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Ammonium sulfate and ammonium chloride are widely used in daily production and life. They can be used as nitrogen fertilizer in agricultural fields and as raw materials for other chemical products in factories. Under the action of precipitation and other factors, these compounds can have a certain impact on water ecology when they enter natural water bodies. In the paper, the acute toxicity of ammonium sulfate and ammonium chloride was investigated using water fleas as indicator organisms. According to GB/T 16125- 2012 test method for acute toxicity of water flea, the 24h-LC50 values of the acute toxicity of ammonium sulfate and ammonium chloride to water flea were 469. 894mg/L and 546. 720mg/L respectively, which were classified as slightly toxic according to the toxicity class.

Molting is a basic physiological behavior of the Oriental river prawn (Macrobrachium nipponense), however, the gene expression patterns and immune mechanisms during the molting process of Oriental river prawn are unclear. In the current study, the gene expression levels of the hepatopancreas of the Oriental river prawn at different molting stages (pre-molting, Prm; mid-molting, Mm; and post-molting, Pom) were detected by mRNA sequencing. A total of 1721, 551, and 1054 differentially expressed genes (DEGs) were identified between the Prm hepatopancreas (PrmHe) and Mm hepatopancreas (MmHe), MmHe and Pom hepatopancreas (PomHe) and PrmHe and PomHe, respectively. The results showed that a total of 1151 DEGs were annotated into 316 signaling pathways, and the significantly enriched immune-related pathways were "Lysosome", "Hippo signaling pathway", "Apoptosis", "Autophagy-animal", and "Endocytosis". The qRT-PCR verification results of 30 randomly selected DEGs were consistent with RNA-seq. The expression patterns of eight immune related genes in different molting stages of the Oriental river prawn were analyzed by qRT-PCR. The function of Caspase-1 (CASP1) was further investigated by bioinformatics, qRT-PCR, and RNAi analysis. CASP1 has two identical conserved domains: histidine active site and pentapeptide motif, and the expression of CASP1 is the highest in ovary. The expression levels of triosephosphate isomerase (TPI), Cathepsin B (CTSB) and Hexokinase (HXK) were evaluated after knockdown of CASP1. This research provides a valuable basis to improve our understanding the immune mechanisms of Oriental river prawns at different molting stages. The identification of immune-related genes is of great significance for enhancing the immunity of the Oriental river prawn, or other crustaceans, by transgenic methods in the future.

Polo-like kinase 1 (Plk1) has multiple functions in the cell cycle, including in the maturation of centrosomes during the G2/M transition, the separation of centrosomes, and the activation of cyclin-dependent kinase 1 expression and spindle assembly. In this study, we investigated the potential regulatory roles of Plk1 in the reproductive development of the male oriental river prawn (Machrobrachium nipponense). The full cDNA sequence of Mn-Plk1 was 2360 base pairs long, with an open reading frame of 1836 base pairs encoding 611 amino acids. Protein sequence alignment identified a conserved serine/threonine kinase domain and two Polo-boxes. Phylogenetic tree analysis revealed that Mn-Plk1 had the closest evolutionary distance with Plk1s of freshwater prawns and then with those of crustacean species, whereas the evolutionary distance with mollusks was much more distant. Quantitative PCR analysis predicted that Mn-Plk1 plays essential roles in the regulation of gonad development. RNA interference analysis and histological observations showed that expression of insulin-like androgenic gland hormone decreased as the expression of Mn-Plk1 decreased, and fewer than 5% of cells were sperm cells at day 14 in the dsPlk1 injected prawns. This result indicated that Plk1 positively regulated testis development in M. nipponense by affecting the expression of this hormone. Our results highlight the functions of Plk1 in M. nipponense and provide valuable information that can be applied to establish artificial techniques to regulate testis development in this species.

Cyclin A (CycA) plays essential roles in regulating multiple steps of the cell cycle, and it affects gonad development in mammals and invertebrates. Previous RNA interference (RNAi) analysis revealed that knocking-down the expression of CycA in female oriental river prawns (Macrobrachium nipponense) inhibited ovarian development. CycA was also predicted to have regulatory roles in reproductive development of male M. nipponense based on significant changes of Mn-CycA expression after eyestalk ablation. The goal of this study was to investigate the potential functions of CycA in the reproductive development of male M. nipponense using RNAi and histological observations. Quantitative real-time PCR analysis revealed that both single-side and double-side eyestalk ablation stimulated the expressions of Mn-CycA, and the expression was higher in prawns with double-side eyestalk ablation (p < 0.05). Mn-CycA expression was significantly higher in the testis and androgenic gland during the reproductive season than during the non-reproductive season (p < 0.05). In the RNAi analysis, Mn-CycA expression significantly decreased after prawns were injected with dsCycA, and the expression of insulin-like androgenic gland hormone (Mn-IAG) also decreased as Mn-CycA expression decreased. This result indicated that CycA positively regulated the expression of IAG in M. nipponense. Histological observations revealed that the number of sperm decreased dramatically to dsCycA-treated group compared to that of control group on day 14, indicating that knockdown of Mn-CycA expression inhibited testis development by affecting the expression of Mn-IAG in M. nipponense. These results highlighted the functions of CycA in male reproductive development of M. nipponense, which can be applied to future studies of male reproduction in other crustacean species.

Polo-like kinase 1 (

Cyclin A (

Genome assembly can be challenging for species that are characterized by high amounts of polymorphism, heterozygosity, and large effective population sizes. High levels of heterozygosity can result in genome mis-assemblies and a larger than expected genome size due to the haplotig versions of a single locus being assembled as separate loci. Here, we describe the first chromosome-level genome for the eastern oyster,Crassostrea virginica. Publicly released and annotated in 2017, the assembly has a scaffold N50 of 54 mb and is over 97.3% complete based on BUSCO analysis. The genome assembly for the eastern oyster is a critical resource for foundational research into molluscan adaptation to a changing environment and for selective breeding for the aquaculture industry. Subsequent resequencing data suggested the presence of haplotigs in the original assembly, and we developed apost hocmethod to break up chimeric contigs and mask haplotigs in published heterozygous genomes and evaluated improvements to the accuracy of downstream analysis. Masking haplotigs had a large impact on SNP discovery and estimates of nucleotide diversity and had more subtle and nuanced effects on estimates of heterozygosity, population structure analysis, and outlier detection. We show that haplotig-masking can be a powerful tool for improving genomic inference, and we present an open, reproducible resource for the masking of haplotigs in any published genome.

Macrobrachium nipponense, is an important economic indigenous prawn in China, which is widely distributed in freshwater in China, Japan and Southeast Asia. In this study, D-loop region sequences was used to investigate the genetic diversity and population structure of 22 wild populations of M. nipponense through China, containing the major rivers and lakes of China. Totally 475 valid D-loop sequences from 22 populations with a length of 1110bp were obtained, and 348 variation sites and 221 haplotypes were detected. A total of 221 haplotypes were defined in 22 populations. Hap48 was the dominant haplotype, accounting for about 13%. The haplotype diversity (h) was ranged from 0.1630 (BN) ~1.0000 (AR) and the nucleotide diversity π value ranged from 0.001164 (MR) ~ 0.037168 (NR). The pairwise genetic differentiation index (FST) ranged from 0.00344 to 0.91243 and most pair-wised FST was significant (P < 0.05). The lowest FST was displayed in MR and JLR population and the highest was between NDR and NR. The phylogenetic tree of genetic distance based on UPGMA showed that all populations were divided into two branches. The DC, NDR, JLR and MR populations were clustered into one branch. The neutral test and mismatch distribution results showed that M. nipponense populations were not experienced expanding and kept a steady increase. Taken together, a joint resources protection and management strategy for M. nipponense have been suggested based on the results of this study for its sustainable use.

The putamen (Put) is necessary for habitual actions, while the nucleus caudate (Cd) is critical for goal-directed actions. However, compared with the natural reward (such as sucrose)-seeking habit, how drug-related dysfunction or imbalance between the Put and Cd is involved in cocaine-seeking habit, which is not easy to bias behavior to goal-directed actions, is absent. Therefore, in our present study, in comparison with sucrose-habitual behavior, we evaluated the distinctive changes of the two subtypes of dopamine (DA) receptors (D1R and D2R) in cocaine-seeking habitual behavior animals. Moreover, the adaptive changes of Cav1.2 and Cav1.3, as prime downstream targets of D1R and D2R respectively, were also assessed. Our results showed that a similar percentage of the animals exhibited habitual seeking behavior after cocaine or sucrose variable-interval self-administration (SA) training in tree shrews. In addition, compared with animals with non-habitual behavior, animals with cocaine habitual behavior showed higher D1Rs and Cav1.2 expression in the Put accompanied with lower D2Rs and Cav1.3 expression in the Cd. However, after sucrose SA training, animals with habitual behavior only showed lower membrane expression of D2R in the Put than animals with non-habitual behavior. These results suggested that the upregulation of D1Rs-Cav1.2 signaling may lead to hyper-excitability of the Put, and the inactivation of D2Rs-Cav1.3 signaling may result in depressed activity in the Cd. This imbalance function between the Put and Cd, which causes an inability to shift between habits and goal-directed actions, may underlie the compulsive addiction habit.

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.

Sex reversal induced by 17β-estradiol (E2) has shown the potential possibility for monoculture technology development. The present study aimed to determine whether dietary supplementation with different concentrations of E2 could induce sex reversal in M. nipponense, and select the sex-related genes by performing the gonadal transcriptome analysis of normal male (M), normal female (FM), sex-reversed male prawns (RM), and unreversed male prawns (NRM). Histology, transcriptome analysis, and qPCR were performed to compare differences in gonad development, key metabolic pathways, and genes. Compared with the control, after 40 days, feeding E2 with 200 mg/kg at PL25 (PL: post-larvae developmental stage) resulted in the highest sex ratio (female: male) of 2.22:1. Histological observations demonstrated the co-existence of testis and ovaries in the same prawn. Male prawns from the NRM group exhibited slower testis development without mature sperm. RNA sequencing revealed 3702 differentially expressed genes (DEGs) between M vs. FM, 3111 between M vs. RM, and 4978 between FM vs. NRM. Retinol metabolism and nucleotide excision repair pathways were identified as the key pathways for sex reversal and sperm maturation, respectively. Sperm gelatinase (SG) was not screened in M vs. NRM, corroborating the results of the slice D. In M vs. RM, reproduction-related genes such as cathepsin C (CatC), heat shock protein cognate (HSP), double-sex (Dsx), and gonadotropin-releasing hormone receptor (GnRH) were expressed differently from the other two groups, indicating that these are involved in the process of sex reversal. Exogenous E2 can induce sex reversal, providing valuable evidence for the establishment of monoculture in this species.

The Cholesterol 7-desaturase gene plays an important role in insect ecdysone synthesis, but its role in ovarian development has not been reported. In this study, characteristics and the phylogenetic relationship of Cholesterol 7-desaturase were identified by bioinformatics. qPCR showed that the Mn-CH7D gene was highly expressed in the ovary, which was much higher than that in other tissues, and the expression level of Mn-CH7D reached the highest level at the third stage of the ovarian development stage (O-III). During embryonic development, the Mn-CH7D gene expression was highest in the zoea stage. The function of the Mn-CH7D gene was explored by RNA interference. The experimental group was injected with Mn-CH7D dsRNA through the pericardial cavity of M. nipponense, while the control group was injected with the same volume of dsGFP. Statistical analysis of gonadal development and GSI calculation showed that the silencing of Mn-CH7D resulted in the suppression of gonadal development. In addition, the molting frequency of the experimental group was significantly lower than that of the control group during the second molting cycle after silencing Mn-CH7D. On the seventh day after silencing, ecdysone content in the experimental group was significantly reduced. These results demonstrated that the Mn-CH7D gene played a dual role in ovarian maturation and molting of M. nipponense.

Male hybrid oriental river prawns grow significantly faster than hybrid females. In this study, the growth and sex traits of 181 individuals of Macrobrachium nipponense were recorded, and each individual genotype was evaluated using the 2b-RAD sequencing method. The genetic parameters for growth and sex traits were estimated. A genome-wide association analysis (GWAS) of these traits was performed. In total, 18 growth-related SNPs were detected from 12 chromosomes using a mixed linear model. The most significant loci of weight are located on the position of the SNP (102638935, chromosome 13), which can explain 11.87% of the phenotypic variation. A total of 11 significant SNPs were detected on four chromosomes associated with sex trait (three on chromosome 4, one on chromosome 7 and seven on chromosome 17). The heritability of this trait is 0.8998 and belongs to the range of ultra-high heritability. Genetic correlations were prevalent among the 11 traits examined, the genetic coefficient between sex and body weight reached a significant level of −0.23. This study is the first GWAS for sex of binary and growth traits in oriental river prawn. Our results provide a set of markers for the genetic selection of growth traits and help us to further understand the genetic mechanisms of growth in Macrobrachium nipponense.

BackgroundMacrobrachium nipponenseis an economically and ecologically important freshwater prawn that is widely farmed in China. In contrast to other species of marine shrimp,M. nipponensehas a short sexual maturity period, resulting in not only high stocking densities, but also a reduced survival rate and increased risk of hypoxia. Therefore, there is an urgent need to study the molecular mechanisms underlying fast ovary maturation in this species.ResultsComparative transcriptome analysis was performed using hepatopancreatic tissue from femaleM. nipponenseacross five ovarian maturation stages to explore differentially expressed genes and pathways involved in ovarian maturation. In total, 118.01 Gb of data were generated from 15 transcriptomes. Approximately 90.46% of clean reads were mapped from theM. nipponensereference genome. A comprehensive comparative analysis between successive ovarian maturation stages generated 230–5814 differentially expressed genes. Gene Ontology (GO) enrichment was highly concentrated in the “biological process” category in all four comparison groups, and mainly focused on energy synthesis and accumulation, energy decomposition and transport. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that, among 20 significantly enriched KEGG pathways, nine were involved in the synthesis, degradation, and metabolism of carbohydrates, lipids, and other nutrient intermediates, suggesting that the hepatopancreas has an important role in energy supply during ovarian maturation. Furthermore, the “Insect hormone biosynthesis” pathway was found to have a dominant role in the development of the ovary from immaturity to maturity, supporting the hypothesis that ecdysteroid- and juvenile hormone-signaling pathways have an important role in hepatopancreas regulation of ovarian maturation.ConclusionTaken together, this study sheds light on the role of the hepatopancreas in the molecular regulation of ovary maturation inM. nipponense. The present study provided new insights for understanding the mechanisms of reproductive regulation in crustaceans.

Background: The lateral hypothalamic orexin (hypocretin) system has a well-established role in the motivation for reward. This has particular relevance to substance use disorders since orexin-1 receptors play a critical role in alcohol-seeking behavior, acting at multiple nodes in relapse-associated networks. Aims: This study aimed to further our understanding of the role of orexin-1 receptor signaling within the lateral hypothalamus and bed nucleus of the stria terminalis, specifically in context-induced relapse to alcohol-seeking following punishment-imposed abstinence. Methods: We trained inbred male alcohol-preferring rats to self-administer alcohol in one environment or context (Context A) and subsequently punished their alcohol-reinforced lever presses in a different environment (Context B) using contingent foot shock punishment. Finally, we tested rats for relapse-like behavior in either context following systemic, intra-lateral hypothalamus or intra-bed nucleus of the stria terminalis orexin-1 receptor antagonism with SB-334867. Results/outcomes: We found that systemic orexin-1 receptor antagonism significantly reduced alcohol-seeking in both contexts. Intra-lateral hypothalamus orexin-1 receptor antagonism significantly reduced alcohol-seeking in Context A whereas intra-bed nucleus of the stria terminalis orexin-1 receptor antagonism had no effect on alcohol-seeking behavior. Conclusions/interpretation: Our results suggest a role for the orexin-1 receptor system in context-induced relapse to alcohol-seeking. Specifically, intra-lateral hypothalamus orexin microcircuits contribute to alcohol-seeking.

This article presents a study of D-loop sequences to characterize the genetic diversity of wild Macrobrachium nipponense populations in Yixing natural waters including two reservoirs (Hengshan reservoir, HS; Youche reservoir, YC), 3 brooks (Linjin Dang, LJD; Magong Dushan Dang, MDD; Yangshan Dang, YSD) and 3 shallow lakes (Dongjiu lake, DJ; Xijiu lake, XJ; Tuanjiu lake, XJ), and compared the genetic differentiation and population structure with wild populations of Taihu Lake (TH), Yangtze River (YZ), and the main local artificially bred varieties “Taihu No. 2” (TH-2). A 747 bp D-loop sequence fragment was amplified in 321 individuals and the results exhibited a higher content of A+T (80.03%) than C+G (19.97%). A total of 110 haplotypes were identified. The h and π value proved the diversity of these populations was at the same level with high genetic diversity. TH-2 and YZ showed remarkable diversity, and XJ is even better. Fst estimates suggested that YZ and TH-2 were significant differentiation with other Yixing populations (P < 0.05). Three populations from shallow lake (DJ, XJ and TJ) displayed significant differentiated with the left Yixing ones (P < 0.05). The pairwise genetic distance, as well as haplotype network results, also suggested that all these 11 populations did not diverge at the species level (P values of Tajima's D and Fu Fs were relatively greater than 0.1 (P > 0.1) and the nucleotide mismatch distribution analysis showed multiple peaks, giving a conclusion that the populations did not exhibited expansion. All these results suggested that TH-2 and YZ have remarkable diversity, and the germplasm resources and genetic diversity of M. nipponense in Yixing are very good and are suitable for original materials of breeding.

Ferritin plays an essential role in organismic and cellular iron homeostasis in

Identification of important genes, involved in the gonad differentiation and development, plays essential roles in the establishment of the artificial technique to regulate the process of testis development in

Cyclin B (CycB) plays essential roles in cell proliferation and promotes gonad development in many crustaceans. The goal of this study was to investigate the regulatory roles of this gene in the reproductive development of male oriental river prawns (Macrobrachium nipponense). A phylo-genetic tree analysis revealed that the protein sequence of Mn-CycB was most closely related to those of freshwater prawns, whereas the evolutionary distance from crabs was much longer. A quantitative PCR analysis showed that the expression of Mn-CycB was highest in the gonad of both male and female prawns compared to that in other tissues (p < 0.05), indicating that this gene may play essential roles in the regulation of both testis and ovary development in M. nipponense. In males, Mn-CycB expression in the testis and androgenic gland was higher during the reproductive season than during the non-reproductive season (p < 0.05), implying that CycB plays essential roles in the reproductive development of male M. nipponense. An RNA interference analysis revealed that the Mn-insulin-like androgenic gland hormone expression decreased as the Mn-CycB expression decreased, and that few sperm were detected 14 days after the dsCycB treatment, indicating that CycB positively affects testis development in M. nipponense. The results of this study highlight the functions of CycB in M. nipponense, and they can be applied to studies of male reproductive development in other crustacean species.

Projections to the striatum are well-identified. For example, in the ventral striatum, two major inputs to the medial nucleus accumbens shell include the ventral subiculum and basolateral amygdala. However, the chemical phenotype(s) of these projection neurons remain unclear. In this study, we examined amygdalostriatal and corticostriatal connectivity in rats using injections of the retrograde tracer cholera toxin b into the nucleus accumbens shell. To determine the neurotransmitter identity of projection neurons, we combined retrograde tracing with RNAscope in-situ hybridization, using mRNA probes against vesicular transporters associated with glutamatergic (VGluT1 - Slc17a7, VGluT2 - Slc17a6) or GABAergic (VGaT - Slc32a1) neurotransmission. Confocal imaging was used to examine vesicular transporter mRNA expression in the ventral subiculum and basolateral amygdala inputs to the nucleus accumbens shell. Both projections contained mostly VGluT1-expressing neurons. Interestingly, almost a quarter of ventral subiculum to nucleus accumbens shell projections co-expressed VGluT1 and VGluT2 compared to a relatively small number (∼3%) that were co-expressed in basolateral amygdala to nucleus accumbens shell afferents. However, almost a quarter of basolateral amygdala to nucleus accumbens shell projections were VGaT-positive. These findings highlight the diverse proportions of glutamatergic and GABAergic afferents in two major projections to the nucleus accumbens shell and raise important questions for functional studies.

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

The eyestalk of crustacean species contained many neurosecretory structures, affected the process of reproduction, molting, metabolism of glucose and other function in crustaceans. In this study, we aimed to selected important metabolic pathways and candidate genes involved in the male sexual development through performing the long-reads and next generation transcriptome sequencing of androgenic gland after the ablation of eyestalk from Macrobrachium nipponense. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation both of the single-side (SS) and double-side (DS) eyestalk, compared with that of control group (CG). The long-reads transcriptome generated 49,480 non-redundant transcripts. A total of 1,319, 2,092 and 4,351 differentially expressed genes (DEGs) were identified between CG vs SS, SS vs DS and CG vs DS, respectively, indicating the ablation of double-side eyestalk has more important regulatory roles than that of single-side ablation on male sexual development, which was consistent with that of qPCR analysis. Cell cycle, Cellular senescence, Oxidative phosphorylation, Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 GEDs were as the same as that of RNA-seq. The qPCR and RNAi analysis of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has the positive regulatory effected on testis development. This study provided valuable evidences on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustacean as well.

Background Macrobrachium nipponense is an economically important species of freshwater shrimp in China. Unlike other marine shrimps, the ovaries in adult female M. nipponense can mature rapidly and periodically during the reproductive period, but the resulting high stocking densities and environmental deterioration can negatively impact the harvest yield and economic benefits. To better understand ovary development in female M. nipponense, we performed systematic transcriptome sequencing of five different stages of ovarian maturation. Results We obtained 255,966 Gb of high quality transcriptome data from 15 samples. Of the 105,082 unigenes that were selected, 30,878 were successfully annotated. From these unigenes, we identified 17 differentially expressed genes and identified three distinct gene expression patterns related to different biological processes. We found that cathepins, legumains, and cystatin were enriched in the lysosome pathway, and they are related to vitellogenin hydrolysis. Additionally, we found that myosin heavy chain 67 participated in oocyte excretion. Conclusions We provide the first detailed transcriptome data relating to the ovarian maturation cycle in M. nipponense. Our results provide important reference information about the genomics, molecular biology, physiology, and population genetics of M. nipponense and other crustaceans. It is conducive to further solve the problem of M. nipponense rapid ovarian maturation from the aspects of energy supply and cell division.

17β-Estradiol (E2) and 17α-methyltestosterone (MT) are steroid hormones with dramatic regulatory effects on the growth performance and gonadal development of aquatic species. The aim of the present study was to investigate the regulatory effects of E2 and MT on the growth performance and gonadal development of juvenile prawns (Macrobrachium nipponense). Male and female juvenile prawns were separately maintained and fed different concentrations of E2 and MT, respectively. Treatment with both E2 and MT at 500 mg/kg dramatically promoted the growth of male and female juvenile prawns, respectively (p 0.05). The expression levels of vitellogenin and vitellogenin receptor were dramatically decreased with increased MT concentrations, and the lowest expression levels were observed in the group treated with 1000 mg/kg (p

In this study, we identified three neuroparsin (NP) genes in Macrobrachium nipponense: Mn-NP1, Mn-NP2, and Mn-NP3, encoding 99, 100, and 101 amino acid proteins, respectively. Multiple sequence alignments showed that these genes contained 12 cysteine residues, of which 11 were at conserved positions. The total sequence identity between the genes was 47.5%, and they showed a high degree of sequence identity (> 54% similarity) with other crustacean genes. Phylogenetic tree analysis showed that Mn-NPs were clustered at different branches, indicating that Mn-NPs may have different functions. Tissue distribution data revealed that the three genes were present in males and females during the breeding and nonbreeding season, but their expression patterns differed. Mn-NP1 was highly expressed in the breeding season, in the male testis, and highly expressed in the nonbreeding season, in the female ovary. Mn-NP3 exhibited biased female expression in the breeding and nonbreeding season, with dominant expression in the ovary. All Mn-NPs were detected during embryo development, but with different expression patterns. These data indicated that Mn-NP1 may function during embryonic development, and that Mn-NP2 may be expressed during early embryo cell division, and late larval development. Mn-NP3 expression patterns reflected maternal inheritance, and may be associated with ovarian maturation. These expression data suggested that Mn-NP1 and Mn-NP2 are negatively correlated with ovarian development, with inhibition roles during this development. Mn-NP3 may be involved in vitellogenesis.

A previous study identified slow-tonic S2 tropomyosin and slow tropomyosin isoform as sex-related genes in Macrobrachium nipponense. Their functions were analyzed using RNA interference. However, more than half of the specimens died approximately 8–12 h after injection of the respective double-stranded RNAs (dsRNAs), and HE staining indicated that the heart and gills were the most likely tissues responsible for the resultant deaths. In the current study, we conducted a comparative transcriptomic study of the gills and hearts of M. nipponense to identify potential target genes associated with acute death after dsRNA injection. A total of 68,772 annotated unigenes were generated. In the heart, differentially expressed genes (DEGs) were mainly enriched in glycolysis/gluconeogenesis and oxidative phosphorylation, while the most relevant pathways in the gills were lysosome, phagosome, and peroxisome. Ten DEGs were screened out and analyzed under lethal hypoxic stress. Among these, fructose 1, 6-biphosphate-aldolase (FBA), glyceraldehyde 3-phosphate dehydrogenase (GDPDH), alcohol dehydrogenase class-3 (ADC3), ATP-synthase subunit 9 (ATPS9), and acid ceramidase-like (ACL) were all differentially expressed under hypoxic conditions. This study shed light on the lethal mechanism caused by interference with tropomyosin genes in M. nipponense, and identifies the related pathways and key genes that could help to improve stress resistance and tolerance in M. nipponense.

In invertebrates, neuropeptide F (NPF) has many regulatory functions, similar to NPY, its homologous peptide. In this study, two neuropeptide F genes were identified in Macrobrachium nipponense: Mn-NPF1 and Mn-NPF2. Mn-NPF2 shared the same amino acid sequence with Mn-NPF1, except for a 37 amino acid insert in the middle of the NPF region. The quantitative-PCR (qPCR) results indicated that Mn-NPF1 expression was positively correlated with ovarian maturation, whereas Mn-NPF2 had opposing expression patterns. Both Mn-NPFs were poorly expressed at early embryonic stages, but enhanced expression levels were observed up to day 10 after hatching, when the gonads began to differentiate. Ovary in situ hybridization (ISH) analyses showed that both Mn-NPFs were present at all stages, but were differentially localized to distinct compartments. Temperature gradient studies showed that both Mn-NPFs were implicated in the seasonal regulation of reproduction. A double-stranded (ds) RNA-Mn-NPF2 injection led to a significant 38.5% increase in the vitellogenin (VG) transcript (P < 0.05). These results demonstrated that Mn-NPF2 plays an important role in inhibiting ovarian maturation.

In this study, we identified three neuroparsin (NP) genes in Macrobrachium nipponense: Mn-NP1, Mn-NP2, and Mn-NP3, encoding 99, 100, and 101 amino acid proteins, respectively. Multiple sequence alignments showed that these genes contained 12 cysteine residues, of which 11 were at conserved positions. The total sequence identity between the genes was 47.5%, and they showed a high degree of sequence identity (> 54% similarity) with other crustacean genes. Phylogenetic tree analysis showed that Mn-NPs were clustered at different branches, indicating that Mn-NPs may have different functions. Tissue distribution data revealed that the three genes were present in males and females during the breeding and nonbreeding season, but their expression patterns differed. Mn-NP1 was highly expressed in the breeding season, in the male testis, and highly expressed in the nonbreeding season, in the female ovary. Mn-NP3 exhibited biased female expression in the breeding and nonbreeding season, with dominant expression in the ovary. All Mn-NPs were detected during embryo development, but with different expression patterns. These data indicated that Mn-NP1 may function during embryonic development, and that Mn-NP2 may be expressed during early embryo cell division, and late larval development. Mn-NP3 expression patterns reflected maternal inheritance, and may be associated with ovarian maturation. These expression data suggested that Mn-NP1 and Mn-NP2 are negatively correlated with ovarian development, with inhibition roles during this development. Mn-NP3 may be involved in vitellogenesis.

Supplemental material, Supplemental_material for Orexin-1 receptor signaling within the lateral hypothalamus, but not bed nucleus of the stria terminalis, mediates context-induced relapse to alcohol seeking by Erin J Campbell, Mitchell KRI Hill, Xavier J Maddern, Shubo Jin, Terence Y Pang and Andrew J Lawrence in Journal of Psychopharmacology

In this study, a full-length cDNA sequence of SoxE (subgroup E within the Sox family of transcription factors) was cloned from Macrobrachium nipponense and named MnSoxE1. The full-length cDNA of MnSoxE1 is 1748 bp, consisting of a 110 bp 5′ UTR, a 105 bp 3′ UTR, and a 1533 bp ORF that encodes 510 amino acids. Conserved domains showed that MnSoxE1 has a high similarity to the SoxE gene of Penaeus vannamei. Phylogenetic tree analysis classified that MnSoxE1 with the SoxE gene of other arthropods into one clade. These results suggested that MnSoxE1 belongs to the SoxE subgroup. During embryonic development, MnSoxE1 was mainly expressed in the gastrula stage, implicating its involvement in tissue cell differentiation and formation. In the post-larval stages, the expression of MnSoxE1 continued to increase on days 1–10. The expression level in males was significantly higher than that in females. Males are clearly distinguishable from females on post-larval day 25, showing that MnSoxE1 may play a role in promoting early development and germ cell and gonadal differentiation, especially for males. qPCR analysis showed that MnSoxE1 may also be involved in oogonium proliferation during ovary development. Further in situ hybridization analysis revealed that MnSoxE1 was mainly located in oocytes and spermatocytes, especially in sertoli cells, and implies that it may be involved in the development of oocytes and spermatocytes, as well as the maintenance of testes in mature prawns. These results indicate that MnSoxE1 is involved in gonadal differentiation and development in M. nipponense, especially testis development.

The androgenic gland produces hormones that play crucial roles in driving male sexual development. In this study, we investigated the regulatory proteins and pathways affecting male sexual development of M. nipponense, by analyzing the proteomes of their androgenic glands during their reproductive and nonreproductive seasons using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 13 sex-related protein homologs out of 999 total proteins were identified in the proteome of M. nipponense based on comparisons with the published literature. A total of 32 proteins were differentially expressed in the androgenic gland between the reproductive and nonreproductive seasons of M. nipponense. However, only 10 differentially expressed proteins (DEPs) were annotated in the Nr, COG, GO, and KEGG databases. Other non-annotated DEPs may also play vital roles in the male sexual development of M. nipponense. The qPCR analysis indicated that the mRNA expression patterns of the 10 annotated DEPs were consistent with that determined by iTRAQ analysis. Additional qPCR analysis of the 10 DEPs in testes, ovaries, and androgenic glands showed that Gem, Ferritin, and Rev3 were highly expressed in the androgenic gland, implying importance in male sexual development, given the important role of the androgenic gland in male differentiation and development in many crustacean species. Our study provided valuable information about DEPs in androgenic glands between reproductive and nonreproductive seasons for M. nipponense and identified their potential roles in male sexual development, which will improve our understanding of the development process in both M. nipponense and other crustaceans.

Opsins are photoreceptors with important roles in reproductive regulation in birds and fishes. In the present study, we identified an opsin gene from the eyes of the oriental river prawn Macrobrachium nipponense using expressed sequence tag analysis and rapid amplification of cDNA ends. The full-length transcript contained 1382 base pairs, encoding 375 amino acids. It was classified into the long-wavelength opsin group by phylogenetic analysis, and designated Mn-LW. Mn-LW expression demonstrated significant seasonal variation in somatic tissues from both male and female prawns, with the highest expression in the eyes, and expression also shown in the ovary. The expression profiles of Mn-LW in eyes and ovary were positively related to ovarian development. In situ hybridization showed that Mn-LW was present in retinular cells in the eye and oocytes in the ovary. Injection of Mn-LW dsRNA in vivo effectively down-regulated Mn-LW expression levels compared with control levels. Mn-LW dsRNA injection also significantly reduced vitellogenin (Vg) expression, indicating a close relationship between Mn-LW and Vg in ovarian development. These results suggest that Mn-LW may play an important role in Vg synthesis and accumulation during ovarian maturation in M. nipponense.

As an important economical freshwater prawn, Macrobrachium nipponense has difficulty with adapting to hypoxia. In this study, comparative transcriptome analysis was used for the first time to explore the differences between different living states of Macrobrachium nipponense under hypoxia. A total of 94.22 Gb clean reads were obtained and assembled into 54,688 unigenes. A total of 224, 266, and 750 differently expressed genes were found in the comparison of the control and death groups, the control and moribund groups, and the control and survived groups, respectively. Three signal pathways closely related to hypoxia were found by enriching of the signal pathways in three comparison groups. In addition, much attention was focused on the differential genes in these pathways. Oxidative stress related genes, such as 70 kDa heat shock protein, phosphoenolpyruvate carboxykinase and cyclooxygenase were differentially expressed in different comparisons. After comparing with previous studies, cyclooxygenase was found to be an important hypoxia-related gene that is fully involved in the hypoxic response. Interestingly, two new genes with no Nr annotation were found in this manuscript. This manuscript will enrich our understanding of oxidative stress response to hypoxia and provide a theoretical basis for the subsequent solution of apoptosis caused by hypoxia.

The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.

• Gonadotropin-releasing hormone (GnRH) has been proven to promote ovarian development. Exogenous GnRH-a blocks the normal functions of the GnRH receptor, leading to a decrease in ovarian development. In this study, we aimed to select important metabolic pathways and proteins involved in the gonad development of the juvenile Acipenser schrenckii through the measurement of steroid hormones and proteome profiling analysis after GnRH-a treatment. The results showed that 3 μg/kg GnRH-a treatment was proven to have the most significant flare up effect in the juvenile A. schrenckii after 7 d of treatment, based on the measurements of steroid hormones. Five gonads and pituitaries were pooled to form one biological replicate at 7 days after 3 μg/kg GnRH-a treatment and GFP treatment, respectively, and three biological replicates were performed for both groups. Proteome profiling analysis revealed that the number of differentially expressed proteins (DEPs) in the gonad was greater than that in the pituitary, indicating that GnRH-a has more regulatory effects on the gonad than on the pituitary. Moreover, Kyoto encyclopedia of genes and genomes analysis of DEPs indicated that signal transduction and endocrine system metabolic pathways and their co-DEPs played essential roles in the gonad development of A. schrenckii. According to the MRM analysis, protein kinase C was the most upregulated protein in both the gonad and pituitary after GnRH-a treatment, whereas estradiol 17β- dehydrogenase was the most downregulated protein. This study provides the basis for further analysis of the effects of GnRH on gonad development in A. schrenckii.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

Cathepsin L is an important member of the cysteine protease family and has a central role in the development of ovaries in insects and fish. In this study, we analyzed the characteristics of the cathepsin L1 gene in the female oriental river prawn, Macrobrachium nipponense. The cathepsin L1 cDNA was 1173 bp in length, including a 960 bp open reading frame that encoded 319 amino acids. The cathepsin L1 gene was classified into the cathepsin L group and named Mn-CTS L1 by phylogenetic analysis. qRT-PCR analysis indicated that Mn-CTS L1 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different stages of ovarian development, Mn-CTS L1 expression in the hepatopancreas and ovaries peaked before ovarian maturation. Mn-CTS L1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization studies revealed that Mn-CTS L1 was localized in the oocyte of the ovary. Injection of Mn-CTS L1 dsRNA significantly reduced Mn-CTS L1 expression and significantly increased the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CTS L1 dsRNA. These results suggest that Mn-CTS L1 has a key role in ovarian development.