Identification of genes regulated by 20-Hydroxyecdysone in Macrobrachium nipponense using comparative transcriptomic analysis.

BACKGROUND: Macrobrachium nipponense is a freshwater prawn of economic importance in China. Its reproductive molt is crucial for seedling rearing and directly impacts the industry's economic efficiency. 20-hydroxyecdysone (20E) controls various physiological behaviors in crustaceans, among which is the initiation of molt. Previous studies have shown that 20E plays a vital role in regulating molt and oviposition in M. nipponense. However, research on the molecular mechanisms underlying the reproductive molt and role of 20E in M. nipponense is still limited. RESULTS: A total of 240.24 Gb of data was obtained from 18 tissue samples by transcriptome sequencing, with > 6 Gb of clean reads per sample. The efficiency of comparison with the reference transcriptome ranged from 87.05 to 92.48%. A total of 2532 differentially expressed genes (DEGs) were identified. Eighty-seven DEGs associated with molt or 20E were screened in the transcriptomes of the different tissues sampled in both the experimental and control groups. The reliability of the RNA sequencing data was confirmed using Quantitative Real-Time PCR. The expression levels of the eight strong candidate genes showed significant variation at the different stages of molt. CONCLUSION: This study established the first transcriptome library for the different tissues of M. nipponense in response to 20E and demonstrated the dominant role of 20E in the molting process of this species. The discovery of a large number of 20E-regulated strong candidate DEGs further confirms the extensive regulatory role of 20E and provides a foundation for the deeper understanding of its molecular regulatory mechanisms.

Identification of Candidate Male-Reproduction-Related Genes from the Testis and Androgenic Gland of Macrobrachium nipponense, Regulated by PDHE1, through Transcriptome Profiling Analysis.

The previous publication identified that pyruvate dehydrogenase E1 (PDHE1) positively regulated the process of male reproduction in M. nipponense through affecting the expressions of insulin-like androgenic gland hormone. The present study aimed to identify the potential male-reproduction-related genes that were regulated by PDHE1 through performing the transcriptome profiling analysis in the testis and androgenic gland after the knockdown of the expressions of PDHE1 by the injection of dsPDHE1. Both RNA-Seq and qPCR analysis identified the significant decreases in PDHE1 expressions in the testis and androgenic gland in dsPDHE1-injected prawns compared to those in dsGFP-injected prawns, indicating the efficiency of dsPDHE1 in the present study. Transcriptome profiling analysis identified 56 and 127 differentially expressed genes (DEGs) in the testis and androgenic gland, respectively. KEGG analysis revealed that the energy-metabolism-related pathways represented the main enriched metabolic pathways of DEGs in both the testis and androgenic gland, including pyruvate metabolism, the Citrate cycle (TCA cycle), Glycolysis/Gluconeogenesis, and the Glucagon signaling pathway. Thus, it is predicted that these metabolic pathways and the DEGs from these metabolic pathways regulated by PDHE1 may be involved in the regulation of male reproduction in M. nipponense. Furthermore, four genes were found to be differentially expressed in both the testis and androgenic gland, of which ribosomal protein S3 was down-regulated and uncharacterized protein LOC113829596 was up-regulated in both the testis and androgenic gland in dsPDHE1-injected prawns. The present study provided valuable evidence for the establishment of an artificial technique to regulate the process of male reproduction in M. nipponense.

NPC Intracellular Cholesterol Transporter 1 Regulates Ovarian Maturation and Molting in Female Macrobrachium nipponense.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

Role of Mn-LIPA in Sex Hormone Regulation and Gonadal Development in the Oriental River Prawn, Macrobrachium nipponense.

This study investigates the role of lysosomal acid lipase (LIPA) in sex hormone regulation and gonadal development in Macrobrachium nipponense. The full-length Mn-LIPA cDNA was cloned, and its expression patterns were analyzed using quantitative real-time PCR (qPCR) in various tissues and developmental stages. Higher expression levels were observed in the hepatopancreas, cerebral ganglion, and testes, indicating the potential involvement of Mn-LIPA in sex differentiation and gonadal development. In situ hybridization experiments revealed strong Mn-LIPA signaling in the spermatheca and hepatopancreas, suggesting their potential role in steroid synthesis (such as cholesterol, fatty acids, cholesteryl ester, and triglycerides) and sperm maturation. Increased expression levels of male-specific genes, such as insulin-like androgenic gland hormone (IAG), sperm gelatinase (SG), and mab-3-related transcription factor (Dmrt11E), were observed after dsMn-LIPA (double-stranded LIPA) injection, and significant inhibition of sperm development and maturation was observed histologically. Additionally, the relationship between Mn-LIPA and sex-related genes (IAG, SG, and Dmrt11E) and hormones (17ß-estradiol and 17α-methyltestosterone) was explored by administering sex hormones to male prawns, indicating that Mn-LIPA does not directly control the production of sex hormones but rather utilizes the property of hydrolyzing triglycerides and cholesterol to provide energy while influencing the synthesis and secretion of self-sex hormones. These findings provide valuable insights into the function of Mn-LIPA in M. nipponense and its potential implications for understanding sex differentiation and gonadal development in crustaceans. It provides an important theoretical basis for the realization of a monosex culture of M. nipponense.

Genetic diversity and population structure of wild Macrobrachium nipponense populations across China: Implication for population management.

BACKGROUND: Macrobrachium nipponense, is an important economic indigenous prawn and is widely distributed in China. However, most these genetic structure analysis researches were focused on a certain water area, systematic comparative studies on genetic structure of M. nipponense across China are not yet available. METHODS AND RESULTS: 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 473 valid D-loop sequences with a length of 1110 bp were obtained, and 348 variation sites and 221 haplotypes were detected. The haplotype diversity (h) was ranged from 0.1630 (Bayannur) ~ 1.0000 (Amur River) and the nucleotide diversity π value ranged from 0.001164 (Min River) ~ 0.037168 (Nen River). 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 Min River and Jialing River populations and the highest was between Nandu River and Nen River populations. The phylogenetic tree of genetic distance showed that all populations were divided into two branches. The Dianchi Lake, Nandu River, Jialing River and Min River 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. CONCLUSIONS: 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.

Identification of Male Sex-Related Genes Regulated by SDHB in Macrobrachium nipponense Based on Transcriptome Analysis after an RNAi Knockdown.

The oriental river prawn (Macrobrachium nipponense) is a commercially important species in Asia. A previous study showed that the succinate dehydrogenase complex iron sulfur subunit B (SDHB) gene participates in testes development in this species through its effect on the expression of the insulin-like androgenic gland hormone gene. This study knocked-down the Mn-SDHB genes in M. nipponense using RNAi. A transcriptome analysis of the androgenic gland and testes was then performed to discover the male sex-related genes regulated by SDHB and investigate the mechanism of male sexual development in this species. More than 16,623 unigenes were discovered in each sample generated. In the androgenic gland, most of the differentially expressed genes were enriched in the hypertrophic cardiomyopathy pathway, while in the testes, they were enriched in the citrate cycle pathway. In addition, after Mn-SDHB knockdown, five genes were found to be downregulated in the androgenic gland in a series of biological processes associated with phosphorylated carbohydrate synthesis and transformations in the glycolysis/gluconeogenesis pathway. Moreover, a total of nine male sex-related genes were identified including Pro-resilin, insulin-like androgenic gland hormone, Protein mono-ADP-ribosyltransferase PAPR11, DNAJC2, C-type Lectin-1, Tyrosine-protein kinase Yes, Vigilin, and Sperm motility kinase Y-like, demonstrating the regulatory effects of Mn-SDHB, and providing a reference for the further study of the mechanisms of male development in M. nipponense.

Deciphering Molecular Mechanisms Governing the Reproductive Molt of Macrobrachium nipponense: A Transcriptome Analysis of Ovaries across Various Molting Stages.

The relationship between molting and reproduction has received more attention in economically important crustacean decapods. Molting and reproduction are synergistic events in Macrobrachium nipponense, but the molecular regulatory mechanisms behind them are unclear. In the current study, we performed Illumina sequencing for the ovaries of M. nipponense during the molt cycle (pre-molting, Prm; mid-molting, Mm; and post-molting, Pom). A total of 66.57 Gb of transcriptome data were generated through sequencing, resulting in the identification of 105,149 unigenes whose alignment ratio with the reference genome exceeded 87.57%. Differentially expressed genes (DEGs) were annotated through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for gene classification and pathway analysis. A total of twenty-six molt-related DEGs were found, and their expression patterns were examined across various molting stages. The KEGG enrichment analysis revealed that the key pathways involved in regulating the molting process of M. nipponense primarily include the mTOR, insect hormone biosynthesis, TGF-beta, and Wnt signaling pathways. Our transcriptomic data suggest that these pathways crosstalk with each other to regulate the synthesis and degradation of ecdysone throughout the molt cycle. The current study has deepened our understanding of the molecular mechanisms of crustacean molting and will serve as a basis for future studies of crustaceans and other molting animals.

17ß-Estradiol Induced Sex Reversal and Gonadal Transcriptome Analysis in the Oriental River Prawn (Macrobrachium nipponense): Mechanisms, Pathways, and Potential Harm.

Sex reversal induced by 17ß-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.

Function Analysis of Cholesterol 7-Desaturase in Ovarian Maturation and Molting in Macrobrachium nipponense: Providing Evidence for Reproductive Molting Progress.

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.

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.

Hepatopancreas transcriptome analyses provide new insights into the molecular regulatory mechanism of fast ovary maturation in Macrobrachium nipponense.

BACKGROUND: Macrobrachium nipponense is an economically and ecologically important freshwater prawn that is widely farmed in China. In contrast to other species of marine shrimp, M. nipponense has 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. RESULTS: Comparative transcriptome analysis was performed using hepatopancreatic tissue from female M. nipponense across 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 the M. nipponense reference 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. CONCLUSION: Taken together, this study sheds light on the role of the hepatopancreas in the molecular regulation of ovary maturation in M. nipponense. The present study provided new insights for understanding the mechanisms of reproductive regulation in crustaceans.

Transcriptome analysis provides novel insights into the immune mechanisms of Macrobrachium nipponense during molting.

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.

MnHR4 Functions during Molting of Macrobrachium nipponense by Regulating 20E Synthesis and Mediating 20E Signaling.

HR4, a member of the nuclear receptor family, has been extensively studied in insect molting and development, but reports on crustaceans are still lacking. In the current study, the MnHR4 gene was identified in Macrobrachium nipponense. To further improve the molting molecular mechanism of M. nipponense, this study investigated whether MnHR4 functions during the molting process of M. nipponense. The domain, phylogenetic relationship and 3D structure of MnHR4 were analyzed by bioinformatics. Quantitative real-time PCR (qRT-PCR) analysis showed that MnHR4 was highly expressed in the ovary. In different embryo stages, the highest mRNA expression was observed in the cleavage stage (CS). At different individual stages, the mRNA expression of MnHR4 reached its peak on the fifteenth day after hatching (L15). The in vivo injection of 20-hydroxyecdysone (20E) can effectively promote the expression of the MnHR4 gene, and the silencing of the MnHR4 gene increased the content of 20E in M. nipponense. The regulatory role of MnHR4 in 20E synthesis and 20E signaling was further investigated by RNAi. Finally, the function of the MnHR4 gene in the molting process of M. nipponense was studied by counting the molting frequency. After knocking down MnHR4, the molting frequency of M. nipponense decreased significantly. It was proved that MnHR4 plays a pivotal role in the molting process of M. nipponense.

Transcriptome analysis of five ovarian stages reveals gonad maturation in female Macrobrachium nipponense.

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.

Molecular cloning, characterization and functional analysis of two neuropeptide F genes from the oriental river prawn (Macrobrachium 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.

Evaluating expression of autophagy-related genes in oriental river prawn Macrobrachium nipponense as potential biomarkers for hypoxia exposure.

Autophagy, a crucial process for maintaining cellular homeostasis, is under the control of several autophagy-related (ATG) proteins, and is highly conserved in most animals, but its response to adverse environmental conditions is poorly understood in crustaceans. Herein, we hypothesised that autophagy acts as a protective response to hypoxia, and Beclin 1, ATG7 and ATG8 in oriental river prawn (Macrobrachium nipponense) were chosen as potential biomarkers under hypoxia exposure; thus, their full-length cDNA sequences were cloned and characterised. Open reading frames (ORFs) of 1281, 2076 and 360 bp, encoding proteins of 427, 692 and 120 amino acid residues, respectively, were obtained. Phylogenetic analysis demonstrated the three M. nipponense proteins do not form a clade with vertebrate homologs. Protein and mRNA levels were investigated in different tissues and developmental stages, and all three were significantly upregulated in a time-dependent manner in the hepatopancreas following hypoxia stress. Biochemical and morphological analysis of hepatocytes revealed that hypoxia increased the abundance of hepatic autophagic vacuoles and stimulated anaerobic metabolism. RNA interference-mediated silencing of ATG8 significantly increased the death rate of M. nipponense juveniles under hypoxia stress conditions. Together, these results suggest that Beclin 1, ATG7 and ATG8 contribute to autophagy-based responses against hypoxia in M. nipponense. The findings also expand our understanding of the potential role of autophagy as an adaptive response against hypoxia toxicity in crustaceans. The results showed that hepatic ATG8 levels may be directly indicative of acute hypoxia in prawns, and provide insight into the time at which hypoxia exposure occurs. Autophagy-related genes expression pattern seems to be sensitive and good biomarkers of acute hypoxia exposure.

Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense).

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.

Potential Functions of Gem-Associated Protein 2-Like Isoform X1 in the Oriental River Prawn Macrobrachium nipponense: Cloning, qPCR, In Situ Hybridization, and RNAi Analysis.

Gem-associated protein 2-like isoform X1 (GEM) was previously predicted to be involved in the sexual development of male Macrobrachium nipponense. In this study, we analyze the GEM functions in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, and RNA interference (RNAi). The full-length Mn-GEM cDNA sequence was 1018 base pairs (bp) long with an open reading frame of 777 bp encoding 258 amino acids. qPCR analysis of Mn-GEM in different tissues and developmental stages showed that Mn-GEM was highly expressed in the gonad and from post-larval developmental stage day 5 (PL5) to PL15, which indicated that GEM has potential roles in gonad differentiation and development in M. nipponense. In situ hybridization and qPCR analysis of various stages of the reproductive cycle of the testis and ovary indicated that GEM may promote spermatid development and gametogenesis in M. nipponense. After injecting with double-stranded RNA (dsRNA) of Mn-GEM, mRNA expression of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone increased with the decrease of Mn-GEM expression, indicating that GEM has negative effects on the male sexual differentiation and development in M. nipponense. Results of this study highlight the functions of GEM in M. nipponense, which can be applied to future studies of male sexual development in M. nipponense and other crustacean species.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense.

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), ß-act (ß-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and ß-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.