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 the effects of alkalinity exposure on the gills of oriental river prawns, Macrobrachium nipponense.

Macrobrachium nipponense is an important commercial freshwater species in China. However, the ability of alkali tolerance of M. nipponense is insufficient to culture in the major saline-alkali water source in China. Thus, it is urgently needed to perform the genetic improvement of alkali tolerance in this species. In the present study, we aimed to analyse the effects of alkali treatment on gills in this species after 96 h alkalinity exposure under the alkali concentrations of 0 mmol/L, 4 mmol/L, 8 mmol/L, and 12 mmol/L through performing the histological observations, measurement of antioxidant enzymes, metabolic profiling analysis, and transcriptome profiling analysis. The results of the present study revealed that alkali treatment stimulated the contents of malondialdehyde, glutathione, glutathione peroxidase in gills, indicating these antioxidant enzymes plays essential roles in the protection of body from the damage, caused by the alkali treatment. In addition, high concentration of alkali treatment (> 8 mmol/L) resulted in the damage of gill membrane and haemolymph vessel, affecting the normal respiratory function of gill. Metabolic profiling analysis revealed that Metabolic pathways, Biosynthesis of secondary metabolites, Biosynthesis of plant secondary metabolites, Microbial metabolism in diverse environments, Biosynthesis of amino acids were identified as the main enriched metabolic pathways of differentially expressed metabolites, which are consistent with the previous publications, treated by the various environmental factors. Transcriptome profiling analyses revealed that the alkali concentration of 12 mmol/L has more regulatory effects on the changes of gene expression than the other alkali concentrations. KEGG analysis revealed that Phagosome, Lysosome, Glycolysis/Gluconeogenesis, Purine Metabolism, Amino sugar and nucleotide sugar metabolism, and Endocytosis were identified as the main enriched metabolic pathways in the present study, predicting these metabolic pathways may be involved in the adaption of alkali treatment in M. nipponense. Phagosome, Lysosome, Purine Metabolism, and Endocytosis are immune-related metabolic pathways, while Glycolysis/Gluconeogenesis, and Amino sugar and nucleotide sugar metabolism are energy metabolism-related metabolic pathways. Quantitative PCR analyses of differentially expressed genes (DEGs) verified the accuracy of the RNA-Seq. Alkali treatment significantly stimulated the expressions of DEGs from the metabolic pathways of Phagosome and Lysosome, suggesting Phagosome and Lysosome play essential roles in the regulation of alkali tolerance in this species, as well as the genes from these metabolic pathways. The present study identified the effects of alkali treatment on gills, providing valuable evidences for the genetic improvement of alkali tolerance in M. nipponense.

Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

Muscle LIM protein of Macrobrachium nipponense (MnMLP) involved in immune and stress response.

The muscle LIM protein (MLP) is a member of the cysteine and glycine-rich protein (CSRP) family, composed of CSRP1, CSRP2 and CSRP3/MLP. MLP is involved in a multitude of functional roles, including cytoskeletal organization, transcriptional regulation, and signal transduction. However, the molecular mechanisms underlying its involvement in immune and stress responses remain to be elucidated. This study identified an MnMLP in the freshwater crustacean Macrobrachium nipponense. The isothermal titration calorimetry assay demonstrated that recombinant MnMLP was capable of coordinating with Zn2+. Upon challenge by Aeromonas veronii or WSSV, and exposure to CdCl2, up-regulation was recorded in the muscle and intestinal tissues, suggesting its involvement in immune and anti-stress responses. MnMLP protein was predominantly expressed in the cytoplasm of the transfected HEK-293T cells, but after treatment with LPS, Cd2+ or H2O2, the MnMLP was observed to be transferred into the nucleus. The comet assay demonstrated that the overexpression of MnMLP could mitigate the DNA damage induced by H2O2 in HEK-293T cells, suggesting the potential involvement of MnMLP in the DNA repair process. These findings suggest that DNA repair may represent a possible mechanism by which MnMLP may be involved in the host's defense against pathogens and stress.

A pattern recognition receptor interleukin-1 receptor is involved in reproductive immunity in Macrobrachium nipponense ovary.

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene' (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense.

rpoS involved in immune response of Macrobrachium nipponens to Vibrio mimicus infection.

Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Isolation and the pathogenicity characterization of Decapod iridescent virus 1 (DIV1) from diseased Macrobrachium nipponense and its activation on host immune response.

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3, Relish, Gly-Cru2, CTL, MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense, one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1.

First report of Metschnikowia bicuspidata infection in the oriental river prawn (Macrobrachium nipponense, de Haan) in China.

During breeding, some oriental river prawns (Macrobrachium nipponense, de Haan), an important aquaculture species in China, exhibit yellowish-brown body colouration, reduced appetite, and vitality. Diseased prawns revealed characteristic emulsifying disease signs, including whitened musculature, hepatopancreatic tissues, milky haemolymph, and non-coagulation. The present study investigated the causative agent of M. nipponense infection through isolation, histopathology, molecular sequencing, and infection experiments. The pathogenic strain exhibited distinctive white colonies on Bengal red medium, with microscopic examination confirming the presence of yeast cells. Histopathological analysis revealed prominent pathological alterations and yeast cell infiltration in muscles, hepatopancreas and gills. Additionally, 26S rDNA sequencing of the isolated yeast strain LNMN2022 revealed Metschnikowia bicuspidata (GenBank: OR518659) as the causative agent. This strain exhibited a 98.28% sequence homology with M. bicuspidata LNMB2021 (GenBank: OK094821) and 96.62% with M. bicuspidata LNES0119 (GenBank: OK073903). The pathogenicity test confirmed that M. bicuspidata elicited clinical signs in M. nipponense consistent with those observed in natural populations, and the median lethal concentration was determined to be 3.3 × 105 cfu/mL. This study establishes a foundation for further investigations into the host range and epidemiological characteristics of the pathogen M. bicuspidata in aquatic animals and provides an empirical basis for disease management 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.

Functional Study of the Role of the Methyl Farnesoate Epoxidase Gene in the Ovarian Development of Macrobrachium nipponense.

Methyl farnesoate epoxidase (MFE) is a gene encoding an enzyme related to the last step of juvenile hormone biosynthesis. Mn-MFE cDNA has a total length of 1695 bp and an open reading frame (ORF) length of 1482 bp, encoding 493 amino acids. Sequence analysis showed that its amino acid sequence has a PPGP hinge, an FGCG structural domain, and other structural domains specific to the P450 family of enzymes. Mn-MFE was most highly expressed in the hepatopancreas, followed by the ovary and gill, weakly expressed in heart and muscle tissue, and barely expressed in the eyestalk and cranial ganglion. Mn-MFE expression remained stable during the larval period, during which it mainly played a critical role in gonadal differentiation. Expression in the ovary was positively correlated and expression in the hepatopancreas was negatively correlated with ovarian development. In situ hybridization (ISH) showed that the signal was expressed in the oocyte, nucleus, cell membrane and follicular cells, and the intensity of expression was strongest at stage O-IV. The knockdown of Mn-MFE resulted in a significantly lower gonadosomatic index and percentage of ovaries past stage O-III compared to the control group. However, no differences were found in the cumulative frequency of molting between the experimental and control groups. Moreover, the analysis of ovarian tissue sections at the end of the experiment showed differences between groups in development speed but not in subcellular structure. These results demonstrate that Mn-MFE promotes the ovarian development of Macrobrachium nipponense adults but has no effect on molting.

Studies on the Relationships between Growth and Gonad Development during First Sexual Maturation of Macrobrachium nipponense and Associated SNPs Screening.

In this study, we used full-sib families to investigate the association between growth and gonad development during first sexual maturation of M. nipponense. We found that male GSI was significantly negatively correlated with growth traits (p < 0.01) and there were no significant correlations between female GSI (Gonadosomatic index) and growth traits (p > 0.05). HSI (Hepatopancreas index) in both males and females showed no significant correlations with growth traits (p > 0.05). We furthermore investigated the association between the specific allele of Mn-CTS L1 polymorphism and gonad development and growth traits. In total, 35 mutation loci were screened and 16 high-quality single-nucleotide polymorphisms (SNPs) loci were obtained after validation. Four and two SNPs proved to be strongly associated with all growth traits in female and male M. nipponense separately, among which A+118T might be a candidate SNP positively associated with large growth traits. Two and one SNPs were screened, respectively, in males and females to associate with GSI, while three SNPs were detected to associate with female HSI, among which A+1379C may be applied as a potential molecular marker for gene-assisted selection to improve both reproduction speed and growth traits 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.

Temperature-Induced Sex Differentiation in River Prawn (Macrobrachium nipponense): Mechanisms and Effects.

Macrobrachium nipponense is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually results in higher yield. To investigate the impact of temperature on sexual differentiation in M. nipponense, two temperature treatments (26 °C and 31 °C) were conducted. The results showed that compared to the 31 °C treatment (3.20 ± 0.12), the 26 °C treatment displayed a lower female/male ratio (2.20 ± 0.11), which implied that a lower temperature could induce masculinization in M. nipponense. The temperature-sensitive sex differentiation phase was 25-35 days post hatching (DPH) at 26 °C while 15-20 DPH at 31 °C. Transcriptome and qPCR analysis revealed that a lower temperature up-regulated the expression of genes related to androgen secretion, and down-regulated the expressions of genes related to oogonia differentiation. Thirty-one temperature-regulated sex-differentiation genes were identified and the molecular mechanism of temperature-regulated sex differentiation was suggested. The finding of this study indicates that temperature regulation can be proposed as an innovative strategy for improving the culture yield of M. nipponense.

Combined transcriptome and metabolome analysis reveal key regulatory genes and pathways of feed conversion efficiency of oriental river prawn Macrobrachium nipponense.

BACKGROUND: Oriental river prawn Macrobrachium nipponense is an economically important aquaculture species in China, Japan, and Vietnam. In commercial prawn farming, feed cost constitutes about 50 to 65% of the actual variable cost. Improving feed conversion efficiency in prawn culture will not only increase economic benefit, but also save food and protect the environment. The common indicators used for feed conversion efficiency include feed conversion ratio (FCR), feed efficiency ratio (FER), and residual feed intake (RFI). Among these, RFI is much more suitable than FCR and FER during the genetic improvement of feed conversion efficiency for aquaculture species. RESULTS: In this study, the transcriptome and metabolome of hepatopancreas and muscle of M. nipponense from high RFI low RFI groups, which identified after culture for 75 days, were characterized using combined transcriptomic and metabolomic analysis. A total of 4540 differentially expressed genes (DEGs) in hepatopancreas, and 3894 DEGs in muscle were identified, respectively. The DEGs in hepatopancreas were mainly enriched in KEGG pathways including the metabolism of xenobiotics by cytochrome P450 (down-regulated), fat digestion and absorption (down-regulated) and aminoacyl-tRNA biosynthesis (up-regulated), etc. The DEGs in muscle were mainly enriched in KEGG pathways including the protein digestion and absorption (down-regulated), glycolysis/gluconeogenesis (down-regulated), and glutathione metabolism (up-regulated), etc. At the transcriptome level, the RFI of M. nipponense was mainly controlled in biological pathways such as the high immune expression and the reduction of nutrients absorption capacity. A total of 445 and 247 differently expressed metabolites (DEMs) were identified in the hepatopancreas and muscle, respectively. At the metabolome level, the RFI of M. nipponense was affected considerably by amino acid and lipid metabolism. CONCLUSIONS: M. nipponense from higher and lower RFI groups have various physiological and metabolic capability processes. The down-regulated genes, such as carboxypeptidase A1, 6-phosphofructokinase, long-chain-acyl-CoA dehydrogenase, et. al., in digestion and absorption of nutrients, and the up-regulated metabolites, such as aspirin, lysine, et. al., in response to immunity could be potential candidate factors contributed to RFI variation for M. nipponense. Overall, these results would provide new insights into the molecular mechanism of feed conversion efficiency and assist in selective breeding to improve feed conversion efficiency in M. nipponense.

Effects of ß-1,3-glucan on growth, immune responses, and intestinal microflora of the river prawn (Macrobrachium nipponense) and its resistance against Vibrio parahaemolyticus.

In this study, we investigated the impact of ß-1,3-glucan on the immune responses and gut microbiota of the river prawn (Macrobrachium nipponense) in the presence of Vibrio parahaemolyticus stress. Shrimps were fed one of the following diets: control (G1), 0.2% curdlan (G2), 0.1% ß-1,3-glucan (G3), 0.2% ß-1,3-glucan (G4), or 1.0% ß-1,3-glucan (G5) for 6 weeks and then challenged with V. parahaemolyticus for 96 h. Under Vibrio stress, shrimps in G4 exhibited the highest length gain rate, weight gain rate, and survival rate. They also showed increased intestinal muscle thickness and villus thickness compared to the control and 0.2% curdlan groups. The apoptosis rate was lower in G4 than in the control group, and the digestive enzyme activities (pepsin, trypsin, amylase, and lipase), immune enzyme activities (acid phosphatase, alkaline phosphatase, lysozyme, and phenoxidase), and energy metabolism (triglyceride, cholesterol, glycogen, and lactate dehydrogenase) were enhanced. Expression levels of growth-related genes (ecdysone receptor, calmodulin-dependent protein kinase I, chitin synthase, and retinoid X receptor) and immune-related genes (toll-like receptor 3, myeloid differentiation primary response 88, mitogen-activated protein kinase 7, and mitogen-activated protein kinase 14) were higher in G4 than in the control. Microbiota analysis indicated higher bacterial abundance in shrimps fed ß-1,3-glucan, as evidenced by Sob, Chao1, and ACE indices. Moreover, 0.2% ß-1,3-glucan increased the relative abundances of Bacteroidota and Firmicutes while reducing those of Corynebacteriales and Lactobacillales. In summary, ß-1,3-glucan enhances immune enzyme activities, alters immune-related gene expression, and impacts gut microbial diversity in shrimp. These findings provide valuable insights into the mechanisms underlying ß-1,3 glucan's immune-enhancing effects.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

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.

Adverse effects of chronic ammonia stress on juvenile oriental river prawn (Macrobrachium nipponense) and alteration of glucose and ammonia metabolism.

Ammonia is one of the common stress factors in aquaculture. However, the effect of chronic ammonia exposure in juvenile oriental river prawn (Macrobrachium nipponense) is currently unexplored. This study explored the effects of chronic ammonia on juvenile healthy oriental river prawns. Fifty prawns (0.123 ± 0.003 g) were exposed to 0, 5, and 15 mg/L total ammonia nitrogen (TAN) in triplicates for 28 days. The effects of chronic ammonia challenge were evaluated on growth, antioxidant capacity, hepatopancreas and gill morphology, and glucose and ammonia metabolism. The results showed that, the chronic ammonia exposure reduced significantly survival rate and weight gain of prawns. The prawns exposed to 15 mg/L ammonia had induced oxidative stress. However, the prawn exposed to 15 mg/L ammonia had significantly lower aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and acid phosphatase activities in the serum. Furthermore, exposure of prawns to 15 mg/L ammonia increased the activities of hexokinase, pyruvate kinase, pyruvate and lactic acid content, and glutamine synthase activity. However, the prawns exposed to 15 mg/L ammonia, reduced succinic dehydrogenase, 6-phosphogluconic dehydrogenase, phosphoenolpyruvate carboxykinase, glutamate synthase, and glutamate dehydrogenase activities but increased ammonia content in serum. The exposure of ammonia deformed lumen, damaged basement membrane and decreased secretory cells in the hepatopancreas, disordered gill epithelial and pillar cells, and caused gill filament base vacuolation. Our study indicates that chronic ammonia stress impairs growth performance, tissue morphology, induces oxidative stress, and alters glucose and ammonia metabolism in juvenile oriental river prawns.

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.