Characterization and expression profiling of buffalo IFN-lambda family.

Interferon lambda (IFN-λ) is an important type III interferon triggered mainly by viral infection. IFN-λ binds to their heterodimeric receptors and signals through JAK-STAT pathways similar to type I IFN. In this study, we deduced the buffalo IFN-λ sequences through the polymerase chain reaction, and then studied IFN-λ's expression patterns in different tissues, and post induction with poly I:C and live MRSA using RT-qPCR. The full-length sequences of buffalo IFN-λ3, IFN-λ receptors, and a transcript variant of IFN-λ4 were determined. IFN-λ1 is identified as a pseudogene. Virus response elements and a recombination hotspot factor was observed in the regulatory region of IFN-λ. The IFN-λ3 expressed highest in lungs and monocytes but IFN-λ4 did not. The expression of Interferon Lambda Receptor 1 was tissue specific, while Interleukin 10 Receptor subunit beta was ubiquitous. Following poly I:C induction, IFN-λ3 expression was primarily observed in epithelial cells as opposed to fibroblasts, displaying cell type-dependent expression. The cytosolic RNA sensors were expressed highest in endometrial epithelial cells, whereas the endosomal receptor was higher in fibroblasts. 2',5'-oligoadenylate synthetase expressed higher in fibroblasts, myxoma resistance protein 1 and IFN-stimulated gene 56 in epithelial cells, displaying cell-specific antiviral response of the interferon stimulated genes (ISGs). The endometrial epithelial cells expressed IFN-λ3 after live S. aureus infection indicating its importance in bacterial infection. The induction of IFN-λ3 was S. aureus isolate specific at the same multiplicity of infection (MOI). This study elucidates the IFN-λ sequences, diverse expression patterns revealing tissue specificity, and specificity in response to poly I:C and bacterial stimuli, emphasising its crucial role in innate immune response modulation.

Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

Robust identification of interactions between heat-stress responsive genes in the chicken brain using Bayesian networks and augmented expression data.

Bayesian networks represent a useful tool to explore interactions within biological systems. The aims of this study were to identify a reduced number of genes associated with a stress condition in chickens (Gallus gallus) and to unravel their interactions by implementing a Bayesian network approach. Initially, one publicly available dataset (3 control vs. 3 heat-stressed chickens) was used to identify the stress signal, represented by 25 differentially expressed genes (DEGs). The dataset was augmented by looking for the 25 DEGs in other four publicly available databases. Bayesian network algorithms were used to discover the informative relationships between the DEGs. Only ten out of the 25 DEGs displayed interactions. Four of them were Heat Shock Proteins that could be playing a key role, especially under stress conditions, where maintaining the correct functioning of the cell machinery might be crucial. One of the DEGs is an open reading frame whose function is yet unknown, highlighting the power of Bayesian networks in knowledge discovery. Identifying an initial stress signal, augmenting it by combining other databases, and finally learning the structure of Bayesian networks allowed us to find genes closely related to stress, with the possibility of further exploring the system in future studies.

Identification of differentially expressed genes and metabolism signaling pathway in the spleen of broilers supplemented with probiotic Bacillus spp.

Probiotics are essential in the body's nutrients, improving the ratio of meat to meat, immune response, and preventing diseases. In this study, RNA-sequencing (RNA-seq) was used to identify the differentially expressed genes (DEGs), enriched related pathways, and Gene Ontology (GO) terms among blank negative control (NC), supplemented with Bacillus spp. (BS) and commercial probiotic (PC) groups after a 42-day fed supplementation. The results showed that 2005, 1356, and 2189 DEGs were significantly altered in BS vs. NC, PC vs NC, and BS vs PC groups, respectively. On the other hand, 9 DEGs were further validated by qRT-PCR, indicating that the qRT-PCR and RNA-Seq results were more consistent. Therefore, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs showed that the DEGs were mainly enriched to metabolism signalling pathways (alpha-linolenic acid metabolism, linoleic acid metabolism, tryptophan metabolism, tyrosine metabolism, ether lipid metabolism, and metabolic pathway, etc) and immune response pathways (cytokine-cytokine receptor interaction, MAPK signalling pathway, and intestinal immune network for IgA production, neuroactive ligand-receptor interaction etc). These results will provide a better understanding of the role of probiotics in chicken development and provide basic information on the genetic development of chickens.

Multitissue transcriptomics demonstrates the systemic physiology of methionine deficiency in broiler chickens.

Methionine (Met) supplementation is common practice in broilers to support nutrition, yet there are gaps in the understanding of its role in systemic physiology. Furthermore, several different Met sources are available that may have different physiological effects. This study evaluated the mode of action of Met deficiency (no Met-supplementation) and supplementation (0.25% DL- or L-Met, 0.41% liquid methionine hydroxy analog-free acid (MHA-FA)), and of Met source (DL-, L- or MHA-FA) in broiler chickens, via host transcriptomics. Biological pathway activation modeling was performed to predict the likely phenotypic effects of differentially expressed genes (DEGs) in tissue samples from the jejunum, liver and breast obtained at 10, 21 and 34/35 d of age from three experiments in a combined analysis. Animal performance data showed that Met deficiency reduced BW, daily BW gain, daily feed intake, and breast yield, and increased feed conversion ratio in all experiments (P < 0.05). Effects of Met deficiency on gene expression were least evident in the jejunum and most evident in the liver and breast, as evidenced by the number of DEG and activated pathways. Activated pathways suggested Met deficiency was associated with inhibited protein turnover, gut barrier integrity, and adaptive immunity functions in the jejunum, that predicted reduced breast yield. There was an interaction with age; in Met-deficient birds, there were 333 DEGs in the jejunum of starter vs finisher birds suggesting young birds were more sensitive to Met deficiency than older birds. In the liver, Met deficiency activated pathways associated with lipid turnover, amino acid metabolism, oxidative stress, and the immune system, whereas in breast, it activated pathways involved in metabolic regulation, hemostasis, the neuronal system, and oxidative stress, again predicting a negative impact on breast yield. In the starter phase, supplementation with DL-Met compared to MHA-FA inhibited gamma-aminobutyric acid activity and oxidative stress in breast tissue. When data from all tissues were integrated, increased expression of a liver gene (ENSGALG00000042797) was found to be correlated with the expression of several genes that best explained variation due to the Met deficiency in jejunum and breast muscle. Some of these genes were involved in anti-oxidant systems. Overall, the findings indicate that impaired growth performance due to Met deficiency results from an array of tissue-specific molecular mechanisms in which oxidative stress plays a key systemic role. Young birds are more sensitive to Met-deficiency and DL-Met was a preferential source of Met than L- or MHA-FA during the starter phase.

The combined analysis of transcriptomics and metabolomics reveals the mechanisms by which dietary quercetin regulates growth and immunity in Penaeus vannamei.

As a potent antioxidant, the flavonoid compound quercetin (QUE) has been widely used in the farming of aquatic animals. However, there are fewer reports of the beneficial effects, especially in improving immunity of Penaeus vannamei by QUE. The aim of this study was to investigate the effects of dietary QUE on growth, apoptosis, antioxidant and immunity of P. vannamei. It also explored the potential mechanisms of QUE in improving the growth and immunity of P. vannamei. P. vannamei were fed diets with QUE for 60 days. The results revealed that QUE (0.5 or 1.0 g/kg) ameliorated the growth, and the expressions of genes related to apoptosis, antioxidant, and immunity. The differentially expressed genes (DEGs) and differential metabolites (DMs) obtained through transcriptomics and metabolomics, respectively, enriched in pathways related to nutritional metabolism such as lipid metabolism, amino acid metabolism, and carbohydrate metabolism. After QUE addition, especially at 0.5 g/kg, DEGs were enriched into the functions of response to stimulus and antioxidant activity, and the pathways of HIF-1 signaling pathway, C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and FoxO signaling pathway. In conclusion, dietary QUE can ameliorate growth, apoptosis, antioxidant and immunity of P. vannamei, the appropriate addition amount was 0.5 g/kg rather than 1.0 g/kg. Regulations of QUE on nutrient metabolism and immune-related pathways, and bioactive metabolites, were important factors for improving the aforementioned abilities in P. vannamei.

Unraveling the immune functions of large yellow croaker Tmem208 in response to Pseudomonas plecoglossicida: Insights from cloning, expression profiling, and transcriptome analysis.

Pseudomonas plecoglossicida, the causative agent of Visceral White Spot Disease, poses substantial risks to large yellow croaker (Larimichthys crocea) aquaculture. Previous genome-wide association studies (GWAS), directed towards elucidating the resistance mechanisms of large yellow croaker against this affliction, suggested that the transmembrane protein 208 (named Lctmem208) may confer a potential advantage. TMEM proteins, particularly TMEM208 located in the endoplasmic reticulum, plays significant roles in autophagy, ER stress, and dynamics of cancer cell. However, research on TMEM's function in teleost fish immunity remains sparse, highlighting a need for further study. This study embarks on a comprehensive examination of LcTmem208, encompassing cloning, molecular characterization, and its dynamics in immune function in response to Pseudomonas plecoglossicida infection. Our findings reveal that LcTmem208 is highly conserved across teleost species, exhibiting pronounced expression in immune-relevant tissues, which escalates significantly upon pathogenic challenge. Transcriptome analysis subsequent to LcTmem208 overexpression in kidney cells unveiled its pivotal role in modulating immune-responsive processes, notably the p53 signaling pathway and cytokine-mediated interactions. Enhanced phagocytic activity in macrophages overexpressing LcTmem208 underscores its importance in innate immunity. Taken together, this is the first time reported the critical involvement of LcTmem208 in regulating innate immune responses of defensing P. plecoglossicida, thereby offering valuable insights into teleost fish immunity and potential strategies for the selective breeding of disease-resistant strains of large yellow croaker in aquaculture practices.

Akirin2 enhances antibacterial ability via interacting with 14-3-3ζ in V. splendidus-challenged Apostichopus japonicus.

Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5'-untranslated region (UTR), and a 154 bp 3'-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

IFN-υ and its receptor subunits, IFN-υR1 and IL10RB in mallard Anas platyrhynchos.

Type IV interferon (IFN) has been shown to be a cytokine with antiviral activity in fish and amphibian. But, it has not been cloned and characterized functionally in avian species. In this study, type IV IFN, IFN-υ, and its 2 possible receptors, IFN-υR1 and IL10RB, were identified from an avian species, the mallard (Anas platyrhynchos). Mallard IFN-υ has a 531 bp open reading frame (ORF), encoding 176 amino acids (aa), and has highly conserved features as reported in different species, with an N-terminal signal peptide and a predicted multi-helix structure. The IFN-υR1 and IL10RB contain 528 and 343 aa, respectively, with IFN-υR1 protein containing JAK1 and STAT binding sites, and IL10RB containing TYK2 binding site. These 2 receptor subunits also possess 3 domains, the N-terminal extracellular domain, the transmembrane domain, and the C-terminal intracellular domain. Expression analysis indicated that IFN-υ, IFN-υR1 and IL10RB were widely expressed in examined organs/tissues, with the highest level observed in pancreas, blood, and kidney, respectively. The expression of IFN-υ, IFN-υR1 and IL10RB in liver, spleen or kidney was significantly upregulated after stimulation with polyI:C. Furthermore, recombinant IFN-υ protein induced the expression of ISGs, and the receptor of IFN-υ was verified as IFN-υR1 and IL10RB using a chimeric receptor approach in HEK293 cells. Taken together, these results indicate that IFN-υ is involved in the host innate immune response in mallard.

Integrated transcriptome and microbiome analyses of residual feed intake in ducks during high production period.

Residual feed intake (RFI) is a crucial parameter for assessing the feeding efficiency of poultry. Minimizing RFI can enhance feed utilization and reduce costs. In this study, 315 healthy female ducks were individually housed in cages. Growth performance was monitored during the high laying period, from 290 to 325 d of age. The cecal transcriptome and microbiome of 12 ducks with high RFI and 12 with low residual feed intake (LRFI) were analyzed. Regarding growth performance, the LRFI group exhibited significantly lower RFI, feed conversion ratio (FCR), and feed intake (Fi) compared to the HRFI group (p < 0.01). However, there were no significant differences observed in body weight (BW), body weight gain (BWG), and egg mass (EML) between the groups (p > 0.05). Microbiome analysis demonstrated that RFI impacted gut microbial abundance, particularly affecting metabolism and disease-related microorganisms such as Romboutsia, Enterococcus, and Megamonas funiformis. Transcriptome analysis revealed that varying RFI changed the expression of genes related to glucose metabolism and lipid metabolism, including APOA1, G6PC1, PCK1, and PLIN1. The integrated analysis indicated that host genes were closely linked to the microbiota and primarily function in lipid metabolism, which may enhance feeding efficiency by influencing metabolism and maintaining gut homeostasis.

Whole transcriptome sequencing reveals key genes and ceRNA regulatory networks associated with pimpled eggs in hens.

Eggshell is one of the most important indicators of egg quality, and due to low shell strength, pimple eggs (PE) are more susceptible to breakage, thus causing huge economic losses to the egg industry. At the current time, the molecular mechanisms that regulate the formation of pimple eggs are poorly understood. In this study, uterine tissues of PE-laying hens (n = 8) and normal egg (NE) -laying hens (n = 8) were analyzed by whole transcriptome sequencing, and a total of 619 differentially expressed mRNAs (DE mRNAs), 122 differentially expressed lncRNAs (DE lncRNAs) and 21 differentially expressed miRNAs (DE miRNAs) were obtained. Based on the targeting relationship among DE mRNAs, DE lncRNAs and DE miRNAs, we constructed a competitive endogenous RNA (ceRNA) network including 12 DE miRNAs, 19 DE lncRNAs, and 128 DE mRNAs. Considering the large amount of information contained in the network, we constructed a smaller ceRNA network to better understand the complex mechanisms of pimple egg formation. The smaller ceRNA network network contains 7 DE lncRNAs (LOC107056551, LOC121109367, LOC121108909, LOC121108862, LOC112530033, LOC121113165, LOC107054145), 5 DE miRNAs (gga-miR-6568-3p, gga-miR-31-5p, gga-miR-18b-3p, gga-miR-1759-3p, gga-miR-12240-3p) and 7 DE mRNAs (CABP1, DNAJC5, HCN3, HPCA, IBSP, KCNT1, OTOP3), and these differentially expressed genes may play key regulatory roles in the formation of pimpled eggs in hens. This study provides the overall expression profiles of mRNAs, lncRNAs and miRNAs in the uterine tissues of hens, which provides a theoretical basis for further research on the molecular mechanisms of pimpled egg formation, and has potential applications in improving eggshell quality.