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Journal of Virology Feb 2024Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic...
Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including SNAP29, VAMP7, and the novel autophagosome SNARE protein Syx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of SNAP29 to competitively inhibit the binding of Syx12-Qa-SNARE with SNAP29-Qb-SNARE; this in turn disrupts the assembly of the Syx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.
Topics: Animals; Astacoidea; Autophagosomes; Autophagy; Qb-SNARE Proteins; SNARE Proteins; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; White spot syndrome virus 1
PubMed: 38189252
DOI: 10.1128/jvi.01408-23 -
Environmental Pollution (Barking, Essex... Mar 2024Isoprothiolane (IPT) and tricyclazole (TCZ) are widely used in rice farming and recently in combined rice-fish farming. However, co-cultured animals are affected by...
Risk assessment of pesticide compounds: IPT and TCZ cause hepatotoxicity, activate stress pathway and affect the composition of intestinal flora in red swamp crayfish (Procambarusclarkii).
Isoprothiolane (IPT) and tricyclazole (TCZ) are widely used in rice farming and recently in combined rice-fish farming. However, co-cultured animals are affected by these pesticides. To investigate the organismal effects and toxicity of pesticides, crayfish were exposed to 0, 1, 10, or 100 ppt TCZ or IPT for 7 days. Pesticide bioaccumulation, survival rate, metabolic parameters, structure of intestinal flora, and antioxidant-, apoptosis-, and HSP-related gene expression were determined. Pesticide exposure caused bioaccumulation of IPT or TCZ in the hepatopancreas and muscles of crayfish; however, IPT bioaccumulation was higher than that of TCZ. Both groups showed significant changes in hepatopancreatic serum biochemical parameters. Mitochondrial damage and chromosomal agglutination were observed in hepatopancreatic cells exposed to 100 ppt IPT or TCZ. IPT induced more significant changes in serum biochemical parameters than TCZ. The results of intestinal flora showed that Vibro, Flavobacterium, Anaerorhabdus and Shewanella may have potential for use as a bacterial marker of TCZ and IPT. Antioxidant-, apoptosis-, and HSP-related gene expression was disrupted by pesticide exposure, and was more seriously affected by IPT. The results suggest that IPT or TCZ induce hepatopancreatic cell toxicity; however, IPT or TCZ content in dietary crayfish exposed to 1 ppt was below the food safety residue standard. The data indicated that IPT exposure may be more toxic than TCZ exposure in hepatopancreas and intestines and toxicity of organism are alleviated by activating the pathway of stress-response, providing an understanding of pesticide compounds in rice-fish farming and food safety.
Topics: Animals; Antioxidants; Pesticides; Gastrointestinal Microbiome; Astacoidea; Risk Assessment; Chemical and Drug Induced Liver Injury; Thiazoles; Thiophenes
PubMed: 38185353
DOI: 10.1016/j.envpol.2024.123315 -
Fish & Shellfish Immunology Feb 2024The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the...
Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity.
The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-β. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs' expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.
Topics: Humans; Animals; Astacoidea; Amino Acid Sequence; Gene Expression Regulation; Immunity, Innate; RNA Interference; Arthropod Proteins; Mammals; Nuclear Proteins; Intracellular Signaling Peptides and Proteins
PubMed: 38168633
DOI: 10.1016/j.fsi.2023.109350 -
PloS One 2024Burrowing is a common trait among crayfish thought to help species deal with adverse environmental challenges. However, little is known about the microhabitat ecology of...
Burrowing is a common trait among crayfish thought to help species deal with adverse environmental challenges. However, little is known about the microhabitat ecology of crayfish taxa in relation to their burrows. To fill this knowledge gap, we assessed the availability of oxygen inside the crayfish shelter by series of in-vivo and in-silico modelling experiments. Under modeled condition, we found that, except for the entrance region of the 200 mm, a flooded burrow microenvironment became anoxic within 8 h, on average. Multiple 12-hour day-night cycles, with burrows occupied by crayfish for 12 h and empty for 12 h, were not sufficient for refreshing the burrow microenvironment. We then examined the degree to which crayfish species with different propensities for burrowing are tolerant of self-created anoxia. From these experiments, primary and secondary burrowers showed best and most consistent tolerance-exhibiting ≥ 64% survival to anoxia and 25-91% survival of ≥ 9 h at anoxia, respectively. Tertiary burrowers exhibited little to no tolerance of anoxia with 0-50% survival to anoxia and only one species exhibiting survival (2%) of ≥ 9 h at anoxia. Results suggest that moderate to strongly burrowing crayfish can quickly draw down the dissolved oxygen in burrow water but appear to have conserved a legacy of strong tolerance of anoxia from their monophyletic ancestors-the lobsters-whereas tertiary burrowers have lost (or never evolved) this ability.
Topics: Animals; Astacoidea; Decapoda; Water; Hypoxia; Ecology; Oxygen
PubMed: 38165988
DOI: 10.1371/journal.pone.0287888 -
Frontiers in Bioscience (Scholar... Dec 2023The mitochondrial genome is a powerful tool for exploring and confirming species identity and understanding evolutionary trajectories. The genus , which consists of...
Characterization of the Birstein & Vinogradov, 1934 (Decapoda: Astacidea) Mitochondrial Genome Using Genome Skimming and the Phylogenetic Implications within the Astacidea Infraorder.
BACKGROUND
The mitochondrial genome is a powerful tool for exploring and confirming species identity and understanding evolutionary trajectories. The genus , which consists of freshwater crayfish, is recognized for its evolutionary and morphological complexities. However, comprehensive genetic and mitogenomic data on species within this genus, such as , remain scarce, thereby necessitating an in-depth mitogenomic exploration to decipher its evolutionary position and validate its species identity.
METHODS
The mitochondrial genome of was obtained through shallow Illumina paired-end sequencing of total DNA, followed by hybrid assembly using both and reference-based techniques. Comparative analysis was performed using available mitochondrial genomes obtained from National Center for Biotechnology Information (NCBI). Additionally, phylogenetic analyses of 23 representatives from three families within the Astacidea infraorder were employed using the PhyloSuite platform for sequence management and phylogenetic preparation, to elucidate phylogenetic relationships via Bayesian Inference (BI), based on concatenated mitochondrial fragments.
RESULTS
The resulting genome, which spans 16,391 base pairs was investigated, revealing 13 protein-coding genes, rRNAs ( and ), 19 tRNAs, and a putative control region. Comparative analysis together with five other mitogenomes retrieved from GenBank unveiled regions that remained unread due to challenges associated with the genome skimming technique. Protein-coding genes varied in size and typically exhibited common start (ATG) and stop (TAA) codons. However, exceptions were noted in ND5 (start codon: GTG) and ND1 (stop codon: TAG). Landscape analysis was used to explore sequence variation across the five available mitochondrial genomes of .
CONCLUSIONS
Collectively, these findings reveal variable sites and contribute to a deeper understanding of the genetic diversity in this genus alongside the further development of species-specific primers for noninvasive monitoring techniques. The partitioned phylogenetic analysis of Astacidea revealed a paraphyletic origin of Asian cambarids, which confirms the data in recent studies based on both multilocus analyses and integrative approaches.
Topics: Humans; Animals; Phylogeny; Genome, Mitochondrial; Astacoidea; Bayes Theorem; Decapoda; Codon, Terminator
PubMed: 38163955
DOI: 10.31083/j.fbs1504015 -
International Journal of Biological... Feb 2024In biological evolution, gene duplication (GD) generates new genes to facilitate new functions. C-type lectins (CTLs) in crayfish have been extended by GD to expand...
In biological evolution, gene duplication (GD) generates new genes to facilitate new functions. C-type lectins (CTLs) in crayfish have been extended by GD to expand their family members. In this study, four CTL genes generated by GD were identified from Procambarus clarkii (PcLec1-4). Among these four genes, PcLec1 can also generate new isoforms with different numbers of tandem repeats through DNA slip mispairing. PcLec1-4 was widely expressed in multiple tissues. The expression levels of PcLec1-4 were upregulated in the intestine of P. clarkii upon white spot syndrome virus (WSSV) challenge at multiple time points. Further analysis indicated that GATA transcription factor regulated PcLec1-4 expression. RNA interference and recombinant PcLec1-4 protein injection experiments suggested that PcLec1-4 promoted the expression of calreticulin (PcCRT) and negatively regulated the expression of antimicrobial peptides, thereby promoting WSSV replication. This study contributes to the understanding of the function of CTLs produced by GD during WSSV invasion in crustaceans.
Topics: Animals; Calreticulin; White spot syndrome virus 1; Virus Replication; Astacoidea; Lectins, C-Type
PubMed: 38151079
DOI: 10.1016/j.ijbiomac.2023.128996 -
Genes Dec 2023is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 () gene has been found...
is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 () gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the gene in the gonad development of , we designed CDS-specific primers for the () gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of in various tissues were detected using qRT-PCR and in situ hybridization. The effects of knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of in the testis was significantly higher (>40 fold) than that in the ovary ( < 0.01). The in situ hybridization results showed that was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the gene using dsRNA, the expression of in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group ( < 0.05) after 10 days of cultivation following knockdown. The expression levels of the and genes, which are related to gonad development, decreased significantly after gene interference. The results suggest that the gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of .
Topics: Male; Animals; Female; Astacoidea; Phylogeny; Amino Acid Sequence; Polymerase Chain Reaction; Cloning, Molecular
PubMed: 38137012
DOI: 10.3390/genes14122190 -
Comparative Biochemistry and... Feb 2024Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be...
Temperature and hypoxia trigger developmental phenotypic plasticity of cardiorespiratory physiology and growth in the parthenogenetic marbled crayfish, Procambarus virginalis Lyko, 2017.
Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be experimentally difficult. Environmental variables must be carefully controlled and then interindividual genetic differences ruled out as sources of phenotypic variation. We investigated phenotypic variability of cardiorespiratory physiology as well as biometric traits in the parthenogenetically-reproducing marbled crayfish Procambarus virginalis Lyko, 2017, all offspring being genetically identical clones. Populations of P. virginalis were reared from eggs tank-bred at four different temperatures (16, 19, 22 and 25 °C) or two different oxygen levels (9.5 and 20 kPa). Then, at Stage 3 and 4 juvenile stages, physiological (heart rate, oxygen consumption) and morphological (carapace length, body mass) variables were measured. Heart rate and oxygen consumption measured at 23 °C showed only small effects of rearing temperature in Stage 3 juveniles, with larger effects evident in older, Stage 4 juveniles. Additionally, coefficients of variation were calculated to compare our data to previously published data on P. virginalis as well as sexually-reproducing crayfish. Comparison revealed that carapace length, body mass and heart rate (but not oxygen consumption) indeed showed lower, yet notable coefficients of variation in clonal crayfish. Yet, despite being genetically identical, significant variation in their morphology and physiology in response to different rearing conditions nonetheless occurred in marbled crayfish. This suggests that epigenetically induced phenotypic variation might play a significant role in asexual but also sexually reproducing species.
Topics: Animals; Astacoidea; Temperature; Parthenogenesis; Adaptation, Physiological; Hypoxia
PubMed: 38113959
DOI: 10.1016/j.cbpa.2023.111562 -
Fish & Shellfish Immunology Feb 2024This study aimed to evaluate the potential benefits of chitosan oligosaccharide (COS) on red claw crayfish (Cherax quadricarinatus) and explore its underlying...
This study aimed to evaluate the potential benefits of chitosan oligosaccharide (COS) on red claw crayfish (Cherax quadricarinatus) and explore its underlying mechanisms. The crayfish were randomly divided into six groups, and the diets were supplemented with COS at levels of 0 (C0), 0.2 (C1), 0.4 (C2), 0.6 (C3), 0.8 (C4), and 1 (C5) g kg. Treatment with COS significantly improved the growth performance of the crayfish with a higher weight gain rate (WGR) and specific growth rate (SGR) in the C2 group compared to the C0 group. Additionally, the content of crude protein in the crayfish muscles in the C1 group was significantly higher than that of the C0 group. Regarding non-specific immunity, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and alkaline phosphatase (AKP), and the levels of expression of the genes related to immunity (SOD; anti-lipopolysaccharide factor [ALF]; thioredoxin1 [Trx1]; C-type lysozyme, [C-LZM]; and GSH-Px) in the hepatopancreas and hemolymph increased significantly (P < 0.05) after supplementation with 0.4 g kg of COS, while the content of malondialdehyde (MDA) decreased (P < 0.05). The survival rate of C. quadricarinatus increased (P < 0.05) in the C2, C3, C4, and C5 groups after the challenge with Aeromonas hydrophila. This study found that COS has the potential to modulate the composition of the intestinal microbiota and significantly reduce the abundance of species of the phylum Proteobacteria and the genera Aeromonas and Vibrio in the gut of C. quadricarinatus, while the abundance of bacteria in the phylum Firmicutes and the genus Candidatus_Hepatoplasma improved significantly. This study suggests that the inclusion of COS in the diet of C. quadricarinatus can enhance growth, boost immunity, and increase resistance to infection with A. hydrophila, especially when supplemented at 0.4-0.8 g kg.
Topics: Animals; Astacoidea; Chitosan; Gastrointestinal Microbiome; Diet; Dietary Supplements; Superoxide Dismutase; Oligosaccharides; Immunity, Innate; Animal Feed
PubMed: 38104697
DOI: 10.1016/j.fsi.2023.109288 -
Developmental and Comparative Immunology Apr 2024Dual oxidase (Duox) a member of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family can induce the production of reactive oxygen species (ROS). In...
Dual oxidase (Duox) a member of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family can induce the production of reactive oxygen species (ROS). In vertebrates, the duox gene was indicated to be associated with the mucosal immunity. The roles of the duox gene in invertebrates were mainly studied in insects for the function of maintaining intestinal flora balance. In recent years, some studies have reported that Duox is involved in regulating the production of ROS and plays an important role in defending against the intestinal pathogen infection. However, the molecular mechanism has not been fully illuminated. In this study, a duox 2 involved in the production of HO was identified for the first time in P. clarkii. Mature Pc-Duox 2 is a 7-transmembrane protein molecule that includes PHD, FAD, and NAD domains. Pc-duox 2 was mainly expressed in hemocytes and intestinal tissue. Its expression levels were obviously upregulated after intramuscular or oral infection with V. harveyi. In the RNAi assay, the upregulated trends of HO and total ROS levels in crayfish intestine were significantly suppressed when Pc-duox 2 was knocked down. Compared with the slightly affected SOD activity, the upregulated CAT activity was suppressed more obviously in the crayfish intestine. Furthermore, Pc-duox 2 had an important effect on the maintenance of the structural stability of crayfish the intestine. Further research revealed that the knockdown of Pc-duox 2 could cause an obvious suppression in the upregulated levels of Toll signalling pathway-related genes, including Pc-toll 1, Pc-toll 3, Pc-dorsal, Pc-ALF 5, Pc-crustin 1, and Pc-lysozyme. Ultimately, these changes triggered the accelerated death of crayfish. Overall, we speculated that Pc-duox 2 played an important role in antibacterial innate immunity in the crayfish intestine by regulating the total ROS level.
Topics: Animals; Dual Oxidases; Amino Acid Sequence; Astacoidea; Reactive Oxygen Species; Hydrogen Peroxide; Immunity, Innate; Intestines; Anti-Bacterial Agents
PubMed: 38101716
DOI: 10.1016/j.dci.2023.105116