Single-nuclei RNA-seq reveals skin cell responses to Aeromonas hydrophila infection in Chinese longsnout catfish Leiocassis longirostris.

As the primary natural barrier that protects against adverse environmental conditions, the skin plays a crucial role in the innate immune response of fish, particularly in relation to bacterial infections. However, due to the diverse functionality and intricate anatomical and cellular composition of the skin, deciphering the immune response of the host is a challenging task. In this study, single nuclei RNA-sequencing (snRNA-seq) was performed on skin biopsies obtained from Chinese longsnout catfish (Leiocassis longirostris), comparing Aeromonas hydrophila-infected subjects to healthy control subjects. A total of 19,581 single nuclei cells were sequenced using 10x Genomics (10,400 in the control group and 9,181 in the treated group). Based on expressed unique transcriptional profiles, 33 cell clusters were identified and classified into 12 cell types including keratinocyte (KC), fibroblast (FB), endothelial cells (EC), secretory cells (SC), immune cells, smooth muscle cells (SMC), and other cells such as pericyte (PC), brush cell (BC), red blood cell (RBC), neuroendocrine cell (NDC), neuron cells (NC), and melanocyte (MC). Among these, three clusters of KCs, namely, KC1, KC2, and KC5 exhibited significant expansion after A. hydrophila infection. Analysis of pathway enrichment revealed that KC1 was primarily involved in environmental signal transduction, KC2 was primarily involved in endocrine function, and KC5 was primarily involved in metabolism. Finally, our findings suggest that neutrophils may play a crucial role in combating A. hydrophila infections. In summary, this study not only provides the first detailed comprehensive map of all cell types present in the skin of teleost fish but also sheds light on the immune response mechanism of the skin following A. hydrophila infection in Chinese longsnout catfish.

PacBio single-molecule long-read sequencing provides new insights into the complexity of full-length transcripts in oriental river prawn, macrobrachium nipponense.

BACKGROUND: Oriental river prawn (Macrobrachium nipponense) is one of the most dominant species in shrimp farming in China, which is a rich source of protein and contributes to a significant impact on the quality of human life. Thus, more complete and accurate annotation of gene models are important for the breeding research of oriental river prawn. RESULTS: A full-length transcriptome of oriental river prawn muscle was obtained using the PacBio Sequel platform. Then, 37.99 Gb of subreads were sequenced, including 584,498 circular consensus sequences, among which 512,216 were full length non-chimeric sequences. After Illumina-based correction of long PacBio reads, 6,599 error-corrected isoforms were identified. Transcriptome structural analysis revealed 2,263 and 2,555 alternative splicing (AS) events and alternative polyadenylation (APA) sites, respectively. In total, 620 novel genes (NGs), 197 putative transcription factors (TFs), and 291 novel long non-coding RNAs (lncRNAs) were identified. CONCLUSIONS: In summary, this study offers novel insights into the transcriptome complexity and diversity of this prawn species, and provides valuable information for understanding the genomic structure and improving the draft genome annotation of oriental river prawn.

Fish female-biased gene cyp19a1a leads to female antiviral response attenuation between sexes by autophagic degradation of MITA.

From insects to mammals, both innate and adaptive immune response are usually higher in females than in males, with the sex chromosome and hormonal differences considered the main reasons. Here, we report that zebrafish cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a), an autosomal gene with female-biased expression, causes female fish to exhibit a lower antiviral response. First, we successfully constructed an infection model by intraperitoneal injection of spring viremia of carp virus (SVCV) into zebrafish (Danio rerio) and Carassius auratus herpesvirus (CaHV) in gibel carp (Carassius gibelio). Specifically, female fish were more vulnerable to viral infection than males, accompanied by a significantly weaker interferon (IFN) expression. After screening several candidates, cyp19a1a, which was highly expressed in female fish tissues, was selected for further analysis. The IFN expression and antiviral response were significantly higher in cyp19a1a-/- than in cyp19a1a+/+. Further investigation of the molecular mechanism revealed that Cyp19a1a targets mediator of IRF3 activation (MITA) for autophagic degradation. Interestingly, in the absence of MITA, Cyp19a1a alone could not elicit an autophagic response. Furthermore, the autophagy factor ATG14 (autophagy-related 14) was found interacted with Cyp19a1a to either promote or attenuate Cyp19a1a-mediated MITA degradation by either being overexpressed or knocked down, respectively. At the cellular level, both the normal and MITA-enhanced cellular antiviral responses were diminished by Cyp19a1a. These findings demonstrated a sex difference in the antiviral response based on a regulation mechanism controlled by a female-biased gene besides sex chromosome and hormonal differences, supplying the current understanding of sex differences in fish.

Two Duplicated Ptpn6 Homeologs Cooperatively and Negatively Regulate RLR-Mediated IFN Response in Hexaploid Gibel Carp.

Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 (ptpn6) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp (Carassius gibelio) ptpn6 homeologs (Cgptpn6-A and Cgptpn6-B), each of which had three alleles with high identities. Then, relative to Cgptpn6-B, dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that CgSHP1-A (encoded by the Cgptpn6-A gene) and CgSHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of CaTBK1-induced phosphorylation of CaMITA shared by CgSHP1-A and CgSHP1-B, and the autophagic degradation of CaMITA exclusively by CgSHP1-A. Meanwhile, the data support that CgSHP1-A and CgSHP1-B have sub-functionalized and that CgSHP1-A overwhelmingly dominates CgSHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates.

Divergent Antiviral Mechanisms of Two Viperin Homeologs in a Recurrent Polyploid Fish.

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs (Cgviperin-A and Cgviperin-B) in auto-allo-hexaploid gibel carp (Carassius gibelio). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both CgViperin-A and CgViperin-B were found to interact with crucian carp (C. auratus) herpesvirus (CaHV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, CgViperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in CgViperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of CgViperin-A and CgViperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of CgViperin-A and CgViperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes.

Differential interferon system gene expression profiles in susceptible and resistant gynogenetic clones of gibel carp challenged with herpesvirus CaHV.

Interferon (IFN) system plays a vital role in the first line of defense against viruses. In this study, we first identified multiple transcripts of 15 IFN system genes, including PRRs (TLR2, TLR3, RIG-I, and LGP2), PRR-mediated IFN signal pathway (MyD88, MITA, and MAVS), IFN regulatory factors (IRF1, IRF3, IRF7, and IRF9), IFNs (IFNφ1 and IFNφ3), and ISGs (Mx and viperin), and one transcript of TLR9 in de novo transcriptome assembly data of gibel carp head-kidney. Multiple nucleotide alignments and phylogenetic analysis of common region showed that the transcripts of every of the 15 IFN system genes were classified into two homologs with distinctly divergent sequences, indicating that hexaploid gibel carp may be an allopolyploid. During Carassius auratus herpesvirus (CaHV) infection, gibel carp resistant clone H significantly suppressed CaHV replication with markedly less viral loads than those in highly susceptible clone A+ and moderately resistant clone F. Then, qPCR analyses were performed to reveal their differential and dynamic expression changes during CaHV infection in head kidney, spleen and liver among three gibel carp gynogenetic clones. Through qPCR and hierarchical clustering analysis, 8 genes, such as RIG-Is, LGP2s, IRF1-B, IRF3s, IRF7s, IRF9-B, Mxs, and viperins, were identified as candidate resistant-related genes. They remarkably increased their expression in immune tissues of three clones after CaHV infection. Significantly, the up-regulation folds of these genes in clone A+, F and H were related to their resistance ability to CaHV, progressively increasing from susceptible clone to resistant clone at 1 dpi. The positive correlation to the resistance ability suggested that resistant clone H immediately triggered stronger IFN response. IFNφ3 showed a different dynamic change and was sharply induced in moderately resistant clone F at 3 dpi. The other 5 IFN system genes (TLR2, TLR3, TLR9, MyD88, and MITA) maintained a low expression level after CaHV challenge. Interestingly, the A or B copies/homologs of almost these IFN system genes exhibited differential transcript abundance in immune tissue after CaHV challenge, suggesting A or B homologs might occur dominant or biased expression of homeologs during gibel carp evolution. These data provide candidate resistant-related genes for disease-resistance breeding of gibel carp.

Differential expression and functional diversification of diverse immunoglobulin domain-containing protein (DICP) family in three gynogenetic clones of gibel carp.

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A+, F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs.

Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes.

BACKGROUND: Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. RESULTS: To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A+, candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A+, F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A+ and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A+. In contrast to strong immune defense in resistant clone H, susceptible clone A+ showed remarkable up-regulation of genes related to apoptosis or death, indicating that clone A+ failed to resist virus offensive and evidently induced apoptosis or death. CONCLUSIONS: Our study is the first attempt to screen distinct resistances and immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.