Regulation of early spermatogenesis in the giant prawn Macrobrachium rosenbergii by a GCL homolog†.

The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120 896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10 days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations.

Monosex Populations of the Giant Freshwater Prawn Macrobrachium rosenbergii-From a Pre-Molecular Start to the Next Generation Era.

Sexual manipulation in the giant freshwater prawn Macrobrachium rosenbergii has proven successful in generating monosex (both all-male and all-female) populations for aquaculture using a crustacean-specific endocrine gland, the androgenic gland (AG), which serves as a key masculinizing factor by producing and secreting an insulin-like AG hormone (IAG). Here, we provide a summary of the advancements from the discovery of the AG and IAG in decapods through to the development of monosex populations in M. rosenbergii. We discuss the broader sexual development pathway, which is highly divergent across decapods, and provide our future perspective on the utility of novel genetic and genomic tools in promoting refined approaches towards monosex biotechnology. Finally, the future potential benefits of deploying monosex prawn populations for environmental management are discussed.

Ferroptosis precedes apoptosis to facilitate specific death signalling by fatty acids.

Cell death is physiologically induced by specific mediators. However, our power to trigger the process in selected cells is quite limited. The protandric shrimp Hippolyte inermis offers a possible answer. Here, we analyse a de novo transcriptome of shrimp post-larvae fed on diatoms. The sex ratio of diatom-fed shrimps versus shrimps fed on control diets was dramatically altered, demonstrating the disruption of the androgenic gland, and their transcriptome revealed key modifications in gene expression. A wide transcriptomic analysis, validated by real-time qPCR, revealed that ferroptosis represents the primary factor to re-shape the body of this invertebrate, followed by further apoptotic events, and our findings open biotechnological perspectives for controlling the destiny of selected tissues. Ferroptosis was detected here for the first time in a crustacean. In addition, this is the first demonstration of a noticeable effect prompted by an ingested food, deeply impacting the gene networks of a young metazoan, definitely determining its future physiology and sexual differentiation.

Multiple Forms of Neural Cell Death in the Cyclical Brain Degeneration of A Colonial Chordate.

Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration-regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.

Sexual Desire and Erotic Fantasies Questionnaire: The Development and Validation of the Erotic Fantasy Use Scale (SDEF2) on Experience, Attitudes, and Sharing Issues.

BACKGROUND: The investigation of sexual fantasies is a delicate issue within sex research. Most studies have focused on the content of these fantasies, rather than on use, experiences, attitudes, and sharing issues, which are fundamental aspects within sexual therapy. The main aim of the present study was to develop and validate the "Sexual Desire and Erotic Fantasies questionnaire-Part 2. Use of Erotic Fantasies (SDEF2)". METHODS: The SDEF2 was completed by 1773 Italian participants (1105 women, 645 men, and 23 other genders). RESULTS: The final 21-item version presented a five-factor structure (fantasies frequency, fantasies normality, fantasies importance, negative emotions, and sharing and experiencing). The SDEF2 showed good psychometric properties, internal reliability, construct, and discriminant validity, appearing to be able to differentiate between sexually clinical and functional women and men (based on the FSFI and IIEF cut-off scores). CONCLUSIONS: The possibility of assessing fantasies frequency, attitudes, and emotions may be extremely useful both for research and clinical purposes. The current study seems to validate that the SDEF2 is a useful measure of assessing the different aspects related to a fantasizing activity, which was shown to be associated with sexual functioning and satisfaction.

Genes Encoding the Glycoprotein Hormone GPA2/GPB5 and the Receptor LGR1 in a Female Prawn.

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

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

The "IAG-Switch"-A Key Controlling Element in Decapod Crustacean Sex Differentiation.

The androgenic gland (AG)-a unique crustacean endocrine organ that secretes factors such as the insulin-like androgenic gland (IAG) hormone-is a key player in crustacean sex differentiation processes. IAG expression induces masculinization, while the absence of the AG or a deficiency in IAG expression results in feminization. Therefore, by virtue of its universal role as a master regulator of crustacean sexual development, the IAG hormone may be regarded as the sexual "IAG-switch." The switch functions within an endocrine axis governed by neuropeptides secreted from the eyestalks, and interacts downstream with specific insulin receptors at its target organs. In recent years, IAG hormones have been found-and sequenced-in dozens of decapod crustacean species, including crabs, prawns, crayfish and shrimps, bearing different types of reproductive strategies-from gonochorism, through hermaphroditism and intersexuality, to parthenogenesis. The IAG-switch has thus been the focus of efforts to manipulate sex developmental processes in crustaceans. Most sex manipulations were performed using AG ablation or knock-down of the IAG gene in males in order to sex reverse them into "neo-females," or using AG implantation/injecting AG extracts or cells into females to produce "neo-males." These manipulations have highlighted the striking crustacean sexual plasticity in different species and have permitted the manifestation of either maleness or femaleness without altering the genotype of the animals. Furthermore, these sex manipulations have not only facilitated fundamental studies of crustacean sexual mechanisms, but have also enabled the development of the first IAG-switch-based monosex population biotechnologies, primarily for aquaculture but also for pest control. Here, we review the crustacean IAG-switch, a unique crustacean endocrine mechanism, from the early discoveries of the AG and the IAG hormone to recent IAG-switch-based manipulations. Moreover, we discuss this unique early pancrustacean insulin-based sexual differentiation control mechanism in contrast to the extensively studied mechanisms in vertebrates, which are based on sex steroids.

Two Homogametic Genotypes - One Crayfish: On the Consequences of Intersexuality.

In the Australian redclaw crayfish, Cherax quadricarinatus (WZ/ZZ system), intersexuals, although exhibiting both male and female gonopores, are functional males bearing a female genotype (WZ males). Therefore, the occurrence of the unusual homogametic WW females in nature is plausible. We developed W/Z genomic sex markers and used them to investigate the genotypic structure of experimental and native C. quadricarinatus populations in Australia. We discovered, for the first time, the natural occurrence of WW females in crustacean populations. By modeling population dynamics, we found that intersexuals contribute to the growth rate of crayfish populations in the short term. Given the vastly fragmented C. quadricarinatus habitat, which is characterized by drought-flood cycles, we speculate that intersexuals contribute to the fitness of this species since they lead to occasional increment in the population growth rate which potentially supports crayfish population restoration and establishment under extinction threats or colonization events.

From sporadic single genes to a broader transcriptomic approach: Insights into the formation of the biomineralized exoskeleton in decapod crustaceans.

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C. quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.

Ovarian development pattern and vitellogenesis of ridgetail white prawn, Exopalaemon carinicauda.

The ridgetail white prawn Exopalaemon carinicauda has the potential to be used as a model organism in crustacean research because it has a transparent body, available draft genome, and short life cycle. However, their ovarian development pattern remains unclear under laboratory culture conditions. This study investigated the changes of ovarian external feature, ovarian histology, gonadosomatic index (GSI), and hepatosomatic index (HSI), as well as the expression and localization of vitellogenin in the ovary and the hepatopancreas during the first ovarian development cycle of E. carinicauda under laboratory-reared condition. The results demonstrated that (1) the first ovarian development cycle of E. carinicauda could be divided into 5 different stages in which the ovary changes its color from white to yellow during the vitellogenesis process in parallel with increasing GSI. (2) After pubertal molt, most females reached ovarian stage II while the females reached stage V after premating molt. (3) During the ovarian development, GSI increased smoothly and HSI relatively stable during the period of stages I to IV, while GSI increased but HSI decreased significantly from stages IV to V. (4) In situ hybridization (ISH) revealed that EcVg was slightly expressed in the oocyte cytoplasm of previtellogenic oocytes. The positive signal was mainly detected in hepatopancreatic fibrillar cells, and a strong signal was found in the hepatopancreas at stage IV. Moreover, the expression level of EcVg-mRNA in the hepatopancreas is stage-specific, and the hepatopancreas contributes majority of vitellin precursor protein to support the ovarian development of E. carinicauda.

The protandric life history of the Northern spot shrimp Pandalus platyceros: molecular insights and implications for fishery management.

The Northern spot shrimp, Pandalus platyceros, a protandric hermaphrodite of commercial importance in North America, is the primary target species for shrimp fisheries within Southeast Alaska. Fishery data obtained from the Alaska Department of Fish and Game indicate that spot shrimp populations have been declining significantly over the past 25 years. We collected spot shrimps in Southeast Alaska and measured reproductive-related morphological, gonadal and molecular changes during the entire life history. The appendix masculina, a major sexual morphological indicator, is indicative of the reproductive phase of the animal, lengthening during maturation from juvenile to the male phase and then gradually shortening throughout the transitional stages until its complete disappearance upon transformation to a female. This morphological change occurs in parallel with the degeneration of testicular tissue in the ovotestis and enhanced ovarian vitellogenesis. Moreover, we obtained the entire mRNA sequence of the yolk protein precursor, vitellogenin, and monitored its transcript levels throughout the entire shrimp life-cycle. Vitellogenin transcript levels in the hepatopancreas increased in the early transitional stage until reaching a peak prior to extruding eggs. Such transcriptomic analyses, coupled with a comprehensive description of the gonad, external sex characters and timing of the reproductive life history of spot shrimps contribute to a better understanding of the hermaphroditic reproduction process in the cold Southeast Alaskan waters. This knowledge can contribute to a revision of current conservation efforts to maintain wild populations sustainable for both commercial and ecological considerations.

Production of WW males lacking the masculine Z chromosome and mining the Macrobrachium rosenbergii genome for sex-chromosomes.

The cultivation of monosex populations is common in animal husbandry. However, preselecting the desired gender remains a major biotechnological and ethical challenge. To achieve an efficient biotechnology for all-female aquaculture in the economically important prawn (Macrobrachium rosenbergii), we achieved - for the first time - WW males using androgenic gland cells transplantation which caused full sex-reversal of WW females to functional males. Crossing the WW males with WW females yielded all-female progeny lacking the Z chromosome. We now have the ability to manipulate - by non-genomic means - all possible genotype combinations (ZZ, WZ and WW) to retain either male or female phenotypes and hence to produce monosex populations of either gender. This calls for a study of the genomic basis underlying this striking sexual plasticity, questioning the content of the W and Z chromosomes. Here, we report on the sequencing of a high-quality genome exhibiting distinguishable paternal and maternal sequences. This assembly covers ~ 87.5% of the genome and yielded a remarkable N50 value of ~ 20 × 106 bp. Genomic sex markers were used to initiate the identification and validation of parts of the W and Z chromosomes for the first time in arthropods.

The gene encoding the insulin-like androgenic gland hormone in an all-female parthenogenetic crayfish.

Male sexual differentiation in crustaceans is controlled by the androgenic gland (AG), a unique male endocrine organ that, in decapods, is located at the base of the 5th pereiopod. In these animals, the insulin-like androgenic gland hormone (IAG) is the major factor secreted from the AG to induce masculinization and maintain male characteristics. It has, however, recently been proposed that this hormone also plays a role in growth and ovarian development in females. In this study, we tested such a possibility by searching for the IAG gene in the marbled crayfish, a parthenogenetic animal that reproduces asexually to form an all-female genetic clone. Based on the phylogenetic relationship between the marbled crayfish and Procambarus fallax, a gonochoristic species of the same North American Cambaridae family, we searched for the IAG gene in the marbled crayfish and then fully sequenced it. The open reading frame of the gene was found to be completely identical in the two species, and their introns shared over 94% identity. It was also found that, in addition to its expression at the base of the 5th pereiopod and in the testes of male P. fallax crayfish, IAG was expressed in the muscle tissue of P. fallax males and females and even of the parthenogenetic marbled crayfish. These findings provide new insight into possible functions of IAG, in addition to its role as a masculinization-inducing factor, and also constitute the basis for a discussion of the evolutionary relationship between the above two species.

A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture.

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.