Transcriptional regulation of IAG by dsx and foxl-2 in mud crab (Scylla paramamosain).

Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG, we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG, respectively. The results showed that dsx could significantly promote the expression of Sp-IAG, while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx, while significantly increased after interfering with foxl-2, which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG.

Association of anaemia with indoor air pollution among older Indian adult population: multilevel modelling analysis of nationally representative cross-sectional study.

INTRODUCTION: Anaemia is a disease of public health importance with multi-causal pathways. Previous literature suggests the role of indoor air pollution (IAP) on haemoglobin levels, but this has been studied less due to logistic constraints. A high proportion of the population in developing countries, including India, still depends on unclean fuel, which exacerbates IAP. The objective was to study the association between anaemia and IAP among the older Indian adult population (≥ 45 years) as per gender. METHODS: Our study analysed the nationally representative dataset of the Longitudinal Ageing Study in India (LASI 2017-18, Wave-1). We have documented the association of anaemia (outcome variable) with IAP (explanatory variable). To reduce the confounding effects of demographic and socioeconomic; health related and behavioural covariates; propensity score matching (PSM) was conducted. Nested multilevel regression modelling was conducted. States and union territories were categorised cross tabulated as low, middle and high as per anaemia and IAP exposure. P value < 0.05 was considered statistically significant. SATA version 17 was used for analysis. RESULTS: More than half (52.52%) of the participants were exposed to IAP (male (53.55%) > female (51.63%)). The odds of having anaemia was significantly 1.19 times higher (AOR 1.19 (1.09-1.31)) among participants using unclean/ solid fuel. The adjusted odds were significantly higher among participants exposed to pollution-generating sources (AOR 1.30; 1.18-1.43), and household indoor smoking (AOR 1.17 (1.07-1.29). The odds of having anaemia were significantly higher (AOR 1.26; 1.15-1.38) among participants exposed to IAP, which was higher in males (AOR 1.36; 1.15-1.61) than females (AOR 1.21; 1.08-1.35). Empowered Action Group (EAG) states like Uttar Pradesh, Chhattisgarh, Madhya Pradesh, Bihar had both high anaemia and IAP exposure. CONCLUSION: This study established the positive association of anaemia with indoor air pollution among older Indian adults through a nationally representative large dataset. The association was higher among men. Further research is recommended to understand detailed causation and to establish temporality. It is a high time to implement positive intervention nationally to decrease solid/ unclean fuel usage, vulnerable ventilation, indoor smoking, IAP and health hazards associated with these with more focused actions towards EAG states.

Identification of a Putative CFSH Receptor Inhibiting IAG Expression in Crabs.

The crustacean female sex hormone (CFSH) is a neurohormone peculiar to crustaceans that plays a vital role in sexual differentiation. This includes the preservation and establishment of secondary female sexual traits, as well as the inhibition of insulin-like androgenic gland factor (IAG) expression in the androgenic gland (AG). There have been no reports of CFSH receptors in crustaceans up to this point. In this study, we identified a candidate CFSH receptor from the mud crab Scylla paramamosain (named Sp-SEFIR) via protein interaction experiments and biological function experiments. Results of GST pull-down assays indicated that Sp-SEFIR could combine with Sp-CFSH. Findings of in vitro and in vivo interference investigations exhibited that knockdown of Sp-SEFIR could significantly induce Sp-IAG and Sp-STAT expression in the AG. In brief, Sp-SEFIR is a potential CFSH receptor in S. paramamosain, and Sp-CFSH controls Sp-IAG production through the CFSH-SEFIR-STAT-IAG axis.

Insulin-like Androgenic Gland Hormone Induced Sex Reversal and Molecular Pathways in Macrobrachium nipponense: Insights into Reproduction, Growth, and Sex Differentiation.

This study investigated the potential to use double-stranded RNA insulin-like androgenic gland hormone (dsIAG) to induce sex reversal in Macrobrachium nipponense and identified the molecular mechanisms underlying crustacean reproduction and sex differentiation. The study aimed to determine whether dsIAG could induce sex reversal in PL30-male M. nipponense during a critical period. The sex-related genes were selected by performing the gonadal transcriptome analysis of normal male (dsM), normal female (dsFM), neo-female sex-reversed individuals (dsRM), and unreversed males (dsNRM). After six injections, the experiment finally resulted in a 20% production of dsRM. Histologically, dsRM ovaries developed slower than dsFM, but dsNRM spermathecae developed normally. A total of 1718, 1069, and 255 differentially expressed genes were identified through transcriptome sequencing of the gonads in three comparison groups, revealing crucial genes related to reproduction and sex differentiation, such as GnRHR, VGR, SG, and LWS. Principal Component Analysis (PCA) also distinguished dsM and dsRM very well. In addition, this study predicted that the eyestalks and the "phototransduction-fly" photoperiodic pathways of M. nipponense could play an important role in sex reversal. The enrichment of related pathways and growth traits in dsNRM were combined to establish that IAG played a significant role in reproduction, growth regulation, and metabolism. Finally, complete sex reversal may depend on specific stimuli at critical periods. Overall, this study provides valuable findings for the IAG regulation of sex differentiation, reproduction, and growth of M. nipponense in establishing a monoculture.

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.

Transcriptome analysis of the post-larvae of giant freshwater prawn (Macrobrachium rosenbergii) after IAG gene knockdown with microRNA interference.

The insulin-like androgenic gland hormone gene (IAG) of crustaceans plays pivotal roles in the regulation of sex differentiation. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that function as post-transcriptional gene regulators. However, little information about the regulatory relationship between miRNA and Macrobrachium rosenbergii IAG (MrIAG) were exposed. In this study, we used the 3' untranslated region (UTR) of MrIAG to predict potential target sites of miRNAs. The results showed that miR-184 has one target site in the 3'UTR of MrIAG. Dual-luciferase report assay in vitro confirmed that miR-184 can significantly down-regulate MrIAG expression. Besides, we constructed mutant plasmids of 3'UTR of MrIAG. The result displayed that after co-transfection of mutant plasmids and miR-184 agomir, the activity of luciferase was not affected compared to the control. These results indicated that miR-184 could directly regulate MrIAG. In addition, we found that overexpression of miR-184 in M. rosenbergii can lead to significant changes in the transcription level of genes. Compared with control group, we identified 1510 differentially expressed genes (DEGs) in the miR-184 injection group. Some DEGs were involved in sex differentiation, gonad development, growth and molting were found. qRT-PCR verification was performed on eight DEGs randomly, and the results showed that the expression level of sex-, growth-, and metabolism-related genes changed significantly after MrIAG gene knockdown. Collectively, findings from this study suggest that miR-184, by mediating IAG expression, may be involved in many physiological processes in M. rosenbergii. The current study lays a basic understanding for short-term silencing of MrIAG with miR-184, and facilitates miRNA function analysis in M. rosenbergii in future.

Two Short Repeats in the 5' Untranslated Region of Insulin-like Androgenic Gland Factor in Procambarus clarkii (PcIAG) That Regulate PcIAG Expression.

Insulin-like androgenic gland factor (IAG) plays an important role in sex manipulation in decapods. Understanding the molecular regulation mechanism of IAG in Procambarus clarkii (PcIAG) is important for realizing its sex control. In this study, the promoter and gene structure of PcIAG, mRNA, and miRNA expression profiles after interfering with two siRNAs synthesized according to the two short repeats in the 5' untranslated regions (5'UTR) of PcIAG were analyzed, and miRNAs of exosomes were investigated to explore the role of repeated sequences with tandem two short repeats located in the 5'UTR of PcIAG isolated from the androgenic gland (AG) in the regulation of IAG expression. The results showed that the repeated sequences of 5'UTR only occurred completely in the cDNA from AG, and the function of the two repeats was different in regulating the expression of PcIAG, in which the Wnt signaling pathway may be involved. Furthermore, we found that six miRNAs including miR-133, miR-193, miR-34, miR-1, miR-100, and let-7 might be involved in the regulation of the expression of PcIAG, wherein miR-133 might directly be related with the repeated sequences of 5'UTR.

Insulin-like androgenic gland hormone 1 (IAG1) regulates sexual differentiation in a hermaphrodite shrimp through feedback to neuroendocrine factors.

Insulin-like androgenic gland hormone (IAG) is regarded as a key sexual differentiation regulator in gonochoristic crustaceans. However, until now the knowledge concerning its functions in hermaphroditic crustaceans is scanty. Herein, we investigated the function of IAG (Lvit-IAG1) in peppermint shrimp Lysmata vittata, a species that possesses protandric simultaneous hermaphroditism (PSH) reproductive system, which is rare among crustaceans. Lvit-IAG1 was exclusively expressed in the androgenic gland. The qRT-PCR demonstrated that its mRNA expression level was relatively high at the functional male phase but decreased sharply in the subsequent euhermaphrodite phase. Both the short-term and long-term silencing experiments showed that Lvit-IAG1 negatively regulated both the gonad-inhibiting hormone (Lvit-GIH) and crustacean female sex hormone (Lvit-CFSH) expressions in the eyestalk ganglion. Besides, Lvit-IAG1 gene knockdown induced a retarded development of the appendices masculinae (AM) and male gonopores while suppressing the germ cells at the primary spermatocyte stage. Also, Lvit-IAG1 gene silencing hindered ovarian development. This in turn led to small vitellogenic oocytes and decreased expression of vitellogenin and vitellogenin receptor genes in hepatopancreas and ovarian region, respectively. Generally, this study's findings imply that Lvit-IAG1 modulated the male sexual differentiation in PSH species L. vittata, and exhibited negative feedback on Lvit-GIH and Lvit-CFSH genes expression in the species' eyestalk ganglion.

Insight into the Regulatory Relationships between the Insulin-Like Androgenic Gland Hormone Gene and the Insulin-Like Androgenic Gland Hormone-binding Protein Gene in Giant Freshwater Prawns (Macrobrachium rosenbergii).

Giant freshwater prawns (Macrobrachium rosenbergii) are commonly found throughout the world. The size of the male giant freshwater prawn is much larger than that of the female. Therefore, understanding the molecular mechanism that underlies the sexual differentiation of M. rosenbergii is of both commercial and scientific importance. Insulin-like androgenic gland hormone (IAG) plays a key role in the differentiation of sex in M. rosenbergii. Although IAG has been investigated, the regulatory relationship between IAG and its binding protein partner, the insulin-like androgenic gland hormone-binding protein (IAGBP), has not been studied in M. rosenbergii. Here, we cloned and characterized the IAGBP from M. rosenbergii (Mr-IAGBP) for the very first time. Transcriptomic analysis showed that Mr-IAGBP mRNA was detected in a wide array of tissues with the highest expression found in the androgenic gland. The importance of IAG in male development was further demonstrated by an increase in IAG transcripts during the development of the androgenic gland and Mr-IAG was only highly transcribed in the androgenic gland of M. rosenbergii. Interestingly, we found that the Mr-IAG gene expression started during the 20th-day larva after hatching stage (LH20), followed (20th-day post-larval stage, PL20) by a gradual elevation of Mr-IAGBP levels. The levels of both genes peaked at the adult stage. The relationship between Mr-IAGBP and Mr-IAG was further analyzed using RNA interference. The injection of Mr-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mr-IAG, while the amount of Mr-IAGBP mRNA and the translation of IAGBP protein was significantly reduced by the injection of Mr-IAG dsRNA. These results revealed that IAGBP is involved in IAG signaling. Furthermore, our data supports the hypothesis that (IAG and IAGBP)-IAG receptor signaling schemes exist in M. rosenbergii. Our results will provide important information for the further study of determining the sex of M. rosenbergii.

Screening for Dmrt genes from embryo to mature Macrobrachium rosenbergii prawns.

The doublesex and mab-3 related transcription 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.

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway.

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.

Isolation and Tissue Distribution of an Insulin-Like Androgenic Gland Hormone (IAG) of the Male Red Deep-Sea Crab, Chaceon quinquedens.

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5' untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3' UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

Understanding Insulin Endocrinology in Decapod Crustacea: Molecular Modelling Characterization of an Insulin-Binding Protein and Insulin-Like Peptides in the Eastern Spiny Lobster, Sagmariasus verreauxi.

The insulin signalling system is one of the most conserved endocrine systems of Animalia from mollusc to man. In decapod Crustacea, such as the Eastern spiny lobster, Sagmariasus verreauxi (Sv) and the red-claw crayfish, Cherax quadricarinatus (Cq), insulin endocrinology governs male sexual differentiation through the action of a male-specific, insulin-like androgenic gland peptide (IAG). To understand the bioactivity of IAG it is necessary to consider its bio-regulators such as the insulin-like growth factor binding protein (IGFBP). This work has employed various molecular modelling approaches to represent S. verreauxi IGFBP and IAG, along with additional Sv-ILP ligands, in order to characterise their binding interactions. Firstly, we present Sv- and Cq-ILP2: neuroendocrine factors that share closest homology with Drosophila ILP8 (Dilp8). We then describe the binding interaction of the N-terminal domain of Sv-IGFBP and each ILP through a synergy of computational analyses. In-depth interaction mapping and computational alanine scanning of IGFBP_N' highlight the conserved involvement of the hotspot residues Q67, G70, D71, S72, G91, G92, T93 and D94. The significance of the negatively charged residues D71 and D94 was then further exemplified by structural electrostatics. The functional importance of the negative surface charge of IGFBP is exemplified in the complementary electropositive charge on the reciprocal binding interface of all three ILP ligands. When examined, this electrostatic complementarity is the inverse of vertebrate homologues; such physicochemical divergences elucidate towards ligand-binding specificity between Phyla.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.

Crystal structure of the effector protein XOO4466 from Xanthomonas oryzae.

Many Gram-negative bacteria deliver their virulence factors into host cells through a secretion system. Those factors, called effector proteins, are involved in the pathogenicity in host cells by interfering with various cellular events. The phytopathogen Xanthomonas oryzae pv. oryzae uses a type III secretion system to inject its effectors, but the functional roles of these proteins remain largely uncharacterized. Here, we determined a crystal structure of XOO4466, an effector from X. oryzae pv. oryzae, and performed a functional analysis. We determined that XOO4466 is similar in sequence to Xanthomonas outer protein Q, a putative nucleoside hydrolase (NH). The overall structure of XOO4466 is homologous to that of NHs, including a metal-binding site, but differs in its oligomeric state and active site topology. Further analysis indicated that antiparallel ß-strands commonly found in NHs adjacent to the active site loop are replaced in XOO4466 with a short loop, causing the active site loop to adopt a conformation distinct from that of NHs. Thus, the catalytic residues emanating from the respective active site loop of NHs are absent in the putative active site of XOO4466. Consistent with these structural features, a functional assay indicated that XOO4466 does not exhibit NH activity and possibly catalyzes yet unknown reactions.

Destabilization of peptide:MHC interaction induces IL-2 resistant anergy in diabetogenic T cells.

Autoreactive T cells are responsible for inducing several autoimmune diseases, including type 1 diabetes. We have developed a strategy to induce unresponsiveness in these cells by destabilizing the peptide:MHC ligand recognized by the T cell receptor. By introducing amino acid substitutions into the immunogenic peptide at residues that bind to the MHC, the half life of the peptide:MHC complex is severely reduced, thereby resulting in abortive T cell activation and anergy. By treating a monoclonal diabetogenic T cell population with an MHC variant peptide, the cells are rendered unresponsive to the wild type ligand, as measured by both proliferation and IL-2 production. Stimulation of T cells with MHC variant peptides results in minimal Erk1/2 phosphorylation or cell division. Variant peptide stimulation effectively initiates a signaling program dominated by sustained tyrosine phosphatase activity, including elevated SHP-1 activity. These negative signaling events result in an anergic phenotype in which the T cells are not competent to signal through the IL-2 receptor, as evidenced by a lack of phospho-Stat5 upregulation and proliferation, despite high expression of the IL-2 receptor. This unique negative signaling profile provides a novel means to shut down the anti-self response.

An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone.

Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone.

Analysis of mRNA and MicroRNA Expression Profiles of Nervous Tissues and Reproductive Tissues in Male Procambarus clarkii After Silencing IAG.

The insulin-like androgenic gland hormone gene (IAG), primarily expressed in the androgenic gland (AG), plays a crucial role in controlling male sex differentiation and maintaining male secondary sexual characteristics in decapods. In this study, we investigated the mRNA and microRNA expression profiles of male Procambarus clarkii to understand the transcriptomic regulatory mechanism of IAG after the injection of an efficient siRNA (GsiRNA) designed based on IAG. The results revealed that several differentially expressed genes were enriched in reproduction-related pathways, such as the wnt signaling pathway, MAPK signaling pathway, and GnRH signaling pathway. In the testis (Te), the injection of GsiRNA led to the up-regulation of many ovary-related genes and down-regulation of testis-related genes. Moreover, the brain (Br) and abdominal nerve cord (AN) appeared to be involved in the regulation of IAG, with numerous differentially expressed genes found in Br and AN. Notably, the expression of five neuropeptide genes, Crustacean hyperglycemic hormone, pigment-dispersing hormone, red pigment concentrating hormone precursor, corazonin, and gonadotropin-releasing hormone II receptor isoform X1 in Br/AN, was significantly changed. Additionally, three ovary-related miRNAs (miR-263a, miR-263b, miR-133) highly expressed in Te/AG showed significant up-regulation after GsiRNA injection. Furthermore, the long-term interference of GsiRNA was found to inhibit the development of male external sexual characteristics during the juvenile stage and delay it during the adult stage. This research provides valuable insights into the molecular regulatory mechanism and function of IAG in P. clarkii.

RNAi Analysis of Potential Functions of Cyclin B3 in Reproduction of Male Oriental River Prawns (Macrobrachium nipponense).

Cyclin B3 (CycB3) is involved in the metabolic pathway of the cell cycle, playing essential roles in the regulation of cell proliferation and mitosis. CycB3 is also predicted to be involved in the reproduction of male oriental river prawns (Macrobrachium nipponense). In this study, the potential functions of CycB3 in M. nipponense were investigated using quantitative real-time PCR, RNA interference, and histological observations. The full-length DNA sequence of CycB3 in M. nipponense was 2147 base pairs (bp) long. An open reading frame of 1500 bp was found, encoding 499 amino acids. A highly conserved destruction box and two conserved cyclin motifs were found in the protein sequence of Mn-CycB3. Phylogenetic tree analysis revealed that this protein sequence was evolutionarily close to that of CycB3s of crustacean species. Quantitative real-time PCR analysis results suggested that CycB3 was involved in the process of spermiogenesis, oogenesis, and embryogenesis in M. nipponense. RNA interference analysis showed that CycB3 had a positive regulatory relationship with insulin-like androgenic gland hormone (IAG) in M. nipponense. In addition, sperm were rarely observed in the testis of double-stranded CycB3-injected prawns after 14 days of treatment, and sperm abundance was dramatically lower than that in the double-stranded GFP-injected prawns on the same day. This result indicated that CycB3 can regulate the testis reproduction in M. nipponense through inhibiting the IAG expressions. Overall, these results indicated that CycB3 plays essential roles in the regulation of male reproduction in M. nipponense, which may promote the studies of male reproduction in other crustacean species.