Molecular characterization, expression analysis and function identification of Pf_IL-12p35, Pf_IL-23p19 and Pf_IL-12p40 genes in yellow catfish (Pelteobagrus fulvidraco).

The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.

Molecular Characterization of Nine TRAF Genes in Yellow Catfish (Pelteobagrus fulvidraco) and Their Expression Profiling in Response to Edwardsiella ictaluri Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.

Genome-wide identification of genes probably relevant to the adaptation of schizothoracins (Teleostei: Cypriniformes) to the uplift of the Qinghai-Tibet Plateau.

BACKGROUND: Molecular adaptation to the severe environments present during the uplift of the Qinghai-Tibet Plateau has attracted the attention of researchers. The divergence of the three specialization groups of schizothoracins (Primitive, Specialized and Highly Specialized) may correspond to the three phases of plateau uplift. Based on the transcripts of representative species of the three specialized groups and an outgroup, genes in schizothoracins that may have played important roles during the adaptation to new environments were investigated. RESULTS: The contigs of Gymnodiptychus dybowskii and Schizothorax pseudaksaiensis were compared with those of Gymnocypris przewalskii ganzihonensis and the outgroup Sinocyclocheilus angustiporus, and 5,894 ortholog groups with an alignment length longer than 90 nt after deleting gaps were retained. Evolutionary analyses indicated that the average evolutionary rate of the branch leading to the Specialized group was faster than that of the branch leading to the Highly Specialized group. Moreover, the numbers of gene categories in which more than half of the genes evolved faster than the average values of the genome were 117 and 15 along the branches leading to the Specialized and Highly Specialized groups, respectively. A total of 40, 36, and 55 genes were likely subject to positive selection along the branches leading to the Primitive, Specialized and Highly Specialized groups, respectively, and many of these genes are likely relevant to adaptation to the cold temperatures, low oxygen concentrations, and strong ultraviolet radiation that result from elevation. CONCLUSIONS: By selecting representative species of the three groups of schizothoracins and applying next-generation sequencing technology, several candidate genes corresponding to adaptation to the three phases of plateau uplift were identified. Some of the genes identified in this report that were likely subject to positive selection are good candidates for subsequent evolutionary and functional analyses of adaptation to high altitude.

Environmentally Relevant Concentrations of the Flame Retardant Tris(1,3-dichloro-2-propyl) Phosphate Inhibit Growth of Female Zebrafish and Decrease Fecundity.

Bioconcentrations of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) in brain, gonad, and liver as well as effects on fecundity and development of zebrafish (Danio rerio) were determined. Zebrafish (1-month old) were exposed to environmentally relevant concentrations of 29 ± 2.1, 600 ± 21, or 6300 ± 130 ng TDCIPP/L. After 120 days of exposure, TDCIPP accumulated in the brain, gonad, and liver with bioconcentration factors of 460, 38, and 87 in females and 26, 55, and 110 in males, respectively. TDCIPP accumulated to a greater extent in brains of females than those of males. Exposure to 6300 ± 130 ng TDCIPP/L resulted in significantly (P < 0.05) fewer eggs being produced, but the histology of the gonad, plasma concentrations of estradiol and 11-ketotestosterone, and expression of genes involved in hypothalamic-pituitary-gonadal-liver axis were not significantly (P > 0.05) different between individuals exposed to TDCIPP and the unexposed control fish. Exposure to TDCIPP resulted in shorter body length, lighter body mass, and lower gonadal-somatic index in females. These effects were possibly due to down-regulation of expression of genes along the growth hormone/insulin-like growth factor (GH/IGF) axis. Correlations between the production of eggs and developmental parameters or expression of genes along the GH/IGF axis further suggested that environmentally relevant concentrations of TDCIPP could have adverse effects on reproduction, possibly due to the inhibition of the growth of females.

Predictable shifts in diversity and ecosystem function in phytoplankton and zooplankton communities along thermocline stratification intensity continua.

Thermocline stratification is a global threat to water quality in drinking water reservoirs, though its underlying mechanisms are not clear. The impacts of thermocline stratification intensity on biodiversity-ecosystem relationships were assessed using phytoplankton and zooplankton indicators from four stages of stratification in Lake Qiandaohu. There were significant differences in biomass, species diversity, and resource use efficiency (RUEpp = phytoplankton biomass/total phosphorus) for phytoplankton between continuous stratification and mixing periods, but only in FDis and RaoQ diversity indices for zooplankton. Phytoplankton species diversity and RUE were higher in the formative and stable periods, while zooplankton species diversity and zooplankton biomass/phytoplankton biomass (RUEzp) were lower. When combining the data from the four periods, a negative linear pattern was found between phytoplankton Simpson's, functional dispersion (FDis), and Rao's Quadratic (RaoQ) diversity indices, and thermocline depth (TD). Only zooplankton FDis and RaoQ diversity indices were significantly positively related to TD. Phytoplankton RUE was significantly negatively related only to its Pielou's evenness (J) diversity, while zooplankton RUE was significantly negatively related to its J, FDis, and RaoQ diversity indices. The results of structural equation models (SEMs) showed that the R2 of RUE for phytoplankton was much higher than that for zooplankton. Thermocline stratification intensity exerted an indirect positive effect on phytoplankton RUE by affecting species diversity but had a negative effect on zooplankton RUE. These findings underscore the negative influence of thermocline stratification resulting in various biodiversity changes in freshwater ecosystems.

Thermocline stratification favors phytoplankton spatial overlap and species diversity in a subtropical deep reservoir.

Spatial segregation of species along opposing resource gradients is a major research topic in ecology as it enables the coexistence and maintenance of high diversity. Thermocline stratification causes heterogeneous resource distribution, however, the effect of thermocline stratification on phytoplankton dynamic in-depth profiles is still unclear. To understand the underlying mechanism behind the effects of thermocline stratification on phytoplankton species diversity in stratified lakes, the monthly changes in thermocline parameters, deep chlorophyll maxima (DCM) parameters, spatial overlap (SO) among major phytoplankton taxonomic groups, and phytoplankton species diversity were evaluated in Lake Qiandaohu in the Zhejiang Province of China from April 2017 to December 2018. Thermocline depth (TD) was significantly negatively related to thermocline strength (TS). The monthly air temperature was the main driver behind the thermocline and the seasonal thermal-stratification cycle was divided into two stratification stages. Significant linear relationships were observed between the DCM parameters (depth, concentration, and thickness) and TD during the strong stratification period. TD was significantly positively related to phytoplankton species diversity and the SO between Cryptophyta and Chlorophyta as well as between Cryptophyta and Bacillariophyta during weak stratification periods. Significant positive correlations were observed between SO and Shannon diversity during both periods. Structural equation modeling (SEM) showed that air temperature significantly decreased TD and increased species diversity by increasing SO during the strong stratification period. Strong stratification under warming favored the formation of shallower and thinner DCMs, leading to phytoplankton coexistence and maintenance of high species diversity through a preclusion of dominance. This study characterized the temporal dynamics of phytoplankton dynamic in-depth profiles in response to strong stratification from warming.

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids.

BACKGROUND: Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation. METHODS: Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains. RESULTS: MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7. CONCLUSIONS: The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.

Do submerged macrophyte species influence crustacean zooplankton functional group richness and their resource use efficiency in the low-light environment?

During the high grazing of epiphytic zooplankton in submerged macrophyte beds, the changes in crustacean zooplankton functional groups are crucial for stabilizing a clear water state in shallow lakes. However, submerged macrophytes often experience low-light stress due to many ecological processes. It is unclear whether submerged macrophytes alter the zooplankton functional group and their resource use efficiency in the low-light environment. We conducted two mesocosm experiments involving the treatments of low-light and submerged macrophyte species (Vallisneria natans and Potamogeton maackianus). The results show that abiotic factors (e.g., light) were the most important variables in explaining the change in the zooplankton community. Specifically, zooplankton functional group (i.e., pelagic species, plant-associated species, and substrate scrapers) richness and zooplankton species diversity decreased with the decreasing light intensity, especially for low substrate scraper abundance. In addition, structural equation models showed that low-light stress reduced zooplankton resource use efficiency by reducing zooplankton functional group richness and species diversity. Compared to species diversity, zooplankton functional group richness had a greater influence on their resource use efficiency (Zp/Chl-a) in the low-light environment. Our results suggest that the low-light stress reduced zooplankton resource use efficiency by changing their functional group richness. Moreover, the abundance of substrate scrapers shaken from V. natans was higher than that from P. maackianus. Therefore, submerged macrophyte species influence crustacean zooplankton functional group richness and their resource use efficiency in the low-light environment. Selecting appropriate aquatic plant species to assure the high diversity of zooplankton should be considered when conducting lake restoration using submerged macrophytes.

Tris(1,3-dichloro-2-propyl) phosphate causes female-biased growth inhibition in zebrafish: Linked with gut microbiota dysbiosis.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is ubiquitous in aquatic environment, but its effect on intestinal health of fish has yet not been investigated. In the present study, the AB strain zebrafish embryos were exposed to environmentally realistic concentrations (0, 30, 300, and 3000 ng·L-1) of TDCIPP for 90 days, after which the fish growth and physiological activities were evaluated, and the intestinal microbes were analyzed by 16S rRNA gene high-throughput sequencing. Our results manifested that the body length and body weight were significantly reduced in the female zebrafish but not in males. Further analyses revealed that TDCIPP resulted in notable histological injury of intestine, which was accompanied by impairment of epithelial barrier integrity (decreased tight junction protein 2), inflammation responses (increased interleukin 1ß), and disruption of neurotransmission (increased serotonin) in female intestine. Male intestines maintained intact intestinal structure, and the remarkably increased activity of glutathione peroxidase (GPx) might protect the male zebrafish from inflammation and intestinal damage. Furthermore, 16S rRNA sequencing analysis showed that TDCIPP significantly altered the microbial communities in the intestine in a gender-specific manner, with a remarkable increase in alpha diversity of the gut microbiome in male zebrafish, which might be another mechanism for male fish to protect their intestines from damage by TDCIPP. Correlation analysis revealed that abnormal abundances of pathogenic bacteria (Chryseobacterium, Enterococcus, and Legionella) might be partially responsible for the impaired epithelial barrier integrity and inhibition in female zebrafish growth. Taken together, our study for the first time demonstrates the high susceptibility of intestinal health and gut microbiota of zebrafish to TDCIPP, especially for female zebrafish, which could be partially responsible for the female-biased growth inhibition.

Cloning and characterization of the HSP90 beta gene from Tanichthys albonubes Lin (Cyprinidae): effect of copper and cadmium exposure.

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. In order to evaluate the effect of copper (Cu(2+)) and cadmium (Cd(2+)) on the expression of HSP90 from Tanichthys albonubes (designated TaHSP90), the full-length complementary DNA (cDNA) of TaHSP90 was cloned using reverse transcription PCR and rapid amplification of cDNA ends (RACE) techniques. A 2,687-bp sequence was sequenced and consisted of an open reading frame (ORF) of 2,181 bp encoding a polypeptide of 727 amino acids with five HSP90 family signatures. Homologous analysis revealed that TaHSP90 gene shared high similarity with other known HSP90 genes and belonged to HSP90ß subtype. Fluorescent real-time quantitative PCR was used to examine the expression pattern of TaHSP90ß mRNA in different tissues (liver, muscle, gill, fin, eye, ovary, intestine and brain), and the result indicated that TaHSP90ß was widely expressed in all examined tissues at different levels. Sensitivity of TaHSP90ß to copper and cadmium was examined by exposing fish to different concentrations of Cu(2+) (0, 13.50 and 27.00 µg/L) and Cd(2+) (0, 1.15, 2.31 mg/L) for 24, 48, 72 and 96 h, respectively. The copper treatment induced TaHSP90ß expression slight increase only at 24 and 48 h, while cadmium treatment caused slight down-regulation of TaHSP90ß only 72 and 96 h. Our data suggest that the cloning and expression analysis of T. albonubes HSP90ß gene provided useful molecular information of T. albonubes responses in stress conditions and potential ways to monitor the chronic stressors in T. albonubes culture environments.

Dualistic effects of bisphenol A on growth, photosynthetic and oxidative stress of duckweed (Lemna minor).

In this study, we exposed duckweed (Lemna minor), a floating freshwater plant, to BPA at different concentrations (0, 1, 5, 20, and 50 mg/L) for 7 days so as to investigate the effects of BPA on its growth, photosynthesis, antioxidant system, and osmotic substances. It was found that BPA had the acute toxic effects of "low promotion and high inhibition" on growth and photosynthesis. Specifically, BPA at a low concentration (5 mg/L) significantly promoted the plant growth and improved the concentration of photosynthetic pigments (chlorophyll a, b, and total Chl ) of L. minor. However, BPA at a high concentration (50 mg/L) significantly inhibited the plant growth, the Chl content, and the maximal photochemical efficiency (Fv/Fm). Furthermore, BPA with high concentration (50 mg/L) induced ROS accumulation and increased the activities of antioxidant enzymes (SOD, CAT, POD, APX, and GR) and the contents of antioxidant substances (GSH, proline, and T-AOC), which indicated that L. minor might tolerate BPA toxicity by activating an antioxidant defense system. The correlation analysis revealed that the fresh weight of L. minor was significantly and positively correlated with photosynthesis and the contents of soluble protein and sugar, while it was negatively correlated with the content of H2O2. Totally, these results showed that BPA at different concentrations had dualistic effects on the growth of L. minor, which was attributed to the alterations of photosynthesis, oxidative stress, and osmotic regulation systems and provided a novel insight for studying the effects of BPA on aquatic plant physiology.

Molecular and functional characterization of teleost-specific Interleukin-17N in yellow catfish (Pelteobagrus fulvidraco).

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

The efficacy of DNA barcoding in the classification, genetic differentiation, and biodiversity assessment of benthic macroinvertebrates.

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time-consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra- and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance-based (ABGD) and tree-based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high-throughput technologies in the near future.

Organic carbon quantity and quality jointly triggered the switch between dissimilatory nitrate reduction to ammonium (DNRA) and denitrification in biofilters.

The effect of organic carbon (OC) quality and quantity on switch between dissimilatory nitrate reduction to ammonium (DNRA) and denitrification (DEN) was studied in biofilter systems. High OC in matrix could promote significantly nitrate (NO3--N) removal due to the reinforce of DEN. Sodium acetate (SA) addition in influent further fueled NO3--N removal in groups with low OC in matrix but increased ammonium (NH4+-N) and nitrite (NO2--N) accumulation in groups with high OC in matrix. This indicated that high OC combined different species, facilitated the DNRA over DEN. Compared to bagasse, corncob was the better suitable OC source in matrix for DEN due to slow and continuous release of OC. Hence, in order to promote NO3--N removal and decline NH4+-N accumulation in biofilters, it is very important to screen suitable OC source (mixed utilization of multiple C sources is recommended) and regulate its dosage (below 80 mg L-1).

Expression and Function Analysis of Interleukin-17A/F1, 2, and 3 Genes in Yellow Catfish (Pelteobagrus fulvidraco): Distinct Bioactivity of Recombinant IL-17A/F1, 2, and 3.

In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, Pf_IL-17A/F1, 2 and 3 genes were cloned from yellow catfish (Pelteobagrus fulvidraco) and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three Pf_IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of Pf_IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with Edwardsiella ictaluri and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that Pf_IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) Pf_IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the rPf_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the rPf_IL-17A/F1. Furthermore, the rPf_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three Pf_IL-17A/F proteins may play different roles in promoting inflammatory response.

The Dynamic Immune Response of Yellow Catfish (Pelteobagrus fulvidraco) Infected With Edwardsiella ictaluri Presenting the Inflammation Process.

Edwardsiella ictaluri is a highly destructive pathogen in cultured yellow catfish, thus it was very necessary to study the immune response of yellow catfish against bacterial infection. In this study, RNA-Seq technology was used to study the immune response in two distinct tissues of yellow catfish at eight different time points (h) after E. ictaluri infection. The number of differentially expressed genes (DEGs) in the spleen and liver was low at 3 h and 6 h post-infection, respectively. Afterwards, the most number of DEGs in the spleen was detected at 72 h, while the number of DEGs in the liver maintained a high level from 24 h to 120 h. The GO and KEGG enrichment analyses of DEGs at different time points uncovered that cytokines were continuously transcribed at 6 h to 120 h; whereas the liver is the main organ that secretes the components of the complement system, and metabolic regulation was activated from 12 h to 120 h. Moreover, an overview of the inflammation response of yellow catfish was exhibited including pattern-recognition receptors, inflammatory cytokines, chemokines, complements, and inflammation-related signal pathways. The similar expression tendency of nine genes by qRT-PCR validated the accuracy of transcriptome analyses. The different transcriptomic profiles obtained from the spleen and liver will help to better understand the dynamic immune response of fish against bacterial infection, and will provide basic information for establishing effective measures to prevent and control diseases in fish.

Molecular characterization, expression analysis and function identification of Pf_TNF-α and its two receptors Pf_TNFR1 and Pf_TNFR2 in yellow catfish (Pelteobagrus fulvidraco).

Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an important role in the inflammatory response by interacting with its receptor (TNFR). In this study, Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 genes were cloned from yellow catfish (Pelteobagrus fulvidraco), and bioinformatics analyses showed that the three genes were conserved and possessed similar sequence characteristics as those of other vertebrates. The qPCR results showed that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 mRNAs were constitutively expressed in 14 tissues and the lymphocytes of four tissues from healthy adults. The mRNA expression levels of Pf_TNF-α and Pf_TNFR1 genes were significantly up-regulated in the spleen, liver, trunk kidney, head kidney and gill after Edwardsiella ictaluri infection, while the mRNA expression of Pf_TNFR2 was significantly up-regulated in the spleen, and down-regulated in the liver and gill. In the isolated peripheral blood leukocytes (PBLs) of yellow catfish, the expression of Pf_TNF-α mRNA was notably up-regulated and the two Pf_TNFR transcripts were distinctly down-regulated after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly I:C) and phytohaemagglutinin (PHA). After stimulated by recombinant (r) Pf_sTNF protein, the mRNA expressions of various inflammatory factors genes were up-regulated in the PBLs. Meanwhile, rPf_sTNF promoted the phagocytic activity of leukocytes, whereas the activity mediated by rPf_sTNF could be inhibited by rPf_TNFR1CRD2/3 and rPf_TNFR2CRD2/3. The up-regulation of TNF-α and IL-1ß mRNAs expression triggered by rPf_sTNF could be inhibited by MAPK inhibitor (VX-702) and NF-κB inhibitor (PDTC). rPf_sTNF induced the expression of FADD mRNA in PBLs and increased the apoptotic rate of PBLs, and inhibiting the NF-κB and MAPK signal pathways could enhance the apoptosis of PBLs. The results indicate that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 play important roles in the immune response of yellow catfish to bacterial invasion.

Interactions between commensal bacteria and viral infection: insights for viral disease control in farmed animals.

Viral diseases cause serious economic loss in farmed animals industry. However, the efficacy of remedies for viral infection in farmed animals is limited, and treatment strategies are generally lacking for aquatic animals. Interactions of commensal microbiota and viral infection have been studied in recent years, demonstrating a third player in the interaction between hosts and viruses. Here, we discuss recent developments in the research of interactions between commensal bacteria and viral infection, including both promotion and inhibition effect of commensal bacteria on viral pathogenesis, as well as the impact of viral infection on commensal microbiota. The antiviral effect of commensal bacteria is mostly achieved through priming or regulation of the host immune responses, involving differential microbial components and host signaling pathways, and gives rise to various antiviral probiotics. Moreover, we summarize studies related to the interaction between commensal bacteria and viral infection in farmed animals, including pigs, chickens, fish and invertebrate species. Further studies in this area will deepen our understanding of antiviral immunity of farmed animals in the context of commensal microbiota, and promote the development of novel strategies for treatment of viral diseases in farmed animals.

Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection.

In this study, to assess the immunotoxicity of deltamethrin on fish, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.1, 0.3, and 1 µg/L deltamethrin for 28 d. Many immunological parameters and histopathological alterations were determined. The results showed that lymphocyte number was markedly decreased at 0.3 and 1 µg/L treatments, whereas the neutrophil number was strongly increased at 1 µg/L treatments (p < 0.05). Furthermore, lysozyme (LYS), immunoglobulin M (IgM), and complement component 3 (C3) levels at 0.3 and 1 µg/L treatments were markedly reduced, whereas alkaline phosphatase (ALP) activity were marked increased at 1 µg/L treatments (p < 0.05). The transcripts of almost all TLR (Toll-like receptor) signaling pathway-related genes were up-regulated. Histological lesions in the livers, intestines, and gills were observed at all treatments. Then, all remaining fish from controls and deltamethrin-exposed groups were injected with Pseudomonas fluorescens (P. fluorescens) for 48 h. At 24 and 48 h post-injection with P. fluorescens (hpi), the lymphocyte numbers were strongly reduced at 0.3 and 1 µg/L deltamethrin-exposed groups, whereas LYS and C3 levels were strongly reduced at 0.3 and 1 µg/L deltamethrin-exposed groups (p < 0.05). Obvious reduces in IgM levels were also detected at 0.3 and 1 µg/L deltamethrin-exposed groups at 48 hpi (p < 0.05). The transcripts of almost all TLR signaling pathway-related genes were significantly down-regulated, whereas the levels of related microRNAs (miRNAs) were markedly increased at all deltamethrin-exposed groups at 24 and 48 hpi. Moreover, the bacterial load in the liver and the mortality of fish were significantly increased at 1 µg/L deltamethrin-exposed groups at 24 and 48 hpi (p < 0.05). Furthermore, obvious histological damage in the livers, intestines, and gills were observed at all deltamethrin-exposed fish at 48 hpi. Overall, our results demonstrated that environmentally relevant concentration deltamethrin suppressed immunity and rendered the fish vulnerable to P. fluorescens infection, subsequently inducing mortality.

The immune responses of the Chinese rare minnow (Gobiocypris rarus) exposed to environmentally relevant concentrations of cypermethrin and subsequently infected by the bacteria Pseudomonas fluorescens.

In the present study, to assess the immunotoxicity of cypermethrin (CYP) in fish, Chinese rare minnows (Gobiocypris rarus) were exposed to environmentally relevant concentrations (0.15, 0.5, and 1.5 µg/L) of CYP for 28 d and subjected to pathogen challenge trials for 2 d. After 28 d of CYP exposure, the levels of Immunoglobulin M (IgM), Alkaline phosphatase (ALP), and C-reactive protein (CRP) were significantly decreased (p < 0.05) after treatment with 1.5 µg/L CYP. Moreover, an induction of inflammatory cytokine transcripts (tnfa, il-6, il-8, and il-12) was observed (p < 0.05) after treatment with 1.5 µg/L CYP. After challenge with Pseudomonas fluorescens (P. fluorescens), plasma lysozyme (LYS) activity at 24 and 48 hours post-injection (hpi) was significantly decreased in the 0.5 and 1.5 µg/L CYP treatment groups (p < 0.05). Moreover, liver Complement component 3 (C3) and CRP contents at 24 hpi were significantly decreased in the 1.5 µg/L CYP treatment group (p < 0.05), whereas significant decreases in liver C3 and IgM contents were observed at 48 hpi (p < 0.05). Inhibition of expression of Toll-like receptor-nuclear factor kappa B (TLR-NF-kB) signaling pathway-related genes was observed in the CYP treatment groups and resulted in significant down-regulation of inflammatory cytokines (il-1ß and il-12) in the 1.5 µg/L CYP treatment group at 48 hpi (p < 0.05). Interestingly, the mortality in the 0.5 and 1.5 µg/L CYP treatments was significantly increased at 48 hpi (p < 0.05). These results indicated that environmentally relevant concentrations of CYP suppressed the Chinese rare minnow immune system and reduced immune defense against bacterial infection, thereby causing subsequent mortality. Meanwhile, our results demonstrated that a subsequent host resistance challenge is an effective method for determining the immunotoxicity of chemicals (e.g., CYP).