor phase. Growth proceeds by orientated attachment of the nanogranules into platelets which coalesce into single crystals, via progressive relaxation of structural defects. Despite their prismatic morphology, the platelet texture is retained in the final crystals, which are composites of crystal lamellae and interlamellar sheets. Interactions between the macromolecular sheets and the planar face of guanine appear to direct nucleation, favoring platelet formation. These findings provide insights on how organisms control the morphology and optical properties of molecular crystals.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

Macrobrachium prawns are voracious predators of the freshwater snails that host the flatworms responsible for bilharzia (schistosomiasis), a health burden in many African countries. A novel strategy to decrease the disease in Africa involves the use of prawns as biocontrol agents of the snails. Although the endemic African river prawn Macrobrachium vollenhovenii is a natural candidate for aquaculture and biocontrol, efforts to domesticate it have been unsuccessful to date, and it is not available in the large quantities required for aquaculture and biocontrol. The Asian giant prawn Macrobrachium rosenbergii has been cultured worldwide for decades. Recently, novel biotechnologies were developed to create monosex (all-male) non-breeding populations for aquaculture that we suggest are also ideal for biocontrol in Africa. Since the above 2 prawn species are of the same genus, exhibit similar sizes and require a female pre-mating molt prior to egg fertilization, the potential for cross-breeding between the 2 species must be tested. To assure that all-male populations of M. rosenbergii will not pose such an ecological threat, we carried out cross-breeding experiments with M. vollenhovenii. Both interspecies encounters and attempts at artificial insemination revealed that fertilization does not occur between the 2 species. Our results demonstrate both behavioral and physiological pre-zygotic reproductive barriers between these species. We suggest that all-male M. rosenbergii can be used as an aquaculture species and as a biocontrol agent in areas where M. vollenhovenii occurs without concern for hybridization.

In crustacean aquaculture, size dimorphism between males and females is the main key factor determining the advantage of monosex aquaculture over that of mixed populations. This factor is particularly relevant for the freshwater prawn, Macrobrachium rosenbergii, for which intensification of cultures is complicated by a complex social structure in which large dominant males are territorial and inhibit the growth performance of smaller males and females. It has therefore been suggested that all-female mono-culture could be the practice of choice, since females are less aggressive and less territorial and are believed to exhibit a relatively homogenous growth pattern. Here we report the first large-scale comparative field study of all-female and mixed populations under extensive and intensive stocking conditions in earthen ponds. The study was facilitated by application of our novel biotechnology based on a single injection of suspended hypertrophied androgenic gland cells. Under both our intensive and extensive conditions, the all-female cultures showed better performance than the mixed cultures in most key aquaculture parameters including survival rate and yield per hectare. Also, the intensively stocked all-female ponds showed better feed conversion ratio than mixed ponds. Furthermore, while the mean size of the animals did not differ significantly between the two treatments, the all-female populations exhibited significantly higher size uniformity. Our study suggests that for M. rosenbergii, female monosex aquaculture is a sustainable method to yield a homogenous crop.

Crustacean chitin-hemocyanin-calcium mineral complexes were designed as bone biomimetics, with emphasis on their ability to bind or release calcium ions. Chitin scaffolds were prepared by dissolving chitin flakes in LiCl/dimethylacetamide, followed by gel formation and freeze-drying. Some of these scaffolds were modified by incorporation of CaCO3 . In some of the chitin-CaCO3 scaffolds, macroporosity was introduced by HCl treatment. Hemocyanin from the crayfish Cherax quadricarinatus was used to further modify the chitin scaffolds by dip coating. Cytocompatibility, cellular adherence and proliferation of human mesenchymal stem cells (hMSCs) were evaluated in terms of cell number as reflected in lactate dehydrogenase activity. The chitin, chitin-CaCO3 , and porous chitin-CaCO3 scaffolds were all found to facilitate cell attachment. Hemocyanin dip-coating of these scaffolds led to increased initial cell adhesion, enhanced proliferation, and osteogenic differentiation. Since the hemocyanin loading of the scaffolds was impaired by sterilization by gamma-irradiation (as required for biomedical applications), the hemocyanin loading was performed on previously sterilized scaffolds. All scaffolds facilitated osteogenic differentiation of osteoblasts, with the highest cell ALP-activity being found on hemocyanin-modified porous chitin-CaCO3 scaffolds. Thus, chitin-hemocyanin scaffolds enhanced the initial stages of bone cell development and could serve as promising biomaterials for bone regeneration.

Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.

The recent introduction into aquaculture of RNA interference (RNAi) for producing the preferred all-male monosex cultures, which give improved yields, has raised awareness of the need to investigate the consequences of such novel biotechnological manipulations. Here, we present meta-analysis style study on data from observations of three consecutive cultured all-male (ZZ) generations of the giant freshwater prawn Macrobrachium rosenbergii (De Man). Each consecutive generation comprised the progeny of RNAi-manipulated sex-reversed males. The manipulation was achieved through the administration of dsRNA encoding the insulin-like androgenic hormone into males (which transformed them into ‘neofemales,’ ZZ) of the previous all-male generation. Each generation was cultured in a separate earthen pond for a short (~ 4–5 months) growout period. At harvest of each of the generations, the typical M. rosenbergii population structure comprising three male morphotypes was obtained. An anatomical examination of the male reproductive system of a representative specimen of the third all-male generation showed normal reproductive outputs, even though the prawns had been grown without the presence of females (WZ) for three generations. At the molecular level, expression of vital male-specific genes in the third generation of all-male M. rosenbergii culture was demonstrated. Thus, the present study showing the lack of any overtly apparent long-term consequences of the RNAi-based biotechnology provides support for the responsible use of temporal RNAi in aquaculture. Finally, the absence of the W chromosome for three generations raises questions with regard to its role and content with respect to crustacean sexual determination and differentiation.

An unresolved problem in aquaculture ponds is the susceptibility of freshwater fish to parasitic diseases, such as those caused by Centrocestus trematodes, which are transmitted by snails of the Thiaridae family. Two species of this family are common in Tilapia aquaculture ponds in Israel, the endemic Melanoides tuberculata and the invasive Thiara scabra, both being hosts of various disease-causing parasitic trematodes. Promising biocontrol agents are freshwater prawns of the genus Macrobrachium, which are known to be voracious predators of freshwater snails. Prawns in contrast to fish, occupy a benthic niche in the aquaculture pond and may thus be expected to prey on disease-carrying snails, which burrow into the pond bottoms. Prawns also bring the added advantage of constituting a high-value side-product to the pond-produced biomass without the need for additional feed. Following recent biotechnological advances in monosex prawn production, non-reproducing monosex prawn populations are now commercially available for testing as ecologically safe biocontrol agents in aquaculture. Laboratory predation experiments examining the ability of monosex Macrobrachium rosenbergii prawns to prey on Melanoides tuberculata and T. scabra snails showed that even a single prawn weighing >4 g is capable of exterminating dozens of snail hatchlings per day and hence of preventing hatchling recruitment in both snail species. Large prawns exhibited significantly superior predation abilities with respect to large snails of both species, with Melanoides snails being more susceptible to predation than Thiara snails. This study is the first to integrate laboratory studies with field observations on the utility of monosex all-female Macrobrachium rosenbergii prawns as biocontrol agents of damaging snails in fish ponds. These prawns were found to be effective biocontrol agents in Tilapia growout aquaculture ponds in terms of reduction in both snail abundance and rates of fish infection with the snail-borne parasites. The promising results of this biocontrol method call for further study aimed at optimizing the biocontrol power and profit of monosex prawn populations in polyculture with fish as part of wider and more comprehensive ecological risk assessment studies.

Parental care entails physiological costs to the mother. These costs, even if dramatic, are usually reversible and do not result in mortality of the mother. In the spider Stegodyphus lineatus Latreille 1817 (Eresidae), maternal care is extreme and irreversible: mothers regurgitate food for the young and then die when consumed by them (matriphagy). We examined whether the mother's midgut tissues undergo structural changes in preparation for regurgitation and matriphagy. Our histological data show that the midgut diverticula (MD) tissues start to degrade during the egg sac incubation period. When the young emerge from the egg sac, the midgut tissues are partly liquefied and are retained within the MD. The degradation process intensifies when the female feeds her young by regurgitation and liquid tissue is observed within and among the diverticula lobes. The presence of the lumen of a diverticulum during the regurgitation process suggests that degenerated tissues enter the lumen and form the regurgi...

The recent introduction of RNA interference (RNAi) based biotechnology in aquaculture has aroused public awareness regarding safety aspects related to the use of such temporal transcript manipulation. In the present study, we demonstrate use of this biotechnology in large-scale production of all-male freshwater prawn Macrobrachium rosenbergii, relying on a two-phased approach. First, the insulin-like androgenic gland hormone (Mr-IAG) was silenced by dsRNA injections, causing sex-reversal of males into ‘neo-females’ (genetic males with female phenotype, with 86% success in this study). The next step involved mating the neo-females with normal males to produce all-male progeny. The administered exogenous dsRNA was fully cleared from the prawn's tissues after 7 days. Gene silencing through dsMr-IAG appeared to be temporary, with decay seen over time and nearly full expression of Mr-IAG (~ 82%) being restored after 28 days. Assessment of reproduction and fecundity in normal females versus neo-females revealed that in both groups, ~ 80% of the prawns were in a reproductive state and no significant difference was found in their reproductive output, either in terms of brood mass or number. Population structure, as indicated by the normal occurrence of male morphotypes, in both mixed-sex and all-male populations was examined in terms of weight and morphotype frequency distribution. The final harvest and marketing of the all-male prawn offspring occur about 19 months, i.e., one generation after the actual RNAi procedure. Thus, on top of the aquaculture yield advantage, the use of such RNAi procedure in crustacean aquaculture seems to be safe due to its temporary nature.

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye-units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the underlying photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied byin situmicro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close to physiological conditions. We show that the distal mirror consists of three or four layers of sparse plate-like nano-crystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen bonded layers of the molecules results in an extremely high calculated refractive index in the H-bonded plane,n= 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.SignificanceAquatic animals use reflectors in their eyes either to form images or to increase photon capture. Guanine is the most widespread molecular component of these reflectors. Here we show that crystals of isoxanthopterin, a pteridine analogue of guanine, form both the image-forming ‘distal’ mirror and the intensity-enhancing tapetum reflector in the compound eyes of some decapod crustaceans. The crystal structure of isoxanthopterin was determined, providing an explanation for why these crystals are so well suited for efficient reflection. Pteridines were previously known only as pigments and our discovery raises the question of which other organic molecules may be used to form crystals with superior reflective properties either in organisms or in artificial optical devices.

RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

The d oublesex and m ab-3 r elated t ranscription factor (Dmrt) gene family is known to be related to the sexual regulators doublesex of arthropods and mab-3 of annelids and to hold highly conserved functions in sexual determination and differentiation across phyla. Here, we report a study of the Dmrt gene family in the freshwater prawn Macrobrachium rosenbergii, a crustacean whose sexual differentiation has been widely researched. A wide transcriptomic screen, from the embryo to the adult M. rosenbergii, identified five novel Dmrt genes (MroDmrts) and confirmed two known MroDmrts. The seven MroDmrts encode proteins of 275–855 amino acids; each protein contained at least one conserved DNA-binding DM domain, which is typical of Dmrt proteins, and five proteins contained 1–4 transactivation domains (TADs). Importantly, in the embryonic, larval and post-larval stages, MroDmrt genes exhibited time-dependent expression patterns rather than sex-specific expression. In-silico screening of the expression of the MroDmrt genes in adult males revealed the enrichment of MroiDmrt1b and MroiDmrt1c in the androgenic gland (AG) as compared to the eyestalks. In vivo silencing of the androgenic gland insulin-like (IAG) encoding gene significantly decreased the expression of the above two Dmrt genes, while not affecting the expression of control genes, thereby suggesting the possible role of these two genes in the IAG-switch and in sex-differentiation processes.

Vitellogenesis, a key process in oviparous animals, is characterized by enhanced synthesis of the lipoprotein vitellogenin, which serves as the major yolk-protein precursor. In most oviparous animals, and specifically in crustaceans, vitellogenin is mainly synthesized in the hepatopancreas, secreted to the hemolymph, and taken up into the ovary by receptor-mediated endocytosis. In the present study, localization of the vitellogenin receptor and its interaction with vitellogenin were investigated in the freshwater prawn Macrobrachium rosenbergii. The receptor was immuno-histochemically localized to the cell periphery and around yolk vesicles. A receptor blot assay revealed that the vitellogenin receptor interacts with most known vitellogenin subunits, the most prominent being the 79 kDa subunit. The receptor was, moreover, able to interact with trypsin-digested vitellogenin peptides. By combining a novel peptide-array approach with tandem mass spectrometry, eleven vitellogenin-derived peptides that interacted with the receptor were identified. A 3D model of vitellogenin indicated that four of the identified peptides are N-terminally localized. One of the peptides is homologous to the receptor-recognized site of vertebrate vitellogenin, and assumes a conserved β-sheet structure. These findings suggest that this specific β-sheet region in the vitellogenin N-terminal lipoprotein domain is the receptor-interacting site, with the rest of the protein serving to enhance affinity for the receptor. The conservation of the receptor recognition site in invertebrate and vertebrate vitellogenin might have vast implications for oviparous species reproduction, development, immunity, and pest management.

Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements.

Indirect evidence indicates that penaeids may have a W/Z sex heritability mechanism with the androgenic gland (AG) mediating masculine differentiation. The present study evaluated the immune response against grafts and the expression of primary and secondary male sexual characters in Litopenaeus vannamei females implanted with terminal ampoules and associated AGs. Experiments included grafting females at PL34–48. Grafting at PL37 generated few cases of melanization exhibiting dark coloration covering parts or the whole implant while most implants showed white coloration, without any trace of melanization. Histology of melanized implants showed necrotic tissue, encapsulated by black depositions. These melanized capsules were externally surrounded by normal haemocytes and flat haemocytes in the middle. An experiment evaluating possible absorption of the grafts based on recovery, gross coloration, size of implants, and histology showed that Complete terminal ampoule-AG implants, Control-vas deferens implants and Abdominal muscle implants, recovered at day 7 and 15, exhibited gradual and statistically significant decrease in size, which was more evident for muscle implants. Grafts with normal appearance from this experiment remained viable during 7 and 15 days post-implantation; these tissues showed a normal cellular structure with neither signs of melanization nor haemocyte infiltration. Appendices masculinae did not develop in any implanted females or controls. Concerning petasma development, two females implanted with AG showed a male phenotype in their first endopodites, characterized by a straight shape at the distal region, a middle protuberance and absence of setae along the middle curved edge of the structure. Regenerated and intact endopodites of Control-proximal terminal ampoule females showed the typical female phenotype with a slender shape with a curved distal edge, without a middle protuberance, and with setae along the middle curved edge of endopodites. Implanted females showed no evidence of masculinization or abnormal development of oocytes. In the light of the present study the effects of AG transplantation on sex in penaeids should be further evaluated in younger PL stages. Statement of relevance This contribution presents new information on sex reversal technology on Litopenaeus vannamei based on androgenic gland implants. The findings are novel for the family Penaeidae concerning the immunological response to tissue grafting and the plasticity for sex reversal in this world aquaculture species. © 2016 Elsevier B.V La evidencia indirecta indica que los peneidos pueden tener un mecanismo de heredabilidad sexual W / Z con la glándula androgénica (AG) que media la diferenciación masculina. El presente estudio evaluó la respuesta inmune contra los injertos y la expresión de caracteres sexuales masculinos primarios y secundarios en hembras Litopenaeus vannamei implantadas con ampollas terminales y AG asociados. Los experimentos incluyeron mujeres injertadas en PL34-48. El injerto en PL37 generó pocos casos de melanización que exhiben coloración oscura que cubre partes o el implante completo, mientras que la mayoría de los implantes mostraron coloración blanca, sin ningún rastro de melanización. La histología de los implantes melanizados mostró tejido necrótico, encapsulado por depósitos negros. Estas cápsulas melanizadas estaban rodeadas externamente por hemocitos normales y hemocitos planos en el medio. Un experimento que evaluó la posible absorción de los injertos en función de la recuperación, la coloración gruesa, el tamaño de los implantes y la histología demostró que los implantes de ampolla terminal AG completa, los implantes Control-deferentes y los implantes musculares abdominales, recuperados en los días 7 y 15, exhibieron gradualmente y disminución estadísticamente significativa del tamaño, que fue más evidente para los implantes musculares. Los injertos con apariencia normal de este experimento permanecieron viables durante 7 y 15 días después de la implantación; estos tejidos mostraron una estructura celular normal sin signos de melanización ni infiltración de hemocitos. Los apéndices masculinos no se desarrollaron en mujeres o controles implantados. Con respecto al desarrollo de petasma, dos mujeres implantadas con AG mostraron un fenotipo masculino en sus primeros endopoditos, caracterizado por una forma recta en la región distal, una protuberancia media y ausencia de setas a lo largo del borde curvo medio de la estructura. Los endopoditos regenerados e intactos de las hembras de ampolla terminal proximal de control mostraron el fenotipo femenino típico con una forma delgada con un borde distal curvo, sin una protuberancia media y con setas a lo largo del borde curvo medio de los endopoditos. Las hembras implantadas no mostraron evidencia de masculinización o desarrollo anormal de ovocitos. A la luz del presente estudio, los efectos del trasplante de AG sobre el sexo en peneidos deben evaluarse más a fondo en las etapas PL más jóvenes. Universidad Nacional, Costa Rica Aquaculture

In assessing the relationship between calcium supplementation and maintaining bone mass or reducing the risk of fracture, the effectiveness of calcium supplementation has never been decisive. Freshwater crayfish rely on amorphous calcium carbonate (ACC), an instable polymorph of calcium carbonate, as the main mineral in the exoskeleton and in the temporary storage organ, the gastrolith. Inspired by the crayfish model, we have previously shown an increase in calcium bioavailability in rats administered with synthetic stable ACC vs. crystalline calcium carbonate (CCC). The current study compared the effects of amorphous calcium derived from either gastrolith or synthetic ACC with those of crystalline calcium, found in commercial CCC or calciumcitrate supplements, in a bone loss prevention model. Rats were subjected to either sham or ovariectomy (OVX) operation (n~20/ group) followed by administration of food pellets supplemented with 0.5% calcium from either source over 12 weeks. Micro-computed tomography (μCT) and histomorphometric analyses revealed bone loss prevention by both gastrolith and ACC treatments, manifested by an increase in morphometric bone parameters, compared to both CCC- and calcium citrate-treated groups. Both gastrolith and ACC treatments resulted in bone formation in the tibia cancellous bone, indicated by dynamic histomorphometry parameters, compared to either the CCC or calcium citrate treatments. Levels of urine deoxypyridinoline (DPD), suggested an anti-resorptive effect of ACC, which was also the only treatment that led to a significant increase in vertebral mechanical strength, as supported by μCT analysis of topology and orientation parameters of the vertebral trabeculae. To our knowledge, such levels of bone loss prevention by calcium supplements have never been reported. These findings thus suggest the potential of both natural (crayfish gastrolith) and, to a greater extent, synthetic ACC sources for the prevention of metabolic bone disorders and possibly of osteoporotic processes.

Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.

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.

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.

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.

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. Temporal silencing of a newly identified insulin-like gene from prawn androgenic gland inhibits primary spermatogenesis, male secondary sex characteristics, and growth.

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.

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 cu- ticle 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 trans- ported 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 elec- tron 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 chiti- nous lamella. Measurements of pH and calcium levels dur- ing gastrolith deposition showed that calcium concentra- tions 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.

Crustaceans, like most mineralized invertebrates, adopted calcium carbonate mineralization for bulk skeleton reinforcement. Here, we show that a major part of the crustacean class Malacostraca (which includes lobsters, crayfishes, prawns and shrimps) shifted toward the formation of calcium phosphate as the main mineral at specified locations of the mandibular teeth. In these structures, calcium phosphate is not merely co-precipitated with the bulk calcium carbonate but rather creates specialized structures in which a layer of calcium phosphate, frequently in the form of crystalline fluorapatite, is mounted over a calcareous “jaw”. From a functional perspective, the co-existence of carbonate and phosphate mineralization demonstrates a biomineralization system that provides a versatile route to control the physico-chemical properties of skeletal elements. This system enables the deposition of amorphous calcium carbonate, amorphous calcium phosphate, calcite and apatite at various skeletal locations, as well as combinations of these minerals, to form graded composites materials. This study demonstrates the widespread occurrence of the dual mineralization strategy in the Malacostraca, suggesting that in terms of evolution, this feature of phosphatic teeth did not evolve independently in the different groups but rather represents an early common trait.

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. Biological significance The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification).

In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish Cherax quadricarinatus, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes.