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.

Histopathological observation and comparative transcriptome analysis reveal immune response mechanisms to Aeromonas dhakensis infection in Macrobrachium rosenbergii.

The Macrobrachium rosenbergii industry is threatened by various Aeromonas, resulting in high mortality of adult prawns. However, there are few studies on the immune response of M. rosenbergii infected with Aeromonas dhakensis. In this study, we observed the hepatopancreas and gills histopathologically, performed a comparative transcriptome analysis of the hepatopancreas, and analyzed the candidate gene expression of immune-related genes in the hemolymph, hepatopancreas, and gills of M. rosenbergii that had been infected with A. dhakensis. Histopathology revealed the hepatopancreas was successively inflamed, followed by cellular vacuolation, lumen deformation, and finally tissue erosion; partial and severe inflammation of the gills occurred successively, and eventually the gill tissue atrophy and the gill filaments detached from the gill arch. Transcriptome analysis showed that a total of 77,742 unigenes and 8664 differentially expressed genes (DEGs), and the immune-related DEGs were mainly enriched in lysosome and phagosome pathways. In addition, 4 immune-related candidate genes (RhoA, CASP9, PKC, and DSCIGN) based on KEGG and PPI analysis were monitored at 6, 12, and 24h post injection (hpi) in hepatopancreas, hemolymph and gills. Their spatio-temporal expression results indicated that A. dhakensis have activated the immune system of M. rosenbergii. The present study may provide new information on the complex immune mechanism of M. rosenbergii.

The Dynamics of Gene Expression Unraveling the Immune Response of Macrobrachium rosenbergii Infected by Aeromonas veronii.

To further investigate the immune response of Macrobrachium rosenbergii against Aeromonas veronii, comparative transcriptomic analyses of the M. rosenbergii hepatopancreas were conducted on challenge and control groups at 6, 12, and 24 h post-infection (hpi), independently. A total of 51,707 high-quality unigenes were collected from the RNA-seq data, and 8060 differentially expressed genes (DEGs) were discovered through paired comparisons. Among the three comparison groups, a KEGG pathway enrichment analysis showed that 173 immune-related DEGs were considerably clustered into 28 immune-related pathways, including the lysosome, the phagosome, etc. Moreover, the expression levels of the four key immune-related genes (TOLL, PAK1, GSK3ß, and IKKα) were evaluated at various stages following post-infection in the hepatopancreas, hemolymph, and gills. Both PAK1 and GSK3ß genes were highly up-regulated in all three tissues at 6 hpi with A. veronii; TOLL was up-regulated in the hepatopancreas and hemolymph but down-regulated in the gill at 6 hpi, and IKKα was up-regulated in hemolymph and gill, but down-regulated in the hepatopancreas at 6 hpi. These findings lay the groundwork for understanding the immune mechanism of M. rosenbergii after contracting A. veronii.

Integrated Transcriptomic and Metabolomic Analyses Reveal Low-Temperature Tolerance Mechanism in Giant Freshwater Prawn Macrobrachium rosenbergii.

Water temperature, as an important environmental factor, affects the growth and metabolism of aquatic animals and even their survival. The giant freshwater prawn (GFP) Macrobrachium rosenbergii is a kind of warm-water species, and its survival temperature ranges from 18 °C to 34 °C. In this study, we performed transcriptomic and metabolomic analyses to clarify the potential molecular mechanism of responding to low-temperature stress in adult GFP. The treatments with low-temperature stress showed that the lowest lethal temperature of the GFP was 12.3 °C. KEGG enrichment analyses revealed that the differentially expressed genes and metabolites were both enriched in lipid and energy metabolism pathways. Some key genes, such as phosphoenolpyruvate carboxykinase and fatty acid synthase, as well as the content of the metabolites dodecanoic acid and alpha-linolenic acid, were altered under low-temperature stress. Importantly, the levels of unsaturated fatty acids were decreased in LS (low-temperature sensitive group) vs. Con (control group). In LT (low-temperature tolerant group) vs. Con, the genes related to fatty acid synthesis and degradation were upregulated to cope with low-temperature stress. It suggested that the genes and metabolites associated with lipid metabolism and energy metabolism play vital roles in responding to low-temperature stress. This study provided a molecular basis for the selection of a low-temperature tolerant strain.

Alterations of the Gut Microbiota and Metabolomics Associated with the Different Growth Performances of Macrobrachium rosenbergii Families.

To investigate the key gut microbiota and metabolites associated with the growth performance of Macrobrachium rosenbergii families, 16S rRNA sequencing and LC-MS metabolomic methods were used. In this study, 90 M. rosenbergii families were bred to evaluate growth performance. After 92 days of culture, high (H), medium (M), and low (L) experimental groups representing three levels of growth performance, respectively, were collected according to the weight gain and specific growth rate of families. The composition of gut microbiota showed that the relative abundance of Firmicutes, Lachnospiraceae, Lactobacillus, and Blautia were much higher in Group H than those in M and L groups. Meanwhile, compared to the M and L groups, Group H had significantly higher levels of spermidine, adenosine, and creatinine, and lower levels of L-citrulline. Correlation analysis showed that the abundances of Lactobacillus and Blautia were positively correlated with the levels of alpha-ketoglutaric acid and L-arginine. The abundance of Blautia was also positively correlated with the levels of adenosine, taurine, and spermidine. Notably, lots of metabolites related to the metabolism and biosynthesis of arginine, taurine, hypotaurine, and fatty acid were upregulated in Group H. This study contributes to figuring out the landscape of the gut microbiota and metabolites associated with prawn growth performance and provides a basis for selective breeding.

Iron overload facilitates neonatal hypoxic-ischemic brain damage via SLC7A11-mediated ferroptosis.

Oxidative damage and cell death are involved in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Ferroptosis is a newly identified mode of cell death that results from the oxidative damage induced by excessive iron. In HIBD, iron accumulates in brain tissues due to the massive destruction of red blood cells and increased permeability of the blood brain barrier vasculature, which can trigger ferroptosis. Ferroptosis is implicated in various diseases involving neuronal injury; however, the roles of iron and ferroptosis in HIBD have not been identified. In the present study, we investigated the role of iron overload in neuronal ferroptosis both in HIBD rat models and in oxygen- and glucose-deprived (OGD) SH-SY5Y cells. We observed that iron deposition in the cerebral cortex was significantly increased in HIBD rats. Features of ferroptosis such as shrunken mitochondria, increased MDA (malondialdehyde) levels, and reduced solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression were observed in the cerebral cortex of HIBD rats. Administration of an iron chelator in HIBD rats upregulated SLC7A11 expression and alleviated neuronal ferroptosis in cerebral cortex tissue. Additionally, overexpression of SLC7A11 in SH-SY5Y cells increased cell viability and attenuated OGD-induced ferroptosis. Our results demonstrate that iron overload induces neuronal ferroptosis by inhibiting SLC7A11 expression in HIBD. Inhibition of neuronal ferroptosis may be a promising strategy to alleviate brain damage in HIBD.

Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein for Toll-like receptor (TLR)-mediated signaling pathways and plays an important role in immune response. In this study, the full-length cDNA of MyD88 from Macrobrachium rosenbergii (MRMyD88) was cloned. The MRMyD88 cDNA is 1758 bp long and contains a 1398-bp open reading frame. Multiple sequence alignment and phylogenetic analysis revealed that the amino acid sequence of MRMyD88 shared high identity with the known MyD88 proteins. The MRMyD88 mRNA was widely expressed in all examined tissues, with highest level in intestine, followed by gonad and pleopod. Furthermore, the MRMyD88 promoter region, spanning 1622 bp, contains several transcription factor-binding sites, including nine GATA-1 box motifs. Electrophoretic mobility shift assay showed that Gfi-1, SRF, and Oct-1 bind to the upstream region of MRMyD88. Additionally, the results showed that the expression levels of TLR1, TLR2 and TLR3 were different in response to Vibrio anguillarum, Lactobacillus plantarum and Aeromonas hydrophila infections. However, these bacteria significantly increased the expression levels of MyD88 and prophenoloxidase. These data suggest that the TLR-mediated signaling pathway is MyD88-dependent in response to pathogenic and probiotic bacteria in M. rosenbergii.

Genetic parameters and selection response for the harvest body weight of the giant freshwater prawn (Macrobrachium rosenbergii) in a breeding program in China.

A multi-trait selective breeding program of Macrobrachium rosenbergii was initiated in China in 2015. In this program, the M. rosenbergii resources were widely collected from four countries, the origin of the founders was verified with 16 microsatellites and the pedigree was reconstructed, and the optimum contribution selection was used to make the mating design. In this study, we evaluated the genetic parameters and selection response for the harvest body weight (HBW) of M. rosenbergii after being communally reared for 95-109 days. The data were collected from two generations that comprised 25,212 progenies from 150 sires and 198 dams. The residual maximum-likelihood methodology was employed to evaluate the variance components, by fitting an animal model. The accuracy of estimated breeding values increased by 0.38% after pedigree reconstruction using microsatellite markers. The estimated heritability (h2) for HBW was moderate (0.212 ± 0.049) and the common environmental coefficient (c2) was low (0.063 ± 0.017) when all the data were used for the analysis. Within generations, h2 was moderate to high (0.198 ± 0.080 to 0.338 ± 0.049). c2 could only be estimated in G1, which was 0.055 ± 0.030. The average HBW of males was significantly larger than that of females (P < 0.01). h2 estimated for female HBWs were higher than that for males within generations, while h2 estimated for female HBWs were lower than that for males across generations. But they were not significantly different (P > 0.05). The genetic correlations between sexes were moderate to high within each generation (0.529 to 0.763). Two methods were used to estimate the realized response. One method was calculated from the differences between the least squares means of the selected population HBW and that of control population HBW, which was 14.01%. The other method was calculated from the differences between the EBVs of the selected population HBW and that of control population HBW, which was 11.52%. The predicted responses derived from two sets of genetic parameters acquired from within- and across- generation datasets were 11.68% and 10.67%, respectively. The present study provides valuable information for breeding programs of M. rosenbergii.

[Sequences analysis of jlFABP2 and the correlation between polymorphisms and body weight gain in Cyprinus carpio var. jian].

Two replicate intestine fatty acid binding protein genes (jlFABP2a and jlFABP2b) were cloned from Cyprinus carpio var. jian using PCR. Both ORFs were 399 bp in length sharing 92.2% similarity with each other, and 88.0% and 90.5% with their counterpart in zebrafish, respectively. The gene structure of jlFABP2s was same as other FABPs, which contained four exons and three introns. Sequences and lengths of introns between 2a and 2b. were obviously different Phylogenetic tree displayed that two jlFABP2s corresponded to one zebrafish FABP2 which matches the fact that the chromosome number of common carp was twice of zebrafish. Real time-PCR showed that jlFABP2 genes mainly expressed in intestine and the expression level was very significantly higher than other tissues such as brain, liver, muscle, kidney, and gonad (P<0.01). The expression level of jlFABP2a was significantly (male, P<0.05) or very significantly (females, P<0.01) higher than 2b in intestine; and 2b was expressed slightly higher than 2a in other tissues. It seemed that 2a expressed specifically in intestine, while 2b expressed ubiquitously. Twelve and four SNP loci were found at jlFABP2a and 2b introns through comparison sequences from 8 individuals, respectively. Genotypes of I1-A15G, I1-A99G, I2-C487T, and I3-A27T on jlFABP2a were detected using PCR-RFLP in selection population of C. carpio var. jian. The SNP genotypes and individual weight gain correlation indicated that four SNPs were significantly (P<0.05 or P<0.01) associated with adult weight gain. Diplotype analysis displayed that individuals with genotype AGGGCCXX or AGGGXXAT grew faster than other individuals by 15%. The individuals with these two genotypes only occupied 9% in total selection populations, indicating the presence of large selection space. The 4 SNPs detected in this experiment can be used in C. carpio var. Jian growth selection breeding plan.