Identification of the genetic characteristics of copy number variations in experimental specific pathogen-free ducks using whole-genome resequencing.

BACKGROUND: Specific pathogen-free ducks are a valuable laboratory resource for waterfowl disease research and poultry vaccine development. High throughput sequencing allows the systematic identification of structural variants in genomes. Copy number variation (CNV) can explain the variation of important duck genetic traits. Herein, the genome-wide CNVs of the three experimental duck species in China (Jinding ducks (JD), Shaoxing ducks (SX), and Fujian Shanma ducks (SM)) were characterized using resequencing to determine their genetic characteristics and selection signatures. RESULTS: We obtained 4,810 CNV regions (CNVRs) by merging 73,012 CNVs, covering 4.2% of the duck genome. Functional analysis revealed that the shared CNVR-harbored genes were significantly enriched for 31 gene ontology terms and 16 Kyoto Encyclopedia of Genes and Genomes pathways (e.g., olfactory transduction and immune system). Based on the genome-wide fixation index for each CNVR, growth (SPAG17 and PTH1R), disease resistance (CATHL3 and DMBT1), and thermoregulation (TRPC4 and SLIT3) candidate genes were identified in strongly selected signatures specific to JD, SM, and SX, respectively. CONCLUSIONS: In conclusion, we investigated the genome-wide distribution of experimental duck CNVs, providing a reference to establish the genetic basis of different phenotypic traits, thus contributing to the management of experimental animal genetic resources.

Unveiling the Influence of Copy Number Variations on Genetic Diversity and Adaptive Evolution in China's Native Pig Breeds via Whole-Genome Resequencing.

Copy number variations (CNVs) critically influence individual genetic diversity and phenotypic traits. In this study, we employed whole-genome resequencing technology to conduct an in-depth analysis of 50 pigs from five local swine populations [Rongchang pig (RC), Wuzhishan pig (WZS), Tibetan pig (T), Yorkshire (YL) and Landrace (LR)], aiming to assess their genetic potential and explore their prospects in the field of animal model applications. We identified a total of 96,466 CNVs, which were subsequently integrated into 7112 non-redundant CNVRs, encompassing 1.3% of the swine genome. Functional enrichment analysis of the genes within these CNVRs revealed significant associations with sensory perception, energy metabolism, and neural-related pathways. Further selective scan analyses of the local pig breeds RC, T, WZS, along with YL and LR, uncovered that for the RC variety, the genes PLA2G10 and ABCA8 were found to be closely related to fat metabolism and cardiovascular health. In the T breed, the genes NCF2 and CSGALNACT1 were associated with immune response and connective tissue characteristics. As for the WZS breed, the genes PLIN4 and CPB2 were primarily linked to fat storage and anti-inflammatory responses. In summary, this research underscores the pivotal role of CNVs in fostering the diversity and adaptive evolution of pig breeds while also offering valuable insights for further exploration of the advantageous genetic traits inherent to China's local pig breeds. This facilitates the creation of experimental animal models tailored to the specific characteristics of these breeds, contributing to the advancement of livestock and biomedical research.

Dietary nano-selenium alleviates heat stress-induced intestinal damage through affecting intestinal antioxidant capacity and microbiota in rainbow trout (Oncorhynchus mykiss).

Heat stress-induced intestinal damage is a key event in fish pathology. Nano-selenium (nano-Se) shows remarkably high biological activity and low toxicity, making it an ideal and ecological Se formulation; however, to date, the protective effects of nano-Se against heat stress-induced intestinal injury and pertinent molecular mechanisms remain unknown. Herein, rainbow trout (Oncorhynchus mykiss) were fed either a basal diet or basal diet + 5 mg/kg nano-Se. Samples were collected before (18 °C for 9 days; CG18 and Se18 groups) and after (24 °C for 8 h; CG24 and Se24 groups) heat stress treatment. On heat stress exposure, intestinal villus height, muscularis thickness, and goblet cell number decreased, and expression of tight junction proteins (ZO-1, occludin, and claudin-8d) was downregulated; dietary supplementation with nano-Se alleviated these effects. Furthermore, in the presence of nano-Se, catalase activity was elevated, and expression of diverse heat shock proteins (Hsp70b, Hsp90α, and Hsp30), selenoproteins (Gpx1a, Gpx1b1, and Trx), and anti-inflammatory cytokine (TGF-ß) was upregulated. In contrast, nano-Se supplementation significantly alleviated the increase of the expression of pro-inflammatory cytokines (IL-1ß and TNF-α) and the malondialdehyde content. We also observed that heat stress markedly increased the relative abundance of Actinobacteria, Firmicutes, Methylobacterium, Akkermansia, and Deinococcus and decreased that of Proteobacteria; nano-Se supplementation restored these changes, making their distribution similar to that in the control group. Overall, our findings suggest that nano-Se plays a protective role against heat stress-induced intestinal damage in rainbow trout by promoting the recovery of antioxidant enzyme activity, enhancing protein repair, alleviating inflammatory responses, and restoring intestinal microbiota composition.

Comprehensive proteomic analysis to elucidate the anti-heat stress effects of nano-selenium in rainbow trout (Oncorhynchus mykiss).

Because of the continuous intensification of global warming, extreme climate fluctuations, and high-density farming, cold-water rainbow trout (Oncorhynchus mykiss) are exposed to conditions of heat stress, which has severely impacted their survival and yield. Nano-selenium (nano-Se) shows higher biological activity and lower toxicity and has emerged as an ideal and ecological Se formulation. Herein rainbow trout were fed either a basal diet (control group) or basal diet plus 5 mg/kg nano-Se (treatment group). Samples were collected before (18 °C for 9 days; CG18, Se18) and after (24 °C for 8 h; CG24, Se24) heat stress. The DIA/SWATH approach was then applied to compare changes at the proteome level. We found 223 and 269 differentially abundant proteins in the CG18-CG24 and Se18-Se24 groups, respectively, which mainly included apoptosis-, heat stress-, and lipid-related proteins. In comparison with the CG18-CG24 group, the Se18-Se24 group showed higher abundance of molecular chaperone, such as Hsp70, Hsp90a.1, Hspa8, Hsp30, DNAJA4, Dnajb1, Bag2 and Ahsa1; on nano-Se supplementation, the heat stress-induced decline in the abundance of the selenoprotein MsrB2 was partially restored. Furthermore, nano-Se supplementation downregulated the abundance of lipid-related (CYP51, EBP, DHCR7, DHCR24, and APOB) and pro-apoptotic (caspase-8 and Bad) proteins. Protein-protein interaction analyses suggested that nano-Se inhibits apoptosis by upregulating the expression of Hsp70, Hsp90a.1, Hspa8, and Dnajb1; further, Hsp70/Hspa8 and MsrB2 appear to play a synergistic role in antioxidant defense under heat stress. Overall, our findings provide novel insights into nano-Se-mediated tolerance of heat stress, demonstrating that nano-Se exerts its anti-heat stress effects in rainbow trout by promoting protein repair, enhancing recovery of antioxidant enzyme activity, and alleviating lipid metabolism and apoptosis.

Protective effects of different concentrations of selenium nanoparticles on rainbow trout (Oncorhynchus mykiss) primary hepatocytes under heat stress.

Heat stress leads to altered expression of associated heat shock proteins (HSPs), which are critical molecular chaperones related to cellular function in living organisms. Selenium nanoparticles (SeNPs), a nanocomposite form of Se, have a protective effect against heat stress-induced cellular damage. In this study, primary rainbow trout hepatocytes were isolated to identify the protective function of SeNPs in rainbow trout hepatocytes. Experiments were divided into five groups and SeNPs were added at concentrations of 0, 2.0, 3.0, 5.0 and 8.0 µg/mL and incubated at 18 â„ƒ for 4, 8, 12, 24 and 48 h respectively. Hepatocyte viability, GSH-Px and SOD activity were enhanced and MDA content was reduced following the addition of SeNPs. Expression of GSH-P1 and genes related to HSPs (including HSP70a, HSP60, HSP90ß, HSP10 and HSP47) were significantly increased and the optimal concentration of SeNPs for adding to hepatocytes was identified as 5.0 µg/mL. Adding 5.0 µg/mL SeNPs following heat stress (24 â„ƒ) increased hepatocyte viability, GSH-Px and SOD activity, while MDA levels first decreased and then increased. Expression of GSH-P1 and genes related to HSPs (including HSP70a, HSP60, HSP90ß, HSP10 and HSP47) were significantly higher than controls. In summary, SeNPs and slight heat stress synergistically enhanced the expression of GSH-P1 and HSPs and protected hepatocytes from heat stress damage, suggesting that SeNPs is a potential hepatocyte protective therapeutic agent.

Dietary nanoselenium supplementation for heat-stressed rainbow trout: effects on organizational structure, lipid changes, and biochemical parameters as well as heat-shock-protein- and selenoprotein-related gene expression.

Nanoselenium (nano-Se) shows unique protective effects against environmental heat stress in rainbow trout as a selenium source additive and free radical scavenger. Accordingly, we investigated the effects of supplementation with different levels of nano-Se (0, 5, and 10 mg/kg) and before and after heat stress (24°C) for different treatment times on the dynamic changes of rainbow trout liver tissue structure, lipid changes, biochemical properties, and gene expression. The results showed that, under heat stress, the fish supplementation of 5 mg/kg nano-Se significantly increased liver glutathione peroxidase (GPx) activity and upregulated expression levels of HSP70b, HSP90a1, GPx1a, and Trx mRNAs, while liver alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) levels as well as tissue structure damage and lipid accumulation were decreased. Combining the trends for the above indicators indicated that stress began to increase significantly at 8 h. It can be concluded that supplementation with 5 mg/kg nano-Se effectively alleviates stress damage in rainbow trout. Furthermore, stress at 24°C for 8 h can be thought of as a critical time point for the study of heat stress in rainbow trout, with significant changes in response but no serious damage. Thus, these results provide a reference for the addition of nano-Se to rainbow trout feed and provide theoretical and practical guidance for enhancing the resistance of rainbow trout to heat stress.

Integrated analysis of the responses of a circRNA-miRNA-mRNA ceRNA network to heat stress in rainbow trout (Oncorhynchus mykiss) liver.

BACKGROUND: With the intensification of global warming, rainbow trout (Oncorhynchus mykiss) suffer from varying degrees of thermal stimulation, leads to mass mortality, which severely restrict the development of aquaculture. Understanding the molecular regulatory mechanisms of rainbow trout under heat stress is useful to develop approaches to relieve symptoms. RESULTS: Changes in nonspecific immune parameters revealed that a strong stress response was caused in rainbow trout at 24 °C, so we performed multiple transcriptomic analyses of rainbow trout liver under heat stress (HS, 24 °C) and control conditions (CG, 18 °C). A total of 324 DEcircRNAs, 105 DEmiRNAs, and 1885 DEmRNAs were identified. A ceRNA regulatory network was constructed and a total of 301 circRNA-miRNA and 51 miRNA-mRNA negative correlation pairs were screened, and three regulatory correlation pairs were predicted: novel_circ_003889 - novel-m0674-3p - hsp90ab1, novel_circ_002325 - miR-18-y - HSPA13 and novel_circ_002446 - novel-m0556-3p - hsp70. Some target genes involved in metabolic processes, biological regulation or response to stimulus were highly induced at high temperatures. Several important pathways involved in heat stress were characterized, such as protein processing in the ER, the estrogen signaling pathway, and the HIF-1 signaling pathway. CONCLUSIONS: These results extend our understanding of the molecular mechanisms of the heat stress response and provide novel insight for the development of strategies that relieve heat stress.

Evaluating genetic diversity and identifying priority conservation for seven Tibetan pig populations in China based on the mtDNA D-loop.

OBJECTIVE: Tibetan pigs, an excellent species unique to China, face serious threats, which in turn affects the development and utilization of the outstanding advantages of plateau hypoxia adaptability and reduces their genetic diversity. Therefore, a discussion of measures to conserve this genetic resource is necessary. The method, based on genetic diversity, genetic divergence and total genetic contribution rate of population, reflects the priority conservation order and varies depending on the three different purposes of conservation. METHODS: We analyzed mitochondrial DNA control region (D-loop) variation in 1,201 individuals from nine Tibetan pig populations across five provinces and downloaded 564 mtDNA D-loop sequences from three indigenous pig breeds in Qinghai, Sichuan, and Yunnan Provinces distributed near the Tibetan pigs. RESULTS: We analyzed three different aspects: Changdu Tibetan pigs have the highest genetic diversity, and from the perspective of genetic diversity, the priority conservation is Changdu Tibetan pigs. Hezuo Tibetan pigs have the highest genetic contribution, so the priority conservation is Hezuo Tibetan pigs in the genetic contribution aspect. Rkaze Tibetan pigs were severely affected by indigenous pig breeds, so if considering from the perspective of introgression, the priority conservation is Rkaze Tibetan pigs. CONCLUSION: This study evaluated genetic diversity and comprehensively assessed conservation priority from three different aspects in nine Tibetan pig populations.

The domestication event of the Tibetan pig revealed to be in the upstream region of the Yellow River based on the mtDNA D-loop.

OBJECTIVE: Evidence from previous reports indicates that pig domestication in East Asia mainly occurred in the Mekong region and the middle and downstream regions of the Yangtze River. Further research identified two new origin centers for domestic pigs in the Tibetan Plateau and the islands of Southeast Asia. However, due to the small sample size of Tibetan pigs, details of the origin and spread of Tibetan pigs has not yet been established. METHODS: We analyzed mitochondrial DNA control region (D-loop) variation in 1,201 individuals from nine Tibetan pig populations across five provinces. Comprehensive Tibetan pig samples were taken to perform the most detailed analysis of Tibetan pigs to date. RESULTS: The result indicate that Rkaze pigs had the lowest level of diversity, while Changdu pigs had the highest diversity. Interestingly, these two populations were both in the Tibetan Plateau area. If we calculate diversity in terms of each province, the Tibetan Plateau area had the lowest diversity, while the Chinese province of Gansu had the highest diversity. Diversity gradient analysis of major haplotypes suggested three domestication centers of Tibetan pigs in the Tibetan Plateau and the Chinese provinces of Gansu and Yunnan. CONCLUSION: We found two new domestication centers for Tibetan pigs. One is in the Chinese province of Gansu, which lies in the upstream region of the Yellow River, and the other is in the Chinese province of Yunnan.

Population Genetic Assessment Model Reveals Conservation Priorities for Gymnocypris Species Resources on the Qinghai-Tibetan Plateau.

The Qinghai-Tibetan Plateau (QTP) has nurtured a rich diversity of species because of its unique geographical and environmental conditions. Gymnocypris species (subfamily Schizopygopsinae) are primitive fishes that live in the special environment of the plateau, and their evolution and distribution are inseparable from the historical changes of the QTP. Recently, the resources of Gymnocypris species have been decreasing due to habit deterioration and the intensification of human activities. Therefore, the scientific conservation of the genetic resources of Gymnocypris species is urgently required. In this study, we established two models for the priority conservation assessment of germplasm resources of Gymnocypris species on the basis of the genetic diversity and phylogenetic relationships of 674 individuals from eight Gymnocypris species populations. The results show that the Gymnocypris potanini (GPO), Gymnocypris eckloni (GE), and Gymnocypris przewalskii (GPR) populations are the most genetically diverse in terms of combined genetic diversity values and should be prioritized for conservation. In terms of genetic contribution, the GPO, GE, and GPR populations have a positive impact on maintaining the distinctiveness and diversity of the entire Gymnocypris species population and should be prioritized for conservation. However, in terms of different evolutionary clades, the Gymnocypris namensis, Gymnocypris waddellii, Gymnocypris dobula, and GE populations in clade A should be given priority for protection, the GE population in clade B should be given priority, and the GPR population in clade C should be given priority. In conclusion, the two models and assessment of conservation priorities will provide a scientific basis for the conservation of Gymnocypris species.

Transcriptome sequencing reveals the effect of selenium nanoparticles on primary hepatocytes of rainbow trout.

Heat stress is one of the important threats in rainbow trout culture, and selenium nanoparticles (SeNPs) have an important role in combating heat stress and enhancing immunity. In this study, to enable rainbow trout to survive at higher temperatures, we added 5 µg/mL SeNPs to hepatocytes to study the resistance effect and immune effect of SeNPs against heat stress, thus enabling rainbow trout to adapt to summer temperatures (Average 26 °C) in Lanzhou, Gansu Province, China. Therefore, we investigated the transcriptome expression profile of hepatocytes spiked with SeNPs when exposed to heat stress. A total of 234 differentially expressed genes (DEGs) were firmly established in SeNPs-added group when exposed to heat stress compared to non-SeNPs-added group. Of these DEGs, 156 were up-regulated and 78 were down-regulated. These DEGs were grouped into different Gene Ontology (GO) functional terms and enriched in 75 significantly different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, of which approximately-one-third (19) were associated with immunity. STRING was used to identify 39 key immune DEGs belonging to 5 immune pathways (C-type lectin receptor signaling pathway, FoxO signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Rachidonic acid metabolism). These pathways interact extensively and formed a complex network to regulate heat stress. These results provided an important basis for future elucidation of the role of SeNPs in heat stress resistance and immune enhancement in rainbow trout.

Effect of selenium nanoparticles on alternative splicing in heat-stressed rainbow trout primary hepatocytes.

Alternative splicing (AS) is a ubiquitous post-transcriptional regulatory mechanism in eukaryotes that generates multiple mRNA isoforms from a single gene, increasing diversity of mRNAs and proteins that are essential for eukaryotic developmental processes and responses to environmental stress. Results showed that a total of 37,463 AS events were identified in rainbow trout hepatocytes. In addition, a total of 364 differential alternative splicing (DAS) events were identified in hepatocytes under selenium nanoparticles (SeNPs) and 3632 DAS events were identified under a combination of SeNPs and heat stress (24 °C). Gene Ontology (GO) enrichment showed that some subcategories "immune effector processes", "response to stimuli" and "antioxidant activity" were associated with immunity, abiotic stimuli and antioxidants. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that differentially expressed genes (DEGs) were significantly enriched in spliceosomes by adding SeNPs in heat-stressed hepatocytes. Splicing factor family (SRSF3, SRSF7, SRSF9, U2AF1 and U2AF2) and pre-RNA splicing factors (ACIN1 and PPRF18) were significantly upregulated and promoted AS. Furthermore, addition of SeNPs activated the phosphatidylinositol signaling system and upregulated the related genes PI4KA, DGKH, ITPK1 and Ocrl, and thus attenuated the inflammatory response to heat stress and enhanced resistance to heat stress by activating the adherent plaque kinase-PI3K-Akt signaling pathway and calcium channels. Those findings suggested that AS could be an essential regulatory mechanism in adaptation of rainbow trout to heat-stressed environments.

Potential role of miR-8159-x in heat stress response in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) is a representative species of cold-water fish. Elevated temperatures during summer often result in significant high mortality rates. MicroRNAs (miRNAs) are class of small non-coding RNAs that play a crucial role as post-transcriptional regulators in various biological processes. Emerging evidence suggests that miRNAs are important regulators role during heat stress. Analyzing previously obtained miRNA-sequencing data, we observed substantial down regulation of miR-8159-x in the liver tissue of heat stressed rainbow trout. In this study, we conducted a dual luciferase reporter assay to validate that miR-8159-x target, a key gene involved in heat stress in rainbow trout. By examining the expression patterns of miR-8159-x and hsp90a1 in the liver tissue at 18 °C (CG) and 24 °C (HS) groups, we propose that miR-8159-x may negatively regulate hsp90a1. Furthermore, in vitro hepatocyte assay, transfection with miR-8159-x mimics significantly reduced the expression level of hsp90a1, whereas transfection with a miR-8159-x inhibitor yielded the opposite effect. Additionally, overexpression of miR-8159-x inhibited cell proliferation and induced apoptosis in normal rainbow trout hepatocytes. We further investigated the effects of miR-8159-x overexpression or inhibition on the mRNA and protein levels of the target gene hsp90a1 under heat stress conditions. In conclusion, our findings suggest that miR-8159-x participates in the biological response to heat stress by targeting hsp90a1. These results contribute to a better understanding of the molecular mechanisms underlying heat stress in rainbow trout and provide valuable insights for future research.

Nano-selenium Antagonizes Heat Stress-Induced Apoptosis of Rainbow Trout (Oncorhynchus mykiss) Hepatocytes by Activating the PI3K/AKT Pathway.

The cold-water fish rainbow trout (Oncorhynchus mykiss) shows poor resistance to heat, which is the main factor restricting their survival and yield. With the advancement of nanotechnology, nano-selenium (nano-Se) has emerged as a key nano-trace element, showing unique advantages, including high biological activity and low toxicity, for studying the response of animals to adverse environmental conditions. However, little is still known regarding the potential protective mechanisms of nano-Se against heat stress-induced cellular damage. Herein, we aimed to investigate the mechanism underlying the antagonistic effects of nano-Se on heat stress. Four groups were assessed: CG18 (0 µg/mL nano-Se, 18 °C), Se18 (5.0 µg/mL nano-Se, 18 °C), CG24 (0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture), and Se24 (5.0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture). We found that after heat treatment (CG24 group), T-AOC, GPx, and CAT activities in rainbow trout hepatocytes showed a decrease of 36%, 33%, and 19%, respectively, while ROS and MDA levels showed an increase of 67% and 93%, respectively (P < 0.05). Meanwhile, the mRNA levels of the apoptosis-related genes caspase3, caspase9, Cyt-c, Bax, and Bax/Bcl-2 in the CG24 group were 41%, 47%, 285%, 65%, and 151% higher than those in the CG18 group, respectively, while those of PI3K and AKT were 31% and 17% lower, respectively (P < 0.05). Besides, flow cytometry analysis showed an increase in the level of apoptotic cells after heat exposure. More importantly, we observed that nano-Se cotreatment (Se24 group) remarkably attenuated heat stress-induced effects (P < 0.05). We conclude that heat stress induces oxidative stress and apoptosis in rainbow trout hepatocytes. Nano-Se ameliorates heat stress-induced apoptosis by activating the PI3K/AKT pathway. Our results provide a new perspective to improve our understanding of the ability of nano-Se to confer heat stress resistance.

Effects of Astaxanthin on Growth Performance, Gut Structure, and Intestinal Microorganisms of Penaeus vannamei under Microcystin-LR Stress.

Microcystin-LR (MC-LR) are biologically active cycloheptapeptide compounds that are released by cyanobacteria during water blooms and are extensively found in aquatic ecosystems. The Penaeus vannamei is a significant species in global aquaculture. However, the high level of eutrophication in aquaculture water frequently leads to outbreaks of cyanobacterial blooms, posing a significant threat to its sustainable cultivation. Astaxanthin (AX) is commonly utilized in aquaculture for its physiological benefits, including promoting growth and enhancing immune function in cultured organisms. This study aimed to examine the protective effect of astaxanthin on P. vannamei exposed to microcystin-induced stress. The experiment consisted of three groups: one group was fed formulated feed containing MC (100 µg/kg), another group was fed formulated feed containing MC (100 µg/kg) + AX (100 mg/kg), and the third group was fed basic feed (control group). After 15 days of feeding, the specific growth rate (SGR) was significantly higher in the MCAX group (2.21% day-1) compared to the MC group (0.77% day-1), and there was no significant difference between the MCAX group (2.21% day-1) and the control group (2.24% day-1). Similarly, the percent of weight gain (PWG) was also significantly higher in the MCAX group (14.61%) compared to the MC group (13.44%) and the control group (16.64%). Compared to the control group, the epithelial cells in the MC group suffered severe damage and detachment from the basement membrane. However, in the MCAX group, although there was still a gap between the intestinal epithelial cells and the basement membrane, the overall intestinal morphology was slightly less impaired than it was in the MC group. The analysis of the intestinal microbiota revealed a significant disparity in the community composition (chao 1 and ACE) between the MC and MCAX groups. When comparing the various bacterial genera, the MC group exhibited an increase in Vibrio abundance, whereas the MCAX group showed a decrease in both Shewanella and Vibrio abundance. The results indicate that AX has a positive impact on the growth performance and resistance of P. vannamei against MC by regulating the composition of the intestinal microbiota. AX can be utilized to mitigate the detrimental effects of MC in aquaculture practices. This function could be attributed to the role of AX in preserving the structural integrity of the intestinal mucosa and regulating the composition of the intestinal microbiota.

Whole-genome resequencing to unveil genetic characteristics and selection signatures of specific pathogen-free ducks.

Specific pathogen-free ducks are important high-grade laboratory animals, with a key role in research related to poultry biosecurity, production, and breeding. However, the genetic characteristics of experimental duck varieties remain poorly explored. Herein we performed whole-genome resequencing to construct a single nucleotide polymorphism genetic map of the genomes of 3 experimental duck varieties [Jinding ducks (JD), Shaoxing ducks (SX), and Fujian Shanma ducks (SM)] to determine their genetic characteristics and identify selection signatures. Subsequent analyses of population structure and genetic diversity revealed that each duck variety formed a monophyletic group, with SM showing richer genetic diversity than JD and SX. Further, on exploring shared selection signatures, we found 2 overlapping genomic regions on chromosome Z of all experimental ducks, which comprised immune response-related genes (IL7R and IL6ST). Moreover, growth and skeletal development (IGF1R and GDF5), meat quality (FoxO1), and stress resistance (HSP90B1 and Gpx8-b) candidate gene loci were identified in strongly selected signatures specific to JD, SM, and SX, respectively. Our results identified the population genetic basis of experimental ducks at the whole-genome level, providing a framework for future molecular investigations of genetic variations and phenotypic changes. We believe that such studies will eventually contribute to the management of experimental animal resources.

Competing endogenous RNA (ceRNA) in a non-model animal: Non-coding RNAs respond to heat stress in rainbow trout (Oncorhynchus mykiss) through ceRNA-regulated mechanisms.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a typical cold-water fish. With global warming and extreme heat, high summer temperatures are the biggest threat to rainbow trout farming. Rainbow trout initiate stress defense mechanisms in response to thermal stimuli, and competing endogenous RNA (ceRNA) regulation of target genes (mRNAs) mediated by non-coding RNAs (microRNAs [miRNAs], long non-coding RNAs) may be the main strategy for responding to thermal stimuli and enhancing adaptation. RESULTS: We screened the LOC110485411-novel-m0007-5p-hsp90ab1 ceRNA relationship pairs for affect heat stress in rainbow trout and validated their targeting relationships and functions based on preliminary high-throughput sequencing analysis results. The transfection of exogenous novel-m0007-5p mimics and inhibitors into primary rainbow trout hepatocytes effectively bound and inhibited the target genes hsp90ab1 and LOC110485411 without significant effects on hepatocyte viability, proliferation, and apoptosis. The inhibitory effect of novel-m0007-5p overexpression on hsp90ab1 and LOC110485411 under heat stress was time-efficient. Similarly, small interfering RNAs (siRNAs) affected hsp90ab1 mRNA expression by silencing LOC110485411 expression time-efficiently. CONCLUSIONS: In conclusion, we found that in rainbow trout, LOC110485411 and hsp90ab1 can bind competitively to novel-m0007-5p via 'sponge adsorption' and that interference with LOC110485411 affects hsp90ab1 expression. These results provide potential for anti-stress drug screening in rainbow trout.

Metabonomics analysis reveals the protective effect of nano­selenium against heat stress of rainbow trout (Oncorhynchus mykiss).

Nano­selenium (nano-Se) shows high biological activity and low toxicity, and has emerged as an ideal antioxidant. Our goal was to determine the underlying mechanism of nano-Se-mediated heat stress tolerance in rainbow trout (Oncorhynchus mykiss). Using liquid chromatography-mass spectrometry (LC-MS) metabolomics, histomorphology, and conventional biochemical assays, we investigated the physiological responses of heat-stressed rainbow trout to nano-Se. Fish were fed to two levels nano-Se at 18 °C for 9 days: CG18 (0 mg/kg) and Se18 (5 mg/kg). The water temperature of all groups was increased to 24 °C and maintained for 8 h (CG24, Se24). The results showed that most glycerophospholipids and CoA levels were decreased in CG18-CG24, and pathway enrichment analysis showed that it mainly interfered with glycerophospholipids and fatty acid metabolism. Meanwhile, hematoxylin and eosin and Oil Red O staining showed significant damage to CG18-CG24, which was ameliorated by Se18-Se24. The results combining analysis of antioxidant enzymes and heat shock proteins further support the notion that nano-Se supplementation inhibited galactose metabolism and activated the glutamate-glutamine metabolic pathway as the key metabolic strategy against heat stress. These results could establish heat stress defense strategies and increase our understanding of the mechanism of nutrient participation in fish's response to adverse environments. SIGNIFICANCE: Global warming is affecting the distribution and survival of cold-water fish worldwide, through seasonal water temperature increases and an increase in the frequency of extreme heat wave events. Surprisingly, Nano­selenium (Nano-Se) with its outstanding advantages of high biological activity and low toxicity, making it a good Se nutrient supplement and free radical scavenger, and also an ideal and ecological way to supplement Se. How to utilize the metabolome to better address the complexity of the interactions that may occur with Nano-Se during the process of heat stress resistance is an important challenge. In the present study, this is the first publicly available metabonomics study of the anti-heat stress effect of Nano-Se as a nutrient on rainbow trout liver. These data indicated that Nano-Se effectively alleviated stress damage in rainbow trout, in which heat stress interfered with the metabolism of glycerophospholipids and fatty acids significantly, causing liver cell membrane damage and lipid metabolism disorders in rainbow trout. Meanwhile, supplementation of Nano-Se downregulated galactose metabolism and activated glutamate and glutamine metabolic pathways, which seems to be a key metabolic strategy to combat heat stress. The results provide a scientific basis for the development of an anti-heat-stress feed for rainbow trout that help maintain their health, productivity and welfare under unfavorable heat conditions.

RNA-seq Analysis Reveals Alternative Splicing Under Heat Stress in Rainbow Trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) is one of the most economically important cold-water farmed species in the world, and transcriptomic studies in response to heat stress have been conducted and will be studied in depth. Alternative splicing (AS), a post-transcriptional regulatory process that regulates gene expression and increases proteomic diversity, is still poorly understood in rainbow trout under heat stress. In the present study, 18,623 alternative splicing events were identified from 9936 genes using RNA transcriptome sequencing technology (RNA-Seq) and genomic information. A total of 2731 differential alternative splicing (DAS) events were found among 2179 differentially expressed genes (DEGs). Gene ontology analysis revealed that the DEGs were mainly enriched in cellular metabolic process, cell part, and organic cyclic compound binding under heat stress. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis displayed that the DEGs were enriched for 39 pathways, and some key pathways, such as lysine degradation, are involved in the regulation of heat stress in liver tissues of rainbow trout. The results were validated by qRT-PCR, confirming reliability of our bioinformatics analysis.

Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver responses to chronic heat stress using DIA/SWATH.

Aquaculture of rainbow trout (Oncorhynchus mykiss) is severely hampered by high temperatures in summer, and understanding the regulatory mechanisms controlling responses to chronic heat stress may assist the development of measures to relieve heat stress. In the present study, biochemical parameters revealed a strong stress response in rainbow trout at 24 °C, including activation of stress defence and immune systems. Liver proteome analysis under heat stress (24 °C) and control (18 °C) conditions using DIA/SWATH identified precursors (90,827), peptides (67,028), proteins (6770) and protein groups (5124), among which 460 differentially abundant proteins (DAPs; q-value < 0.05, fold change >1.5), 201 and 259 were up- and down-regulated, respectively. Many were related to heat shock proteins (HSPs), metabolism and immunity. Gene Ontology (GO) analysis showed that some DAPs induced at high temperature were involved in regulating cell homeostasis, metabolism, adaptive stress and stimulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified metabolic pathways, protein processing in endoplasmic reticulum, PPAR signalling, and complement and coagulation cascades. Protein-protein interaction (PPI) network analysis indicated that HSP90b1 and C3 may cooperative to affect cell membrane integrity under heat stress. Our findings assist the development of strategies to relieve heat stress in rainbow trout.