The physiological response of oriental river prawn Macrobrachium nipponense to starvation-induced stress.

Environmental stresses play critical roles in the physiology of crustaceans. Food deprivation is an important environmental factor and a regular occurrence in both natural aquatic habitats and artificial ponds. However, the underlying physiological response mechanisms to starvation-caused stress in crustaceans are yet to be established. In the present study, the hepatopancreas tissue of Macrobrachium nipponense was transcriptome analyzed and examined for starvation effects on oxidative stress, DNA damage, autophagy, and apoptosis across four fasting stages (0 (control group), 7, 14, and 21 days). These results indicated that a ROS-mediated regulatory mechanism is critical to the entire fasting process. At the initial stage of starvation (fasting 0 d ~ 7 d), ROS concentration increased gradually, activating antioxidant enzymes to protect the cellular machinery from the detrimental effects of oxidative stress triggered by starvation-induced stress. ROS content production (hydrogen peroxide and superoxide anion) then rose continuously with prolonged starvation (fasting 7 d ~ 14 d), reaching peak levels and resulting in autophagy in hepatopancreas cells. During the final stages of starvation (fasting 14 d ~ 21 d), excessive ROS induced DNA damage and cell apoptosis. Furthermore, autophagolysosomes and apoptosis body were further identified with transmission electron microscopy. These findings lay a foundation for further scrutiny of the molecular mechanisms combating starvation-generated stress in M. nipponense and provide fishermen with the theoretical guidance for adopting fasting strategies in M. nipponense aquaculture.

Integrated microRNA-mRNA analysis reveals a possible molecular mechanism of enteritis susceptibility in Litopenaeus vannamei.

Enteritis is one of the main diseases affecting Pacific whiteleg shrimp (Litopenaeus vannamei) in recent years, and it has resulted in huge losses to the aquaculture industry. Prior to this study, the molecular mechanism underlying enteritis in L. vannamei was unclear, and comprehensive multi-omics analysis had not been conducted. In this study, 1209 differentially expressed genes (DEGs) were identified from the hepatopancreas of L. vannamei with and without enteritis. Kyoto Encyclopedia of Genes and Genomes analysis showed that genes were significantly enriched in immune, metabolic, and endocrine regulatory pathways. Forty-eight significantly different microRNAs (miRNAs) were identified in the miRNA-Seq analysis. Further functional annotation analysis showed that the regulatory pathway of target gene enrichment of differentially expressed miRNAs was consistent with DEGs. Through miRNA-mRNA integration analysis, 47 meaningful miRNA-mRNA pairs were obtained, of which melanogenesis and pancreatic secretion were considered key pathways. Subsequent miRNA-mRNA interaction network analysis revealed that mja-miR-6493-3p, Mja-miR-6494, novel-198, novel-272, novel-261, novel-200, novel-183, novel-184, novel-237, and novel-192 may be key miRNAs involved in the regulation of these two signaling pathways. Finally, the RAS signaling pathway was found to inhibit the translation level of proteins in the hepatopancreas. These results suggest that target gene integration analysis of mRNA-miRNA can reveal the molecular mechanism underlying enteritis in L. vannamei and also provide valuable new insights for resisting enteritis.

Different responses to risperidone treatment in Schizophrenia: a multicenter genome-wide association and whole exome sequencing joint study.

Risperidone is routinely used in the clinical management of schizophrenia, but the treatment response is highly variable among different patients. The genetic underpinnings of the treatment response are not well understood. We performed a pharmacogenomic study of the treatment response to risperidone in patients with schizophrenia by using a SNP microarray -based genome-wide association study (GWAS) and whole exome sequencing (WES)-based GWAS. DNA samples were collected from 189 patients for the GWAS and from 222 patients for the WES after quality control in multiple centers of China. Antipsychotic response phenotypes of patients who received eight weeks of risperidone treatment were quantified with percentage change on the Positive and Negative Syndrome Scale (PANSS). The GWAS revealed a significant association between several SNPs and treatment response, such as three GRM7 SNPs (rs141134664, rs57521140, and rs73809055). Gene-based analysis in WES revealed 13 genes that were associated with antipsychotic response, such as GPR12 and MAP2K3. We did not identify shared loci or genes between GWAS and WES, but association signals tended to cluster into the GPCR gene family and GPCR signaling pathway, which may play an important role in the treatment response etiology. This study may provide a research paradigm for pharmacogenomic research, and these data provide a promising illustration of our potential to identify genetic variants underlying antipsychotic responses and may ultimately facilitate precision medicine in schizophrenia.

From Structural Design to Functional Construction: Amine Molecules in High-Performance Formamidinium-Based Perovskite Solar Cells.

Formamidinium (FA) based perovskites are considered as one of the most promising light-absorbing perovskite materials owing to their narrower band gap and better thermal stability compared to conventional methylammonium-based perovskites. Constant improvement by using various additives stimulates the potential application of these perovskites. Amine molecules with different structures have been widely used as typical additives in FA-based perovskite solar cells, and decent performances have been achieved. Thus, a systematic review focusing on structural regulation and functional construction of amines in FA-based perovskites is of significance. Herein, we analyze the construction mechanism of different structural amines on the functional perovskite crystals. The influence of amine molecules on specific perovskite properties including defect conditions, charge transfer, and moisture resistance are evaluated. Finally, we summarize the design rules of amine molecules for the application in high-performance FA-based perovskites and propose directions for the future development of additive molecules.

Transcriptome analysis of the potential molecular mechanism of enteritis in intestine and hepatopancreas of Litopenaeus vannamei.

The prawn, Litopenaeus vannamei (L. vannamei), is the most widely farmed species in the world but the incidence of enteritis in L. vannamei has increased in recent years. However, the pathogenesis of enteritis remains unclear. In this study, high-throughput sequencing was used to analyze the hepatopancreatic and intestinal transcriptome of healthy and enteritis-affected individuals from the same pond. In total, 1209 and 1608 differently-expressed genes (DEGs) were detected in the hepatopancreatic and intestinal transcriptomes, respectively. Significantly changed genes were enriched in the intestinal immune network for IgA Production, Lysosomes, Sphingolipid Metabolism and the Peroxisome Signaling Pathway. Expression of the integrin α4ß7 gene was significantly increased in the intestine of L. vannamei with enteritis, while expression of 38 DEGs associated with the lysosome was significantly down-regulated. Furthermore, the expression of sphingolipid metabolism-related enzymes and superoxide dismutase (SOD) genes was also significantly decreased, indicating that abnormal autoimmune function, weak intestinal resistance to external pathogenic microbial invasion, and self-healing ability were important factors associated with enteritis in L. vannamei. In addition, the expression of trypsin and pancreatic lipase was decreased in the hepatopancreas of L. vannamei with enteritis. This study provided new insights into the possible molecular pathogenesis of enteritis in L. vannamei.

Cloning, functional and regulation analysis of a novel male reproduction-related protein gene from the oriental river prawn Macrobrachium nipponense.

Gonadogenesis processes in crustaceans are complex. There, however, has been a large amount of research focused on regulation of female gonad (ovary) development in crustaceans, however, there has been little focus on the male gonad (testis). In the current study, a novel male reproduction-related protein gene (Mn-MRP) was identified from Macrobrachium nipponense. The relative abundance of Mn-MRP mRNA transcript in tissues and at different developmental stages were investigated. The relative abundance of Mn-MRP mRNA transcript was larger in the testis than other tissues, and during the testis maturation stage than at other developmental stages, suggesting Mn-MRP may have important functions in reproduction processes. The RNA interference (RNAi) was used to further investigate the Mn-MRP biological function. Silencing of the Mn-MRP gene effectively decreased the abundance of the sperm gelatinase (Mn-SG) mRNA transcript, implying the protein encoded by this gene may have functions in sperm activity during the fertilization process. Further studies with RNAi and eyestalk ablation confirmed that gonad inhibiting hormone gene (Mn-GIH) is a negative regulator of Mn-MRP, and that the insulin-like androgenic gland hormone gene (Mn-IAG) is a positive regulator. There, therefore, was cloning of the Mn-MRP gene, and investigation of its potential biological function, as well as elucidation of the positive/negative regulators in current study. The results from this study provide for a greater understanding of regulatory mechanisms of male reproduction in crustaceans.

MicroRNA transcriptome analysis of oriental river prawn Macrobrachium nipponense in responding to starvation stress.

Food deprivation or fasting is an important environmental factor, and a regular occurrence in both natural aquatic habitats and artificial ponds. However, the potential immunoregulatory mechanisms underlying starvation stress in crustaceans remain unclear. MicroRNAs (miRNAs) are a new class of non-coding RNAs that can regulate various biological processes, such as stress and immune responses. In the present work, miRNAs related to starvation stress responses and immune properties were identified and characterised in oriental river prawn Macrobrachium nipponense using high-throughput sequencing and bioinformatics analyses. Twelve small RNA libraries from hepatopancreas tissue were sequenced across four fasting stages lasting 0, 7, 14 or 21 days. In total, 550 miRNAs were identified including 198 putative novel miRNAs and 352 conserved miRNAs belonging to 57 families. Moreover, compared with expression levels at 0 days, 27, 27 and 43 miRNAs were differentially expressed (DE-miRNAs) at 7, 14 and 21 days, respectively. Among these, four DE-miRNAs (ame-miR-190-5p, dme-miR-307a-3p, hme-miR-2788-3p and novel_68) were co-expressed at all three timepoints. Furthermore, 661 target genes regulated by these DE-miRNAs were identified, and associated functional annotations were derived by GO enrichment and KEGG pathway analyses, which showed that most DE-miRNAs were mainly participated in metabolic processes and immune responses. Furthermore, 26 host DE-miRNAs potentially participated in interactions with white spot syndrome virus (WSSV) were identified by predicting and analysing target genes from WSSV. The further WSSV challenge under starvation stress showed that dme-miR-307a-3p played a part in the antiviral responses against WSSV. Our results demonstrate that dme-miR-307a-3p may play vital regulatory roles in responding to starvation stress and WSSV infection. The findings contribute new insight into the molecular mechanisms associated with immune responses to environmental stress in crustaceans.

Molecular characteristics and abundance of insulin-like androgenic gland hormone and effects of RNA interference in Eriocheir sinensis.

Insulin-like androgenic gland hormone (IAG) has an important function in sexual differentiation and somatic growth in crustaceans. In this study, there was cloning of the full-length sequence of IAG from Eriocheir sinensis (Es-IAG). The full-length Es-IAG gene was 1392 base pairs long and encoded a protein of 151 amino acid residues. The precursor peptide included a signal peptide, and the protein was a protein that is secreted from the cell in which it is produced with no transmembrane domain. Amino acid sequence alignment indicated there was the greatest homology between E. sinensis and Chaceon quinquedens (47 %), followed by Callinectes sapidus (44 %). Results from analysis of the relative abundances of Es-IAG mRNA transcript at different developmental stages indicated that Es-IAG may have an important endocrine function in early embryonic development, and that Stages I through Ⅲ may be an important period for sexual differentiation in juvenile E. sinensis. In vivo treatment with siRNA-391 resulted in a 66.7 % lesser relative abundance of the Es-IAG mRNA transcript. Three treatments with siRNA-391 to inhibit Es-IAG production during Stages Ⅲ to Ⅴ period resulted in about 10 % of male crabs being transformed into "neo-females." These results provide the basis for further research into the sexual differentiation mechanism and monosex breeding of E. sinensis.

A Simple Genome Walking Strategy to Isolate Unknown Genomic Regions Using Long Primer and RAPD Primer.

BACKGROUND: Genome walking is a DNA-cloning methodology that is used to isolate unknown genomic regions adjacent to known sequences. However, the existing genome-walking methods have their own limitations. OBJECTIVES: Our aim was to provide a simple and efficient genome-walking technology. MATERIAL AND METHODS: In this paper, we developed a novel PCR strategy (termed SLRA PCR) that uses a single long primer (SLP), a set of gene specific primers (GSP), and a random amplified polymorphic DNA (RAPD) primer for genome walking. SLRA PCR consists of two processes: the first amplification using SLP, and three successive rounds of nested PCR amplified by GSP and RAPD primer. The novelty of the approach lies in the use of long primers (SLP and GSP) and same annealing and extension temperature 68℃ in combination. This method offers higher amplification efficiency, superior versatility, and greater simplicity compared with conventional randomly primed PCR methods for genome walking. RESULTS: The promoter regions and the first introns of the insulin-like androgenic gland hormone (IAG) gene and the hemocyanin gene of Macrobrachium nipponense were cloned using SLRA PCR, respectively. CONCLUSIONS: This genome walking strategy can be applied to a wide range of genomes.

Molecular insights into ovary degeneration induced by environmental factors in female oriental river prawns Macrobrachium nipponense.

The oriental river prawn, Macrobrachium nipponense, is an important breeding species in China. The ovary development of this prawn is regulated by the genetic factors and external environmental factors and has obvious seasonal regularity. However, the molecular mechanism of regulating ovary degradation in M. nipponense remains unclear. To address this issue, we performed transcriptome sequencing and gene expression analyses of eyestalks, cerebral ganglia (CG) and thoracic ganglia (TG) of female M. nipponense between the full ovary stage and degenerate ovary stage. Differentially expressed genes enrichment analysis results identified several important pathways such as "phototransduction-fly," "circadian rhythm-fly" and "steroid hormone biosynthesis secretion." In the period of ovarian degeneration, the expressions of Tim, Per2 and red pigment concentration hormone (RPCH) were significantly decreased in the eyestalk, CG and TG. And expression of 7 genes in the steroid synthesis pathway, including steryl-sulfatase, cytochrome P450 family 1 subfamily A polypeptide 1, estradiol 17ß-dehydrogenase 2, glucuronosyltransferase, 3-oxo-5-alpha-steroid 4-dehydrogenase 1, estradiol 17-dehydrogenase 1 and estrone sulfotransferase was significantly decreased in the CG. Food and light signals affect the expression of clock genes and thereby decrease the expression of RPCH and the estradiol synthesis-related genes in the nervous system, which may be the main cause of ovarian degeneration in M. nipponense. The results will contribute to a better understanding of the molecular mechanisms of ovarian development regulation in crustaceans.

Molecular and functional analysis of the insulin-like peptides gene in the oriental river prawn Macrobrachium nipponense.

The insulin-like peptide (ILP) family is a group of evolutionarily conserved proteins that control body size and organ growth in metazoans. In the current study we describe, for the first time, the Mn-ILP gene in the oriental river prawn Macrobrachium nipponense. Full-length of the Mn-ILP cDNA was 1630 bp, encoding 174 amino acids. The deduced amino acid sequence of Mn-ILP had the typical features of ILP proteins, including two cleavage sites and six conserved cysteines. To define the function of Mn-ILP, the expression ofthe Mn-ILP gene in different growth stages of prawns of both sexes, in male prawns of different sizes, and in prawns at different stages of the molt cycle was analyzed by qRT-PCR. Mn-ILP expression was significantly higher 1) in the rapid growth stage than in the other stages of male prawns; 2) in the normal growth stage than in the gonad development stage of female prawns; 3) in big male prawns than in small male prawns; and 4) in the intermolt stage than in the other stages of the molt cycle in prawns of the same size. Further, silencing Mn-ILP expression by RNAi effectively slowed down the growth speed of M. nipponense. Thus, Mn-ILP appears to have an important role in the growth and development process of M. nipponense.

Transcriptional responses to starvation stress in the hepatopancreas of oriental river prawn Macrobrachium nipponense.

Various crustaceans are farmed using aquaculture, and food deprivation or fasting can occur due to changing of environmental or management strategies. However, the molecular mechanisms underlying responses to starvation in crustaceans remain unclear. To address this, 12 hepatopancreas transcriptomes were compared for oriental river prawn (Macrobrachium nipponense) from four fasting stages (0, 7, 14 and 21 d). Gene Ontology functional annotation and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes were subsequently performed. During the early stages of starvation (0-7 d), drug metabolism via the cytochrome P450 pathway and metabolism of xenobiotics by the cytochrome P450 pathway were enriched, suggesting that they metabolised compounds generated under starvation stress. As starvation proceeded (7-14 d), the retinol (vitamin A) metabolism pathway was activated, based on three up-regulated genes (CYP3, ADH and UGT), along with the two p450 pathways. Meanwhile, vitamin A was gradually consumed. As acute starvation was reached (14-21 d), vitamin A deficiency decreased the mRNA expression levels of IGF-I that is involved in the mTOR signalling pathway, which ultimately affected the growth and development of M. nipponense. Our results implicate drug/xenobiotic metabolism by cytochrome P450s in adaptation to starvation stress. Furthermore, metabolic cascades (CYP and retinol pathways) and growth (mTOR signalling) pathways are clearly triggered in crustaceans during starvation. The findings expand our understanding of the genes associated with hepatopancreas functioning in M. nipponense, and the underlying molecular mechanisms that govern the responses of crustaceans to starvation stress.

Comparative transcriptology reveals effects of circadian rhythm in the nervous system on precocious puberty of the female Chinese mitten crab.

In juvenile Chinese mitten crabs, ovarian maturation occurring in the first year is known as precocity, and can cause huge economic losses to crab breeding. To discover the molecular mechanisms underlying the regulation of the nervous system of female precocious crabs, eyestalk, brain, and thoracic ganglion transcriptome data were obtained in normal and precocious crabs via high-throughput sequencing technology. A total of 81, 4276, and 22,684 differentially-expressed genes were obtained from the eyestalk, brain, and thoracic ganglion groups, respectively. Functional analysis showed that these genes were significantly enriched in the categories of nutrition metabolism, immunity, endocrine regulation, and circadian rhythm. In precocious eyestalk, the expression of vrille was up-regulated significantly and the ribosome endocrine function decreased, which may result in the decline of gonad-inhibiting hormone secretion. In precocious brains, the expression of period2 with the function of delaying clock phase was down-regulated significantly. In precocious thoracic ganglion, expression changes in circadian rhythm-related genes were very complex, and the precocity of female crabs may be the concrete reflex of integrated actions of many endocrine hormones such as estradiol, ecdysteroid, and juvenile hormone, among others. In addition, we found that the mRNA of vitellogenin was highly expressed in the thoracic ganglion. This study discovered some clock genes and related molecular regulatory mechanisms by the RNA-sequence, which would provide foundational information to further study precocity in female Chinese mitten crabs.

Comparative transcriptional analysis and RNA interference reveal immunoregulatory pathways involved in growth of the oriental river prawn Macrobrachium nipponense.

A source of premium animal protein, crustaceans are widely distributed and cultivated around the world. Short-term or long-term starvation events occur frequently owing to natural environment changes or manual management strategies in the life cycle of crustaceans. The result induced by starvation is that somatic growth of crustaceans will be retarded, while the immune mechanism is activated in this process. The aim of this study was to investigate whether the immune regulatory pathways are involved in the growth of crustaceans. Twelve muscle tissue transcriptomes of the oriental river prawn Macrobrachium nipponense were sequenced across four fasting stages lasting 0, 7, 14 and 21 d. The results showed that three immune-related pathways were involved in the growth of M. nipponense by regulating actin expression inducing the chemokine signaling pathway, the leukocyte transendothelial migration pathway and the FcR-mediated phagocytosis pathway. Furthermore, we employed RNA interference (RNAi) to further verify the effects that genes involved in the pathways had on regulating growth of M. nipponense. Comparative transcriptional analysis and RNA interference reveal that VASP and WAVE positively regulated the expression of actin; however, WASP negatively regulated the expression of actin. This is the first report that the immune regulatory pathways play key roles in the growth of crustaceans. Our results will not only provide an entirely new understanding of the immune mechanism of crustaceans from a unique angle but also further enrich and develop the theory of growth and developmental biology in crustaceans.

Comparative Transcriptome Analysis Reveals Related Regulatory Mechanisms of Androgenic Gland in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is one of the most commercially important aquaculture species in China. The androgenic gland (AG) of crustaceans plays pivotal roles in the regulation of male differentiation and in maintaining the male sexual characteristics. In order to reveal related mechanisms in AG, we compared transcriptomes of AG between proliferation and secretion phase. A total of 72,000 unigenes and 4,027 differentially expressed genes were obtained. Gene ontology enrichment analysis indicated that biological processes and metabolic pathways related to protein synthesis and secretion such as transcription, translation, and signal transduction were significantly enriched. Critical genes such as IAG, SXL, TRA-2, SRY, FTZ-F1, FOXL2, and FEM-1 were identified and potentially involved in maintaining the testis development and spermatogenesis. Ribosomes pathway revealed the cause of insulin-like androgenic gland hormone secretion increase. Three insulin-like receptors were thought to be associated with growth and spermatogenesis. In the neuroactive ligand-receptor interaction pathway, the expression of octopamine receptor, 5-HT receptor 1, and melatonin receptor was significantly changed, which revealed the key regulation mechanism of aggressive and mating behavior of males. Comparative transcriptome analysis provided new insights into the genome-wide molecular mechanisms of AG development and the regulatory mechanisms of male development.