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.

Effects of chronic abamectin stress on growth performance, digestive capacity, and defense systems in red swamp crayfish (Procambarus clarkii).

Abamectin is a globally used pesticide, which is one of 16-member macrocyclic lactones compound. As an environmental contaminant, pesticide residues pose a great threat to the health and survival of aquatic animals. Procambarus clarkii is one of the most important economic aquatic animals in China. It is necessary to explore the toxic mechanism of abamectin to P. clarkii. In this study, the toxic mechanism of abamectin to P. clarkii was investigated by 0, 3 and 6 µg/L abamectin stress for 28 days. The digestive-, antioxidant- and immune- related enzymes activities, genes expression levels, and histological observations were analytical indicators of growth performance, digestive capacity, and defense systems. The results in this study showed that with abamectin concentration increasing, the growth of P. clarkii was stunted significantly, and the mortality rate increased significantly. With exposure time and abamectin concentration increasing, the expression levels of related genes, the activities of digestive-, antioxidant-, and immune- related enzymes decreased ultimately. Moreover, through histological observation, it was found that with abamectin concentration increasing, the hepatopancreas, muscle, and intestine were damaged. As elucidated by the results, once abamectin exists in the environment for a long time, even low doses will threaten to healthy growth and survival of P. clarkii. This study explored the potential toxicity and the toxic mechanism of abamectin to P. clarkii, and provides a theoretical basis for further study on the toxicity of pesticides to aquatic animals.

Toxic effects of nanopolystyrene and cadmium on the intestinal tract of the Chinese mitten crab (Eriocheir sinensis).

Nanopolystyrene (NP) and cadmium (Cd) are ubiquitous contaminants in aquatic systems. The present study aimed to investigate the toxic effects of exposure to ambient concentrations of NP and/or Cd on the intestinal tract of the Chinese mitten crab (Eriocheir sinensis). Exposure to NP and/or Cd induced oxidative stress, as evidenced by a significant increase in lipid peroxide content (LPO), total antioxidant capacity (T-AOC), and peroxidase activity (POD), and significant decreases in superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities in E. sinensis. In addition, exposure to NP and/or Cd imbalanced the homeostasis of the intestinal microbiota, as demonstrated by the significantly increased abundance of Spiroplasma. Transcriptomic and metabolomic analyses were performed to investigate the mechanisms underlying intestinal toxicity. Our results showed that ferroptosis, ABC transporters, phosphotransferase system, apoptosis, and leukocyte transendothelial migration were disturbed after exposure to NP and/or Cd. In particular, Cd exposure affected mucin type O-glycan biosynthesis, purine metabolism, and neuroactive ligand-receptor interaction. Intriguingly, co-exposure to NP and Cd might mitigate intestinal toxicity by decreasing oxidative stress and affecting these pathways. Taken together, our study clearly demonstrates that exposure to NP and/or Cd at environmentally relevant concentrations causes intestinal toxicity in E. sinensis.

Effects of Metamifop on Defense Systems in Monopterus albus.

The effects of herbicides on non-target organisms in paddy fields have become a popular research topic. As a widely used herbicide, it is necessary to explore the potential toxicity of metamifop in non-target organisms, especially aquatic animals, in co-culture mode. In the present study, we evaluated the effects of metamifop (0, 0.2, 0.4, 0.6, and 0.8 mg/L) on the defense system (antioxidation, immunity, and apoptosis) in Monopterus albus. Reactive oxygen species (ROS) production, malondialdehyde (MDA) content, and protein carbonylation (PCO) increased significantly (p < 0.05) with the increasing metamifop concentration, resulting in oxidative damage. In the antioxidant system, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly (p < 0.05) in the 0.2 mg/L treatment group compared with the control group, and decreased in 0.4, 0.6, and 0.8 mg/L treatment groups. Glutathione peroxidase (GPX) activity decreased significantly (p < 0.05) with the increasing metamifop concentration. In the immune system, white cell number (WCN) increased significantly (p < 0.05) in 0.2 mg/L treatment group, and then decreased with the increase in metamifop concentration. Compared with control group, acid phosphatase (ACP) activity not only increased significantly (p < 0.05) in 0.2 mg/L treatment group, but also decreased significantly (p < 0.05) compared with the increase in metamifop concentration. However, in all treatment groups, alkaline phosphatase (AKP) activity was significantly lower than that in the control group (p < 0.05). In the inflammatory response, TNF-α and IL-1ß expression levels in the NF-κB signaling pathway decreased significantly (p < 0.05) with the increase in metamifop concentration, while IL-8 expression level in the same signaling pathway increased significantly (p < 0.05) in treatment groups. The expression levels of genes related to apoptosis showed that apoptosis was promoted after exposure to metamifop. The results of the present study show that metamifop induced oxidative damage via a high level of ROS production, and then inhibited or damaged the defense systems of M. albus.

Transcriptomic analysis of the antioxidant responses and immunomodulatory effects of dietary melatonin in red swamp crayfish (Procambarus clarkii).

This study aimed to investigate the effects of dietary melatonin (MT) levels on the antioxidant capacity, immunomodulatory, and transcriptional regulation of red swamp crayfish. Six experimental diets with different levels of MT (0, 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg diet) were fed to juvenile crayfish for 60 d. The transcriptome data of the control group and the group supplemented with dietary MT at 165.1 mg/kg were obtained using RNA-seq. In total, 3653 differentially expressed genes (2082 up-regulated and 1571 down-regulated) were identified. Pathways and genes related to antioxidant immune and growth performance were verified by qRT-PCR. The total hemocyte count, phagocytosis rate, and respiratory burst were significantly increased in the MT (165.1 mg/kg) group compared to the control group. Analysis of antioxidant immune-related enzymes in the hepatopancreas demonstrated that dietary MT (165.1 mg/kg) significantly increased activities of catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase and significantly decreased aspartate aminotransferase and alanine aminotransferase activity. At the transcriptional level, dietary MT up-regulated expression levels of genes associated with antioxidant immune and development, which included toll-like receptors, Crustin, C-type lectin, and so on. To conclude, MT could be used as a supplement in crayfish feed to increase immunity and antioxidant capacity and according to the broken line regression, the ideal MT concentration was the 159.02 mg/kg. Overall, this study demonstrates the role of melatonin in the antioxidant responses and immunomodulatory of Procambarus clarkii, laying the foundation for the development of melatonin as a feed additive in the aquaculture of this species.

Effects of abamectin on nonspecific immunity, antioxidation, and apoptosis in red swamp crayfish (Procambarus clarkii).

Abamectin, a pesticide of 16-member macrocyclic lactones, is widely applied in agriculture. As an important environmental factor, pesticides pose a great threat to defense system in aquatic animals. Procambarus clarkii is one of the most important economic aquatic animals in China. It is necessary to explore the defense mechanism of P. clarkii to abamectin. In this study, P. clarkii were exposed to 0, 0.2, 0.4, 0.6 mg/L abamectin, immune- and antioxidant-related enzymes activities, genes expression levels, and histological observations were used to analyze the defense capacity of P. clarkii to abamectin. With increasing abamectin concentration, reactive oxygen species (ROS) level and malondiadehyde (MDA) content increased significantly. Meanwhiile, acid phosphate (ACP), alkaline phosphatase (AKP) activities, total haemocyte counts (THC), and Crustin expression level decreased significantly, superoxide dismutase (SOD), catalase (CAT) activities, total antioxidant capacity (T-AOC), and GPX expression level also decreased significantly. Hematoxylin & eosin (H&E) observation showed that with increasing abamectin concentration, hepatopancreas were damaged, especially membrane structure. Through TUNEL observation and apoptosis-related genes (PcCTSL, Bcl-2, Bax, BI-1, PcCytc, caspase-3) expression levels, with increasing abamectin concentration, apoptosis rate increased significantly. Results of this study indicated that abamectin caused oxidative damage to P. clarkii, resulting in damage to defense system, suppression of nonspecific immunity and antioxidation, and promotion of apoptosis. It provided theoretical basis for healthy P. clarkii culture, and for further study on defense mechanism of aquatic animals to pesticides.

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.

Impact of Titanium Dioxide-Graphene Oxide (TiO2-GO) Composite Nanoparticle on the Juveniles of the Giant River Prawn, Macrobrachium rosenbergii: Physio-Biochemistry and Transcriptional Response.

Nanomaterials are used in many fields, resulting in inevitably releasing into the aquatic environment. The presence of nanomaterials, including TiO2-GO in the aquatic environment, can be toxic to aquatic organisms. However, few studies have focused on the effects of TiO2-GO composite nanoparticle on crustaceans. In the present study, the giant river prawn Macrobrachium rosenbergii juveniles were exposed to two concentrations of TiO2-GO composite nanoparticle (0.1 and 0.5 mg/L). The effects of TiO2-GO composite exposure on activities of digestive and antioxidant-related enzymes and expressions of growth and immune-related genes at the transcriptome were studied. The results showed that the survival rate and growth performance were not negatively affected by TiO2-GO composite at the two exposure levels. Nevertheless, exposure to TiO2-GO composite causes an effect on the activities of digestive and antioxidant enzymes in the juvenile prawns. The enzyme activities of CAT, SOD, GSH-Px, AMS, TPS, and LPS in the 0.1 mg/L TiO2-GO composite experimental group were markedly reduced than those in the control group. Additionally, the expression level of genes involved in growth and immunity was significantly affected by TiO2-GO composite. After exposure to the 0.1 mg/L TiO2-GO composite, the mRNA expression level of MSTN was significantly increased, but the level of EcR, Raptor, and CaBP was significantly decreased. However, the mRNA levels of the CTL, TLR, JAK, and STAT were significantly increased after exposure to the 0.5 mg/L concentration of TiO2-GO composite. Furthermore, to understand the molecular mechanism of M. rosenbergii under TiO2-GO composite exposure, RNA-Seq was employed to analyze the changes of the muscle and hepatopancreas transcriptome. Compared with the control group, we identified 5166 and 4784 differentially expressed genes (DEGs) in the muscle and hepatopancreas, respectively (p < 0.05). Based on gene ontology and KEGG analysis, significant differences were observed in the DEGs involved in activity and binding, metabolism, immune response, and environmental information processing. These results showed that exposure to TiO2-GO composite nanoparticle led to the changes of enzyme activity and gene expression, suggesting that TiO2-GO composite existing in aquatic environments would disrupt the physiology of M. rosenbergii. This study will serve as a foundation for subsequent research into the evaluation of nanomaterial toxicity on crustacean species.

Effects of titanium dioxide (TiO2)/activated carbon (AC) nanoparticle on the growth and immunity of the giant freshwater prawn, Macrobrachium rosenbergii: potential toxicological risks to the aquatic crustaceans.

Due to their unique physicochemical characteristics, nanomaterials exhibit many excellent properties and functions, leading to their applications in numerous fields. The large-scale production and widespread application of nanomaterials have inevitably resulted in their release into the environment, especially the water environment. Several studies have confirmed that exposure to nanomaterials can be toxic to aquatic organisms. However, few studies have focused on the effects of nanomaterial exposure on growth and immunity in crustaceans. In the present study, juvenile Macrobrachium rosenbergii were exposed to different concentrations of titanium dioxide (TiO2)/activated carbon (AC) composite nanomaterial (0.1 and 0.5 mg/L) for 45 days. The effects of nanoparticle exposure on digestion and antioxidant-related enzyme activities, as well as the expression of growth and immunity-related genes and signaling pathway, were evaluated. Our results show that in response to low concentration of TiO2/AC nanoparticle (0.1 mg/L), most of the enzyme activities related to digestion and antioxidation (TPS, LPS, AMS, SOD, and CAT) were diminished. On the contrary, the GSH-Px activity increased under the 0.1 mg/L group of TiO2/AC nanoparticle concentration. Additionally, the level of digestive and antioxidant enzyme activities we detected was increased when exposed to 0.5 mg/L TiO2/AC nanoparticle. By comparison to the expression level of growth-related genes in the control group, MSTN, CaBP, E75, Raptor, EcR, and EGF were significantly inhibited at 0.1 and 0.5 mg/L concentrations of TiO2/AC nanoparticle, whereas the expression level of genes (TLR, JAK, STAT, PPAF, ACP, and AKP) related to immunity was increased when exposed to different concentrations of TiO2/AC nanoparticle. Compared with the control group (0 mg/L concentration), 5166 DEGs were identified in the TiO2/AC nanoparticle group, and a large number of DEGs were involved in molting, energy metabolism, stress tolerance, and germ cell development. Moreover, KEGG analysis revealed that many DEGs were assigned into signaling pathways related to metabolic growth and immune stress. These results showed that exposure to TiO2/AC nanoparticle will result in the changes of enzyme activity and routine mRNA expression, suggesting that TiO2/AC nanoparticle which existed in aquatic environment might affect the physiology of M. rosenbergii. This study will provide significant information for the evaluation of nanomaterial toxicity on aquatic crustaceans.

RNA-seq Provides Novel Insights into Response to Acute Salinity Stress in Oriental River Prawn Macrobrachium nipponense.

The oriental river prawn Macrobrachium nipponense is an important aquaculture species in China, Vietnam, and Japan. This species could survive in the salinity ranging from 7 to 20 ppt and accelerate growth in the salinity of 7 ppt. To identify the genes and pathways in response to acute high salinity stress, M. nipponense was exposed to the acute high salinity of 25 ppt. Total RNA from hepatopancreas, gills, and muscle tissues was isolated and then sequenced using high-throughput sequencing method. Differentially expressed genes (DGEs) were identified, and a total of 632, 836, and 1246 DEGs with a cutoff of significant twofold change were differentially expressed in the hepatopancreas, gills, and muscle tissues, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome pathway enrichment analyses were conducted. These DEGs were involved in the GO terms of cellular process, metabolic process, membrane, organelle, binding, and catalytic activity. The DEGs of hepatopancreas and gill tissues were mainly enriched in PPAR signaling pathway, longevity regulating pathway, protein digestion and absorption, and the DEGs of muscle tissue in arginine biosynthesis, adrenergic signaling in cardiomyocytes, cardiac muscle contraction, and cGMP-PKG signaling pathway. Real-time PCR conducted with fifteen selected DEGs indicated high reliability of digital analysis using RNA-Seq. The results indicated that the M. nipponense may regulate essential mechanisms such as metabolism, oxidative stress, and ion exchange to adapt the alternation of environment, when exposed to acute high salinity stress. This work reveals the numbers of genes modified by salinity stress and some important pathways, which could provide a comprehensive insight into the molecular responses to high salinity stress in M. nipponense and further boost the understanding of the potential molecular mechanisms of adaptation to salinity stress for euryhaline crustaceans.

Transcriptome analysis of the post-larvae of giant freshwater prawn (Macrobrachium rosenbergii) after IAG gene knockdown with microRNA interference.

The insulin-like androgenic gland hormone gene (IAG) of crustaceans plays pivotal roles in the regulation of sex differentiation. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that function as post-transcriptional gene regulators. However, little information about the regulatory relationship between miRNA and Macrobrachium rosenbergii IAG (MrIAG) were exposed. In this study, we used the 3' untranslated region (UTR) of MrIAG to predict potential target sites of miRNAs. The results showed that miR-184 has one target site in the 3'UTR of MrIAG. Dual-luciferase report assay in vitro confirmed that miR-184 can significantly down-regulate MrIAG expression. Besides, we constructed mutant plasmids of 3'UTR of MrIAG. The result displayed that after co-transfection of mutant plasmids and miR-184 agomir, the activity of luciferase was not affected compared to the control. These results indicated that miR-184 could directly regulate MrIAG. In addition, we found that overexpression of miR-184 in M. rosenbergii can lead to significant changes in the transcription level of genes. Compared with control group, we identified 1510 differentially expressed genes (DEGs) in the miR-184 injection group. Some DEGs were involved in sex differentiation, gonad development, growth and molting were found. qRT-PCR verification was performed on eight DEGs randomly, and the results showed that the expression level of sex-, growth-, and metabolism-related genes changed significantly after MrIAG gene knockdown. Collectively, findings from this study suggest that miR-184, by mediating IAG expression, may be involved in many physiological processes in M. rosenbergii. The current study lays a basic understanding for short-term silencing of MrIAG with miR-184, and facilitates miRNA function analysis in M. rosenbergii in future.

Effects of cadmium on antioxidant and non-specific immunity of Macrobrachium nipponense.

As a non-essential and toxic element, cadmium poses an important threat to aquatic organisms and human food safety. In this study, the effects of cadmium on antioxidant and non-specific immunity of Macrobrachium nipponense were studied from the physiological and biochemical indexes, histology and expression of related genes. These results showed that low concentrations (0.01, 0.02 mg/L) of cadmium have a positive effect on the non-specific immunity of M. nipponense, but high concentration (0.04 mg/L) of cadmium could inhibit or even damage the non-specific immunity of M. nipponense. The cadmium could induce oxidative stress in M. nipponense, and M. nipponense actived the antioxidant defense system to deal with oxidative stress, but high concentration (0.04 mg/L) of cadmium could inhibit the antioxidant defense system of M. nipponense, leading to oxidative damage, and may induce apoptosis in severe case. At the same time, the results of histology showed that cadmium can damage the structure of gill and hepatopancreas tissues of M. nipponense. This study provides theoretical data for evaluating the influences of heavy metal cadmium on M. nipponense and the toxic mechanism of heavy metal cadmium.

Comparative transcriptome analysis of the gills and hepatopancreas from Macrobrachium rosenbergii exposed to the heavy metal Cadmium (Cd2+).

Heavy metal Cadmium (Cd2+) pollution has become a severe environmental problem for aquatic organisms. In crustaceans, gills (Gi) and hepatopancreas (Hp) play a vital role in the toxicology. However, in Macrobrachium rosenbergill, there are few researches about gill and hepatopancreases responding to Cd2+ stress at a molecular level. In this study, transcriptomic analysis was applied to characterize gene expression profiles of gills and hepatopancreas of M. rosenbergill after Cd2+ exposure for 0 h, 3 h and 3 d. Six cDNA libraries (Gi 0 h, Gi 3 h, Gi 3 d, Hp 0 h, Hp 3 h, and Hp 3 d) were constructed and a total of 66,676 transcripts and 48,991 unigenes were annotated. Furthermore, differentially expressed genes (DEGs) were isolated by comparing the Cd2+ treated time-point libraries (3 h and 3 d group) with the control library (0 h group). The results showed that most of the DEGs were down-regulated after Cd2+ exposure and the number of DEGs among gill groups were significantly higher than those among hepatopancreas groups. GO functional and KEGG pathway analysis suggested many key DEGs in response to the Cd2+ stress, such as metallothionein and Hemocyanin. Additionally, a total of six DEGs were randomly selected to further identify their expressional profile by qPCR. The results indicated that these DEGs were involved in the response to Cd2+. This comparative transcriptome provides valuable molecular information on the mechanisms of responding to Cd2+ stress in M. rosenbergii, which lays the foundation for further understanding of heavy metal stress.

Identifying Neuropeptide and G Protein-Coupled Receptors of Juvenile Oriental River Prawn (Macrobrachium nipponense) in Response to Salinity Acclimation.

Neuropeptides and their G protein-coupled receptors (GPCRs) from the central nervous system regulate the physiological responses of crustaceans. However, in crustaceans, our knowledge regarding GPCR expression patterns and phylogeny is limited. Thus, the present study aimed to analyze the eyestalk transcriptome of the oriental river prawn Macrobrachium nipponense in response to salinity acclimation. We obtained 162,250 unigenes after de novo assembly, and 1,392 and 1,409 differentially expressed genes were identified in the eyestalk of prawns in response to low and high salinity, respectively. We used combinatorial bioinformatic analyses to identify M. nipponense genes encoding GPCRs and neuropeptides. The mRNA levels of seven neuropeptides and one GPCR were validated in prawns in response to salinity acclimation using quantitative real-time reverse transcription polymerase chain reaction. A total of 148 GPCR-encoding transcripts belonging to three classes were identified, including 77 encoding GPCR-A proteins, 52 encoding GPCR-B proteins, and 19 encoding other GPCRs. The results increase our understanding of molecular basis of neural signaling in M. nipponense, which will promote further research into salinity acclimation of this crustacean.

Alternative splice variants and differential relative abundance patterns of vasa mRNAs during gonadal development in the Chinese mitten crab Eriocheir sinensis.

Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.

Identification and profiling of microRNAs during gonadal development in the giant freshwater prawn Macrobrachium rosenbergii.

As post-transcriptional regulators, microRNAs (miRNAs) play an important role in growth and reproductive processes. So far, there is limited information regarding crustacean miRNAs. To explore the potential role of miRNAs in the gonadal development of the prawn Macrobrachium rosenbergii, we constructed seven small RNA libraries from ovarian and testicular tissues at various stages using somatic tissue as the control. A total of 1,954 known and 129 novel miRNAs were retrieved. By comparing differentially expressed miRNAs (DEMs) between testes and ovaries, forty-one miRNAs were identified with sex-biased expression patterns, including 17 ovary-biased and 24 testis-biased patterns. Furthermore, the putative target genes of the sex-biased miRNAs, such as cyclin L1, mitogen-activated protein kinase 7 (MAPK 7), heat shock protein (HSP), and zinc finger protein, were significantly enriched in many reproduction-related pathways including the Gonadotropin-releasing hormone (GnRH) pathway, glycolysis, gluconeogenesis pathway, ovarian steroidogenesis, estrogen signaling pathway, MAPK pathway, Wnt pathway, and insulin signaling pathway, implicating potential regulatory roles of miRNAs in reproduction. These data aid in the further investigation of the mechanism of gonadal development and reproductive regulation mediated by miRNA in M. rosenbergii.

Transcriptome analysis of the brain of the Chinese mitten crab, Eriocheir sinensis, for neuropeptide abundance profiles during ovarian development.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

A second generation SNP and SSR integrated linkage map and QTL mapping for the Chinese mitten crab Eriocheir sinensis.

The Chinese mitten crab Eriocheir sinensis is the most economically important cultivated crab species in China, and its genome has a high number of chromosomes (2n = 146). To obtain sufficient markers for construction of a dense genetic map for this species, we employed the recently developed specific-locus amplified fragment sequencing (SLAF-seq) method for large-scale SNPs screening and genotyping in a F1 full-sib family of 149 individuals. SLAF-seq generated 127,677 polymorphic SNP markers, of which 20,803 valid markers were assigned into five segregation types and were used together with previous SSR markers for linkage map construction. The final integrated genetic map included 17,680 SNP and 629 SSR markers on the 73 linkage groups (LG), and spanned 14,894.9 cM with an average marker interval of 0.81 cM. QTL mapping localized three significant growth-related QTL to a 1.2 cM region in LG53 as well as 146 sex-linked markers in LG48. Genome-wide QTL-association analysis further identified four growth-related QTL genes named LNX2, PAK2, FMRFamide and octopamine receptors. These genes are involved in a variety of different signaling pathways including cell proliferation and growth. The map and SNP markers described here will be a valuable resource for the E. sinensis genome project and selective breeding programs.

The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts.

Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance.

A genome-wide association study in catfish reveals the presence of functional hubs of related genes within QTLs for columnaris disease resistance.

BACKGROUND: Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus). RESULTS: A genomic region on linkage group 7 was found to be significantly associated with columnaris resistance. Within this region, five have known functions in immunity, including pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on linkage groups 7, 12, and 14. The resistant genotypes on the QTLs of linkage groups 7 and 12 were found to be homozygous with both alleles being derived from channel catfish. The paralogs of the candidate genes in the suggestively associated QTL of linkage group 12 were found on the QTLs of linkage group 7. Many candidate genes on the four associated regions are involved in PI3K pathway that is known to be required by many bacteria for efficient entry into the host. CONCLUSION: The GWAS revealed four QTLs associated with columnaris resistance in catfish. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the associated QTLs on linkage groups 7 and 12 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway, suggesting its importance for columnaris resistance.