Baohong Xu’s research while affiliated with Hunan Agricultural University and other places

The hybrid F1 offspring of Ctenopharyngodon idella (♂) and Squaliobarbus curriculus (♀) exhibit heterosis in disease resistance and also show abnormal sex differentiation. To understand the mechanism behind gonadal differentiation in the hybrid F1, we analyzed the transcriptomes of C. idella, S. curriculus, and the hybrid F1; screened for genes related to gonad development in these samples; and measured their expression levels. Our results revealed that compared to either C. idella or S. curriculus, the gene expressions in most sub-pathways of the SNARE interactions in the vesicular transport pathway in the hypothalamus, pituitary, and gonadal tissues of their hybrid F1 offspring were significantly up-regulated. Furthermore, insufficient transcription of genes involved in oocyte meiosis may be the main reason for the insufficient reproductive ability of the hybrid F1 offspring. Through transcriptome screening, we identified key molecules involved in gonad development, including HSD3B7, HSD17B1, HSD17B3, HSD20B2, CYP17A2, CYP1B1, CYP2AA12, UGT2A1, UGT1A1, and FSHR, which showed significant differences in expression levels in the hypothalamus, pituitary, and gonads of these fish. Notably, the expression levels of UGT1A1 in the gonads of the hybrid F1 were significantly higher than those in C. idella and S. curriculus. These results provide a scientific basis for further research on the gonadal differentiation mechanism of hybrid F1 offspring.

Background The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp. Results Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression. Conclusions These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts.

The inconsistency between traditional morphological taxonomy and molecular phylogenetic data is a major issue that puzzles the study of fish classification and evolution. Although mitochondrial genes are commonly used in phylogenetic analyses to compare fish species, the mitochondrial evolution and diversity of Schistura are still not well understood. To better understand the evolution of Schistura, we sequenced the mitochondrial genome of Schistura fasciolata and compared it with other species of Schistura. A 16,588 bp circular mitochondrial genome of S. fasciolata was obtained and it contains 13 protein-coding, 22 transfer RNA, and two ribosomal RNA genes, and a non-coding control region. The gene arrangement in the mitochondrial genomes of all Schistura species was consistent. However, we also found that S. fasciolata was not monophyletic. Although mitochondrial genes can be effectively used for Schistura species identification, they may not be suitable for inferring the evolutionary process of Schistura species. These results provide support for the use of mitochondrial genes in identifying Schistura species, and also serve as a warning against mistakenly using them to evaluate the evolution process of Schistura species.

Grass carp (Ctenopharyngodon idella) and barbel chub (Squaliobarbus curriculus)—both Leuciscinae subfamily species—demonstrate differences in grass carp reovirus (GCRV) infection resistance. We infected barbel chubs with type II GCRV and subjected their liver, spleen, head kidney, and trunk kidney samples to investigate anti-GCRV immune mechanisms via RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). We identified 139, 970, 867, and 2374 differentially expressed genes (DEGs) in the liver, spleen, head kidney, and trunk kidney, respectively. Across all four tissues, gene ontology analysis revealed significant immune response-related DEG enrichment, and the Kyoto Encyclopedia of Genes and Genomes analysis revealed pattern recognition receptor (PRR) and cytokine-related pathway enrichment. We noted autophagy pathway enrichment in the spleen, head kidney, and trunk kidney; apoptosis pathway enrichment in the spleen and trunk kidney; and complement- and coagulation-cascade pathway enrichment in only the spleen. Comparative transcriptome analysis between GCRV-infected barbel chubs and uninfected barbel chubs comprehensively revealed that PRR, cytokine-related, complement- and coagulation-cascade, apoptosis, and autophagy pathways are potential key factors influencing barbel chub resistance to GCRV infection. qRT-PCR validation of 11 immune-related DEGs confirmed our RNA-seq data’s accuracy. These findings provide a theoretical foundation and empirical evidence for the understanding of GCRV infection resistance in barbel chub and hybrid grass carp–barbel chub breeding.

The ancestors of chemokines originate in the most primitive of vertebrates, which has recently attracted great interest in the immune functions and the underlying mechanisms of fish chemokines. In the current study, we identified an evolutionarily conserved chemokine, CiCXCL13, from a teleost fish, grass carp. CiCXCL13 was characterized by a typical SCY (small cytokine CXC) domain and four cysteine residues (C34, C36, C61, C77), with the first two cysteines separated by a random amino acid residue, although it shared 24.2–54.8% identity with the counterparts from other vertebrates. CiCXCL13 was an inducible chemokine, whose expression was significantly upregulated in the immune tissues of grass carps after grass carp reovirus infection. CiCXCL13 could bind to the membrane of grass carp head kidney leukocytes and promote cell migration, NO release, and the expression of >15 inflammatory cytokines, including IL-1β, TNF-α, IL-10 and TGF-β1, thus regulating the inflammatory response. Mechanistically, CiCXCL13 interacted with its evolutionarily conserved receptor CiCXCR5 and activated the Akt–NF-κB and p38–AP-1 pathways, as well as a previously unrevealed p38–NF-κB pathway, to efficiently induce inflammatory cytokine expression, which was distinct from that reported in mammals. Zebrafish CXCL13 induced inflammatory cytokine expression through Akt, p38, NF-κB, and AP-1 as CiCXCL13. Meanwhile, the CiCXCL13–CiCXCR5 axis–mediated inflammatory activity was negatively shaped by grass carp atypical chemokine receptor 2 (CiACKR2). The present study is, to our knowledge, the first to comprehensively define the immune function of CXCL13 in inflammatory regulation and the underlying mechanism in teleosts, and it provides a valuable perspective on the evolution and biology of fish chemokines.

Complement factor I (CFI), a complement inhibitor, is well known for regulating the complement system activation by degrading complement component 3b (C3b) in animal serum, thus becoming involved in innate defense. Nevertheless, the functional mechanisms of CFI in the complement system and in host-pathogen interactions are far from being clarified in teleost fish. In the present study, we cloned and characterized the CFI gene, CiCFI, from grass carp (Ctenopharyngodon idella) and analyzed its function in degrading serum C3b and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiCFI was found to be 2121 bp, encoding 706 amino acids with a molecular mass of 79.06 kDa. The pairwise alignments showed that CiCFI shared the highest identity (66.9%) with CFI from Carassius gibelio and the highest similarity (78.7%) with CFI from Danio rerio. The CiCFI protein was characterized by a conserved functional core Tryp_SPc domain with the catalytic triad and substrate binding sites. Phylogenetic analysis indicated that CiCFI and the homologs CFIs from other teleost fish formed a distinct evolutionary branch. Similar with the CFIs reported in mammals, the recombinant CiCFI protein could significantly reduce the C3b content in the serum, demonstrating the conserved function of CiCFI in the complement system in the grass carp. CiCFI mRNA and protein showed the highest expression level in the liver. After GCRV infection, the mRNA expressions of CiCFI were first down-regulated, then up-regulated, and then down-regulated to the initial level, while the protein expression levels maintained an overall downward trend to the late stage of infection in the liver of grass carps. Unexpectedly, the protein levels of CiCFI were also continuously down-regulated in the serum of grass carps during GCRV infection, while the content of serum C3b proteins first increases and then returns to the initial level, suggesting a distinct role of CiCFI in regulating complement activation and fish-virus interaction. Combining our previous results that complement factor D, a complement enhancer, shows continuously up-regulated expression levels in grass carps during GCRV infection, and this study may provide the further essential data for the full picture of complex complement regulation mechanism mediated by Df and CFI of the grass carp during pathogen infection.

Interaction between the chemokine receptor XCR1 and its ligand is closely related to the immune function in animals; however, there are only a few reports on role of XCR1 in the immune system of fish. We aimed to analyze the expression of XCR1 in various organs or tissues of grass carp before and after Grass Carp Reovirus (GCRV) infection to better understand the function of XCR1 in resistance to GCRV infection. We cloned and sequenced the cDNA of grass carp XCR1 and analyzed the molecular structure of XCR1 based its amino acid sequence. Further, we analyzed the relative expression levels of XCR1 in different organs or tissues of male parent grass carp with GCRV resistance (P1) and their first-generation offspring (F1) before and after GCRV infection. Our results show that the total length of cDNA of the grass carp XCR1 gene is 1659 bp and encodes 365 amino acids. XCR1 contains seven conserved transmembrane helical domains. The homologous tertiary structure of XCR1 is similar to its homologs in other species. After artificial GCRV infection, there were significant differences in the expression of the grass carp XCR1 gene in different tissues, at different time points, and between P1 and F1 fish. These results will contribute to our understanding of the role of XCR1 in fish immune responses and contribute to the development of GCRV-resistant grass carp.

Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, CiDf, from grass carp (Ctenopharyngodon idella) and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiDf was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. CiDf harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that CiDf shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami. Phylogenetic analysis indicated that CiDf and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the CiDf gene structure is characterized by four introns and five exons. The incubation of recombinant CiDf protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of CiDf in the AP in promoting C3 cleavage, similar to Dfs in mammals. CiDf mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of CiDf mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of CiDf and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp C. idella.