Characterization of T-cell receptors and immunoglobulin heavy chains loci and identification of T/B cell clusters in teleost.

Bacterial disease is one of the important factors leading to economic losses in the turbot (Scophthalmus maximus) cultivation industry. T lymphocytes are major components of cellular immunity, whereas B lymphocytes produce immunoglobulins (Ig) that are key elements of humoral immune responses against infection. However, the genomic organization of genes encoding T-cell receptors (TCR) and immunoglobulin heavy chains (IgHs) in turbot remains largely unknown. In this study, abundant full-length transcripts of TCRs and IgHs were sequenced by Isoform-sequencing (Iso-seq), and we investigated and annotated the V, D, J and C gene loci of TCRα, TCRß, IgT, IgM and IgD in turbot. Furthermore, through single-cell RNA sequencing (scRNA-seq) of blood leukocytes, we confirmed that these identified TCRs and IgHs were highly expressed in T/B cell clusters, respectively. Meanwhile, we also identified the IgM+IgD+ B and IgT+ B cells with differential gene expression profiles and potential functions. Taken together, our results provide a comprehensive understanding of TCRs and IgHs loci in turbot, which will contribute to evolutionary and functional characterization of T and B lymphocytes in teleost.

Single-Cell Transcriptomic Analysis Reveals Neutrophil as Orchestrator during ß-Glucan-Induced Trained Immunity in a Teleost Fish.

Trained immunity defines long-term memory of innate immunity based on transcriptional, epigenetic, and metabolic modifications of myeloid cells, which are characterized by elevated proinflammatory responses toward homologous or heterologous secondary stimuli in mammals. However, the evidence of trained immunity-associated immune cells and its molecular mechanism in teleost fish remains largely unknown. In this study, we established a trained immunity activation model in turbot (Scophthalmus maximus) and found that administration with ß-glucan induces protection against a bacterial infection. Through single-cell RNA sequencing to annotate 14 clusters of innate and adaptive immune cells, as well as two clusters of blood cells, from head kidney and spleen, respectively, we characterized that neutrophil displays cardinal features of trained immunity by analyzing the expression abundance of trained immunity database-related genes at the single-cell level. Subsequently, through establishing an in vivo training and in vitro neutrophil challenge model, we found that the trained neutrophils exhibit a significant elevation of the IL-1R signaling pathway after Edwardsiella piscicida infection. Furthermore, inhibition of neutrophil's IL-1R signaling pathway through anakinra treatment impaired the heightened production of reactive oxygen, nitrogen species, lactate, as well as the neutrophil extracellular traps formation and bacterial killing ability. Taken together, these findings characterized neutrophil as the orchestrator to express features of trained immunity, and revealed that the IL-1R signaling pathway plays a critical role in induction of trained immunity for bacterial clearance in teleost fish.

Iso-Seq assembly and functional annotation of full-length transcriptome of turbot (Scophthalmus maximus) during bacterial infection.

Turbot (Scophthalmus maximus) is a flatfish, which is not only important in mariculture worldwide with the unique characteristic of body asymmetry, but also as an economically important species in aquaculture. Herein, we performed the first full-length transcriptome sequencing of turbot during the bacterial infection. A total of 307.1 Gb raw reads were obtained and processed with Iso-Seq, generating 187,509 high-quality redundant transcripts with an average length of 3005 base pairs. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis identified 81.5% complete BUSCOs and only 1.7% fragmented BUSCOs, suggesting a transcript structural completeness and functional diversity of this transcriptome. Moreover, the redundant transcripts were collapsed and compared to ENSEMBL reference with Cupcake and SQANTI3. Among 60,476 collapsed transcripts, we identified 12,059 annotated and 1684 novel genes. 42,956 (71.1%) transcripts provided new evidence for splice junctions identification. Furthermore, the untranslated region (UTR) identification was also benefited from the transcriptome. The open read frames prediction was conducted with PASApipeline. 42,118 transcripts were assigned with known function by aligning against Swiss-Prot or functional domain prediction. Taken together, the full-length transcriptome built in this study could provide important resources for immunologic research on turbot.

Identification and characterization of FtsH mediating in vivo colonization and stress adaptation in the fish pathogen Edwardsiella piscicida.

Edwardsiella piscicida is an important pathogenic enteric bacterium of fish. FtsH is a unique membrane-anchored AAA + protease that regulates protein homeostasis in bacteria. In cooperation with modulators HflK and HflC, FtsH is essential in enteric bacteria and controls the response to environmental stresses. Here, we used in vivo pattern analysis of conditional essentiality (PACE) and identified that ftsH and hflK/C were associated with impaired in vivo colonization in Edw. piscicida and attenuated internalization ability of ZF4 cells. The ftsH mutant displayed increased survival during prolonged treatment of starvation and high osmotic stresses in Edw. piscicida. Further analysis showed that the disruption of ftsH resulted in the overproduction of the established substrate LpxC, which is responsible for the synthesis of LPS (lipopolysaccharide), as well as the substrate YfgM, which is involved in high osmolality tolerance during stationary phase. However, the inconsistency in the abilities of the ftsH and hflK/C mutants to achieve YfgM-based osmotic resistance indicated that there might be multiple, while distinctive, pathways controlled by FtsH and the associated modulator proteins HflK/C. This investigation revealed the unique functions of FtsH and its modulator HflK/C in Edw. piscicida.

Characterization of the complete chloroplast genome of sugar maple (Acer saccharum).

Acer saccharum is one ecologically and economically important tree species cultivated widely across the world. In this study we generated the complete chloroplast (cp) genome of A. saccharum via genome-skimming method. The assembled genome is 155,684 base-pairs (bp) in size, with one large single copy region of 85,393 bp and one small single copy region of 18,033 bp separated by two inverted repeats of 26,129 bp. The genome contains a total of 133 genes, including 85 protein-coding genes, 8 rRNAs and 40 tRNAs. Furthermore, phylogenomic estimation strongly supported A. saccharum as a distinct lineage within the monophyletic Acer.