Integration of Gut Microbiota with Transcriptomic and Metabolomic Profiling Reveals Growth Differences in Male Giant River Prawns (Macrobrachium rosenbergii).

The giant freshwater prawn (GFP; Macrobrachium rosenbergii), a tropical species cultured worldwide, has high market demand and economic value. Male GFP growth varies considerably; however, the mechanisms underlying these growth differences remain unclear. In this study, we collected gut and hemolymphatic samples of large (ML), medium (MM), and small (MS) male GFPs and used the 16S rRNA sequencing and liquid chromatography-mass spectrometry-based metabolomic methods to explore gut microbiota and metabolites associated with GFP growth. The dominant bacteria were Firmicutes and Proteobacteria; higher growth rates correlated with a higher Firmicutes/Bacteroides ratio. Serum metabolite levels significantly differed between the ML and MS groups. We also combined transcriptomics with integrative multiomic techniques to further elucidate systematic molecular mechanisms in the GFPs. The results revealed that Faecalibacterium and Roseburia may improve gut health in GFP through butyrate release, affecting physiological homeostasis and leading to metabolic variations related to GFP growth differences. Notably, our results provide novel, fundamental insights into the molecular networks connecting various genes, metabolites, microbes, and phenotypes in GFPs, facilitating the elucidation of differential growth mechanisms in GFPs.

Insights on the virulence and genomic features of Lactococcus garvieae isolated from giant freshwater prawn Macrobrachium rosenbergii (de Man 1879).

Giant freshwater prawn (Macrobrachium rosenbergii (MR)) is a significant aquafarm species commercially cultured in Taiwan. Intensive farming practices have led to the outbreak of Lactococcus garvieae (LG), which causes Lactococcosis in MR. Recently, LG has re-emerged and the number of mortalities in prawn farms has increased in Taiwan. However, there is no preventative strategy described and a lack of knowledge on virulence factors and pathogenesis from LG in MR. The most virulent strain of L. garvieae from M. rosenbergii was screened in vivo among seven isolates selected for infectivity testing injecting 0.1 mL of 108 CFU/mL bacterial concentration. Among the seven isolates screened, L. garvieae 109-6 resulted in 100% mortality within 3 days post-infection. Furthermore, 109-6 L. garvieae LD50 dosage from in MR was found to be 106 CFU/mL. Subsequently, the most virulent strain 109-6 was sequenced using MinIon Nanopore sequencing. Results indicated that the LG genome yielded a protein-coding of 3857 with 59 tRNA and 16 rRNA and no plasmid. Interestingly, the distribution of subsystems in the annotated genome revealed genes related to virulence, defence, and disease among LG 50 genes. Altogether, the virulent strain and its genome data revealed distinctive features of LG, which hinted toward its pathogenicity and could facilitate for better preventive strategies.

Differential expression of microRNAs in giant freshwater prawn (Macrobrachium rosenbergii) during the infection of Vibrio parahaemolyticus.

MicroRNAs (miRNAs) are a category of small non-coding RNAs regarded as vital regulatory factors in various biological processes, especially immune regulation. The differently expressed miRNAs in Macrobrachium rosenbergii after the challenge of Vibrio parahaemolyticus were identified using high-throughput sequencing. A total of 18 known as well as 12 novel miRNAs were markedly differently expressed during the bacterial infection. The results of the target gene prediction and enrichment analysis indicated that a total of 230 target genes involved in a large variety of signaling pathways and biological processes were mediated by the miRNAs identified in the current research. Additionally, the effects of novel-miR-56, a representative differentially expressed miRNA identified in the previous infection experiment, on the immune-related gene expression in M. rosenbergii were explored. The expression of the immune-related genes including Spätzle1(Spz1), Spz4, Toll-like receptor 1 (TLR1), TLR2, TLR3, immune deficiency (IMD), myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor 1 (ALF1), crustin1, as well as prophenoloxidase (proPO) was significantly repressed in the novel-miR-56-overexpressed prawns. The expression of these genes tested in the novel-miR-56-overexpressed M. rosenbergii was still signally lower than the control in the subsequent V. parahaemolyticus challenge, despite the gene expression in each treatment increased significantly after the infection. Additionally, the cumulative mortality of the agomiR-56-treated prawns was significantly higher than the other treatments post the bacterial challenge. These results suggested that novel-miR-56 might function as a negative regulator of the immune-related gene expression of M. rosenbergii in the innate immune defense against V. parahaemolyticus.

Transcriptomics-based analysis of Macrobrachium rosenbergii growth retardation.

Macrobrachium rosenbergii is an economically important crustacean in many parts of the world, but in recent years, growth retardation has become an increasingly serious issue. While the underlying causes remain unclear, this has inevitably impacted on aquaculture and production outputs. In this study, gill, hepatopancreas, and muscle tissue samples from M. rosenbergii, with distinct growth differences, underwent transcriptome sequencing and bioinformatics analyses using high-throughput sequencing. In total, 59,796 unigenes were annotated. Differential expression analyses showed that the most differentially expressed genes (DEGs) were screened in gill tissue (1790 DEGs). In muscle and hepatopancreas tissues, 696 and 598 DEGs were screened, respectively. These DEGs were annotated to Kyoto Encyclopedia of Genes and Genomes pathways, which identified several significantly enriched pathways related to growth metabolism, such as PI3K-AKT, glycolysis/gluconeogenesis, and starch and sucrose metabolism. These results suggest that low growth metabolism levels may be one cause of M. rosenbergii growth retardation. Our data provide support for further investigations into the causes and molecular mechanisms underpinning growth retardation in M. rosenbergii.

Identification and characterization of olfactory gene families in Macrobrachium rosenbergii based on full-length transcripts and genome sequences.

The olfactory gene families include odorant binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). To investigate the molecular function of olfactory perception in Macrobrachium rosenbergii, we integrated the full-length transcripts and whole-genome sequences to identify the olfactory gene families. In this study, a total of 38,955 full-length transcripts with an N50 length of 3383 bp were obtained through PacBio SMRT sequencing. Through the annotation of full-length transcripts and whole-genome sequences, several olfactory gene families were identified, including 18 MrORs, 16 MrIRs, 151 MrIGluRs (ionotropic glutamate receptors), 2 MrVIGluRs (variant ionotropic glutamate receptors) and 3 MrCRs (chemosensory receptors). Notably, the CRs were first identified in prawns and shrimps. Additionally, the olfactory gene families in M. nipponense were identified, comprising 4 MnORs, 21 MnIRs, 79 MnIGluRs, 5 MnVIGluRs, 1 MnGR and 1 MnOBP, using the available whole-genome sequences. Meanwhile, the external morphology of the chemical sensory organs of M. rosenbergii was explored, and the presence of plumose setae (PS), hard thorn setae (HTS), bamboo shoot setae (BSS), soft thorn setae (STS) and aesthetascs (AE) on the antennules, HTS and BSS on the second antennae, and PS on the pereiopods were observed by scanning electron microscope. This study provides valuable insights for future functional studies into the olfactory perception of crustaceans and establishes a theoretical basis for molecular design breeding in M. rosenbergii.

A chromosome-level reference genome assembly and a full-length transcriptome assembly of the giant freshwater prawn (Macrobrachium rosenbergii).

The giant freshwater prawn (Macrobrachium rosenbergii) is a key species in the aquaculture industry in several Asian, African, and South American countries. Despite a considerable growth in its production worldwide, the genetic complexities of M. rosenbergii various morphotypes pose challenges in cultivation. This study reports the first chromosome-scale reference genome and a high-quality full-length transcriptome assembly for M. rosenbergii. We employed the PacBio High Fidelity (HiFi) sequencing to obtain an initial draft assembly and further scaffolded it with the chromatin contact mapping (Hi-C) technique to achieve a final assembly of 3.73-Gb with an N50 scaffold length of 33.6 Mb. Repetitive elements constituted nearly 60% of the genome assembly, with simple sequence repeats and retrotransposons being the most abundant. The availability of both the chromosome-scale assembly and the full-length transcriptome assembly enabled us to thoroughly probe alternative splicing events in M. rosenbergii. Among the 2,041 events investigated, exon skipping represented the most prevalent class, followed by intron retention. Interestingly, specific isoforms were observed across multiple tissues. Additionally, within a single tissue type, transcripts could undergo alternative splicing, yielding multiple isoforms. We believe that the availability of a chromosome-level reference genome for M. rosenbergii, along with its full-length transcriptome, will be instrumental in advancing our understanding of the giant freshwater prawn biology and enhancing its molecular breeding programs, paving the way for the development of M. rosenbergii with valuable traits in commercial aquaculture.

Regulation and Response Mechanism of Acute Low-Salinity Stress during Larval Stages in Macrobrachium rosenbergii Based on Multi-Omics Analysis.

Macrobrachium rosenbergii is an essential species for freshwater economic aquaculture in China, but in the larval process, their salinity requirement is high, which leads to salinity stress in the water. In order to elucidate the mechanisms regulating the response of M. rosenbergii to acute low-salinity exposure, we conducted a comprehensive study of the response of M. rosenbergii exposed to different salinities' (0‱, 6‱, and 12‱) data for 120 h. The activities of catalase, superoxide dismutase, and glutathione peroxidase were found to be significantly inhibited in the hepatopancreas and muscle following low-salinity exposure, resulting in oxidative damage and immune deficits in M. rosenbergii. Differential gene enrichment in transcriptomics indicated that low-salinity stress induced metabolic differences and immune and inflammatory dysfunction in M. rosenbergii. The differential expressions of MIH, JHEH, and EcR genes indicated the inhibition of growth, development, and molting ability of M. rosenbergii. At the proteomic level, low salinity induced metabolic differences and affected biological and cellular regulation, as well as the immune response. Tyramine, trans-1,2-Cyclohexanediol, sorbitol, acetylcholine chloride, and chloroquine were screened by metabolomics as differential metabolic markers. In addition, combined multi-omics analysis revealed that metabolite chloroquine was highly correlated with low-salt stress.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis.

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: -0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

Temporal Transcriptomic Profiling Reveals Dynamic Changes in Gene Expression of Giant Freshwater Prawn upon Acute Saline-Alkaline Stresses.

Bicarbonate and sulfate are among two primary ion constituents of saline-alkaline water, with excessive levels potentially causing metabolic disorders in crustaceans, affecting their molting and interrupting development. As an economically important crustacean species, the molecular adaptive mechanism of giant freshwater prawn Macrobrachium rosenbergii in response to the stress of bicarbonate and sulfate remains unexplored. To investigate the mechanism underlying NaHCO3, Na2SO4, and mixed NaHCO3, Na2SO4 stresses, M. rosenbergii larvae were exposed to the above three stress conditions, followed by total RNA extraction and high-throughput sequencing at eight distinct time points (0, 4, 8, 12, 24, 48, 72, and 96 h). Subsequent analysis revealed 13, 16, and 13 consistently identified differentially expressed genes (DEGs) across eight time points under three stress conditions. These consistently identified DEGs were significantly involved in the Gene Ontology (GO) terms of chitin-based cuticle development, protein-carbohydrate complex, structural constituent of cuticle, carnitine biosynthetic process, extracellular matrix, and polysaccharide catabolic process, indicating that alkaline stresses might potentially impact the energy metabolism, growth, and molting of M. rosenbergii larvae. Particularly, the transcriptome data revealed that DEGs associated with energy metabolism, immunity, and amino acid metabolism were enriched across multiple time points under three stress conditions. These DEGs are linked to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including glycolysis/glucogenesis, amino sugar and nucleotide sugar metabolism, and lysine degradation. Consistent enrichment findings across the three stress conditions support conclusions above. Together, these insights are instrumental in enhancing our understanding of the molecular mechanisms underlying the alkaline response in M. rosenbergii larvae. Additionally, they offer valuable perspectives on the regulatory mechanisms of freshwater crustaceans amid saline-alkaline water development.

Metagenomic dissection of the intestinal microbiome in the giant river prawn Macrobrachium rosenbergii infected with Decapod iridescent virus 1.

Microbiome in the intestines of aquatic invertebrates plays pivotal roles in maintaining intestinal homeostasis, especially when the host is exposed to pathogen invasion. Decapod iridescent virus 1 (DIV1) is a devastating virus seriously affecting the productivity and success of crustacean aquaculture. In this study, a metagenomic analysis was conducted to investigate the genomic sequences, community structure and functional characteristics of the intestinal microbiome in the giant river prawn Macrobrachiumrosenbergii infected with DIV1. The results showed that DIV1 infection could significantly reduce the diversity and richness of intestinal microbiome. Proteobacteria represented the largest taxon at the phylum level, and at the species level, the abundance of Gonapodya prolifera and Solemya velum gill symbiont increased significantly following DIV1 infection. In the infected prawns, four metabolic pathways related to purine metabolism, pyrimidine metabolism, glycerophospholipid metabolism, and pentose phosphate pathway, and five pathways related to nucleotide excision repair, homologous recombination, mismatch repair, base excision repair, and DNA replication were significantly enriched. Moreover, several immune response related pathways, such as shigellosis, bacterial invasion of epithelial cells, Salmonella infection, and Vibrio cholerae infection were repressed, indicating that secondary infection in M. rosenbergii may be inhibited via the suppression of these immune related pathways. DIV1 infection led to the induction of microbial carbohydrate enzymes such as the glycoside hydrolases (GHs), and reduced the abundance and number of antibiotic-resistant ontologies (AROs). A variety of AROs were identified from the microbiota, and mdtF and lrfA appeared as the dominant genes in the detected AROs. In addition, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration were the main antibiotic resistance mechanisms. Collectively, the data would enable a deeper understanding of the molecular response of intestinal microbiota to DIV1, and offer more insights into its roles in prawn resistance to DIVI infection.

Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

OBJECTIVE: We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. METHODS: An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. RESULT: The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. CONCLUSION: These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

Differential expression of neuropeptide F in the digestive organs of female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle.

Neuropeptide F is a key hormone that controls feeding in invertebrates, including decapod crustaceans. We investigated the differential expression of Macrobrachium rosenbergii neuropeptide F (MrNPF) in the digestive organs of female prawns, M. rosenbergii, during the ovarian cycle. By using RT-qPCR, the expression of MrNPF mRNA in the esophagus (ESO), cardia (CD), and pylorus (PY) of the foregut (FG) gradually increased from stage II and peaked at stage III. In the midgut (MG), hindgut (HG), and hepatopancreas (HP), MrNPF mRNA increased from stage I, reaching a maximal level at stage II, and declined by about half at stages III and IV (P < 0.05). In the ESO, CD, and PY, strong MrNPF-immunoreactivities were seen in the epithelium, muscle, and lamina propria. Intense MrNPF-ir was found in the MG cells and the muscular layer. In the HG, MrNPF-ir was detected in the epithelium of the villi and gland regions, while MrNPF-ir was also more intense in the F-, R-, and B-cells in the HP. However, we found little colocalization between the MrNPF and PGP9.5/ChAT in digestive tissues, implying that most of the positive cells might not be neurons but could be digestive tract-associated endocrine cells that produce and secrete MrNPF to control digestive organ functions in feeding and utilizing feed. Taken together, our first findings indicated that MrNPF was differentially expressed in digestive organs in correlation with the ovarian cycle, suggesting an important link between MrNPF, the physiology of various digestive organs in feeding, and possibly ovarian maturation in female M. rosenbergii.

Comparative Transcriptome Analysis of the Hepatopancreas from Macrobrachium rosenbergii Exposed to the Heavy Metal Copper.

The contamination of aquatic ecosystems by the heavy metal copper (Cu) is an important environmental issue and poses significant risks to the physiological functions of aquatic organisms. Macrobrachium rosenbergii is one of the most important freshwater-cultured prawns in the world. The hepatopancreas of crustaceans is a key organ for immune defense, heavy metal accumulation, and detoxification, playing a pivotal role in toxicological research. However, research on the molecular response of the hepatopancreas in M. rosenbergii to Cu exposure is still lacking. In this study, the transcriptomic response in the hepatopancreas of M. rosenbergii was studied after Cu exposure for 3 and 48 h. Compared with the control group, 11,164 (7288 up-regulated and 3876 down-regulated genes) and 10,937 (6630 up-regulated and 4307 down-regulated genes) differentially expressed genes (DEGs) were identified after 3 and 48 h exposure, respectively. Most of these DEGs were up-regulated, implying that gene expressions were largely induced by Cu. Functional enrichment analysis of these DEGs revealed that immunity, copper homeostasis, detoxification, DNA damage repair, and apoptosis were differentially regulated by Cu. Seven genes involved in immunity, detoxification, and metabolism were selected for validation by qRT-PCR, and the results confirmed the reliability of RNA-Seq. All these findings suggest that M. rosenbergii attempts to resist the toxicity of Cu by up-regulating the expression of genes related to immunity, metabolism, and detoxification. However, with the excessive accumulation of reactive oxygen species (ROS), the antioxidant enzyme system was destroyed. As a result, DNA damage repair and the cellular stress response were inhibited, thereby exacerbating cell damage. In order to maintain the normal function of the hepatopancreas, M. rosenbergii removes damaged cells by activating the apoptosis mechanism. Our study not only facilitates an understanding of the molecular response mechanisms of M. rosenbergii underlying Cu toxicity effects but also helps us to identify potential biomarkers associated with the stress response in other crustaceans.

Pathogenicity of Aeromonas veronii Isolated from Diseased Macrobrachium rosenbergii and Host Immune-Related Gene Expression Profiles.

Aeromonas veronii is widespread in aquatic environments and is responsible for infecting various aquatic animals. In this study, a dominant strain was isolated from the hepatopancreas of diseased Macrobrachium rosenbergii and was named JDM1-1. According to its morphological, physiological, and biochemical characteristics and molecular identification, isolate JDM1-1 was identified as A. veronii. The results of artificial challenge showed isolate JDM1-1 had high pathogenicity to M. rosenbergii with an LD50 value of 8.35 × 105 CFU/mL during the challenge test. Histopathological analysis revealed severe damage in the hepatopancreas and gills of the diseased prawns, characterized by the enlargement of the hepatic tubule lumen and gaps between the tubules as well as clubbing and degeneration observed at the distal end of the gill filament. Eight virulence-related genes, namely aer, ompA, lip, tapA, hlyA, flgA, flgM, and flgN, were screened by PCR assay. In addition, virulence factor detection showed that the JDM1-1 isolate produced lipase, lecithinase, gelatinase, and hemolysin. Furthermore, the mRNA expression profiles of immune-related genes of M. rosenbergii following A. veronii infection, including ALF1, ALF2, Crustin, C-lectin, and Lysozyme, were assessed, and the results revealed a significant upregulation in the hepatopancreas and intestines at different hours post infection. This study demonstrates that A. veronii is a causative agent associated with massive die-offs of M. rosenbergii and contributes valuable insights into the pathogenesis and host defense mechanisms of A. veronii invasion.

Regulation of early spermatogenesis in the giant prawn Macrobrachium rosenbergii by a GCL homolog†.

The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120 896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10 days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations.

Metabolomic assessment of African snail (Achatina fulica) meal on growth performance of giant river prawn (Macrobrachium rosenbergii).

This study aimed to investigate the effects of replacing fishmeal (FM) with African giant snail (Achatina fulica) meal (SM) on the growth performance of giant river prawn (Macrobrachium rosenbergii), as well as to analyze the associated metabolomic changes. Six diets were formulated, replacing FM with SM at different inclusion levels ranging from 0 % to 100 %. Growth performance and feed conversion ratio of prawns fed diets with FM replaced by SM up to 80 % were not significantly different from control. In contrast, significantly decreased growth performance and higher feed conversion ratio (FCR) occurred with diets containing 100 % SM. To gain insights into the metabolic regulation of prawns fed different diets, a 1H NMR metabolomics approach was used to assess the metabolic changes in prawns fed diets containing 0 % and 80 % SM. The results revealed up-regulated metabolites significantly involved in several metabolic pathways, including alanine, aspartate, and glutamate metabolism; citrate cycle (TCA cycle); aminoacyl-tRNA biosynthesis; and valine, leucine, and isoleucine biosynthesis. These findings imply that including SM in the diet might modulate the regulation of muscle amino acids and tRNA synthesis, suggesting a potential impact on protein biosynthesis mechanisms. Additionally, alterations in the TCA cycle may reflect changes in carbon utilization, potentially contributing to the growth performance of giant river prawns when fishmeal is replaced with SM without adversely affecting their growth. In conclusion, this study demonstrated that SM could be a promising alternative protein source in aquafeed. The metabolomic approach provides valuable insights into the metabolic changes in prawns fed different diets, aiding in the development of more effective aquafeeds in the future. The study's limitations, such as the simplified diet formulation and the limited scope of the metabolomic analysis, were acknowledged and discussed, highlighting the need for further research to build upon these findings.

Identifying the function of the PI3K-AKT pathway during the pathogenic infection of Macrobrachium rosenbergii.

The phosphoinositide-3-kinase/protein kinase b (PI3K-Akt) pathway is a signalling pathway based on protein phosphorylation and can be activated by a wide range of factors. To investigate the function of the PI3K-AKT signalling pathway in antibacterial immunity, we analysed the gene expression level of three key factors (PI3K, AKT and FoxO) and innate immune factors in immune tissues at different time points after Vibrio parahaemolyticus and Staphylococcus aureus infection. Tissues analysis showed that PI3K, AKT, and FoxO were expressed at high levels in the intestinal, hemocytes and hepatopancreas. Moreover, the expression levels of PI3K, AKT and FoxO can be regulated postinfection by different pathogens. In hemocytes and the intestine, V. parahaemolyticus infection was found to regulate the levels of PI3K, AKT, and FoxO more rapidly; however, an S. aureus infection regulated the levels of these factors more rapidly in the hepatopancreas and gills. Analysis showed that V. parahaemolyticus and S. aureus infection caused changes in the gene expression level of crustin, caspase 3 and NF-κB. Therefore, PI3K-AKT regulates the downstream immune pathway differentially in different immune tissues and participates in the regulation of cell apoptosis and the inflammatory response by activating caspase and NF-κB, respectively, following infection with V. parahaemolyticus and S. aureus.

Selection and characterization of ssDNA aptamer targeting Macrobrachium rosenbergii nodavirus capsid protein: A potential capture agent in gold-nanoparticle-based aptasensor for viral protein detection.

The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.

Diversity of MrTolls and their regulation of antimicrobial peptides expression during Enterobacter cloacae infection in Macrobrachium rosenbergii.

Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.

Transcriptome analysis reveal the effect of freshwater sediments containing 2,3,7,8-tetrachlorodibenzo-p-dioxin on the Macrobrachium rosenbergii hepatopancreas, intestine, and muscle.

This research evaluated the hepatopancreas, intestine, and muscle transcriptome alternation of Macrobrachium rosenbergii, and to confirm the relative glycerophospholipid, cytochrome P450 system, and fatty acid metabolism gene expression in sediments containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of 60 ng/sediment (g) and 700 ng/sediment (g) for 90 days of culture. Transcriptome analysis revealed that the TCDD sediment affected the hepatopancreatic metabolism of xenobiotics in M. rosenbergii via the cytochrome P450 system, drug metabolism-other enzymes, drug metabolism-cytochrome P450, chemical carcinogenesis, and lysosome function. Intestinal analysis also showed a similar phenomenon, but this finding was not observed in the muscle tissue. qPCR analysis indicated that the expression levels of APTG4, LPGAT1, ACHE, GPX4, ECHS1, ATP5B, FABP, and ACC in the hepatopancreatic and intestinal tissues decreased, but those in the muscle tissues did not. In summary, TCDD sediment induced tissue metabolism, especially in the hepatopancreas and intestine. TCDD sediment mainly affected the digestive enzyme gene expression with concentration. These results indicated that the presence of TCDD in the sediment played a major role in the hepatopancreatic and intestinal metabolism system of M. rosenbergii.