Integrated mRNA-miRNA transcriptome profiling of blood immune responses potentially related to pulmonary fibrosis in forest musk deer.

Background: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear. Methods: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR. Results: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation. Conclusion: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.

Akkermansia muciniphila isolated from forest musk deer ameliorates diarrhea in mice via modification of gut microbiota.

BACKGROUND: The forest musk deer, a rare fauna species found in China, is famous for its musk secretion which is used in selected Traditional Chinese medicines. However, over-hunting has led to musk deer becoming an endangered species, and their survival is also greatly challenged by various high incidence and high mortality respiratory and intestinal diseases such as septic pneumonia and enteritis. Accumulating evidence has demonstrated that Akkermannia muciniphila (AKK) is a promising probiotic, and we wondered whether AKK could be used as a food additive in animal breeding programmes to help prevent intestinal diseases. METHODS: We isolated one AKK strain from musk deer feces (AKK-D) using an improved enrichment medium combined with real-time PCR. After confirmation by 16S rRNA gene sequencing, a series of in vitro tests was conducted to evaluate the probiotic effects of AKK-D by assessing its reproductive capability, simulated gastrointestinal fluid tolerance, acid and bile salt resistance, self-aggregation ability, hydrophobicity, antibiotic sensitivity, hemolysis, harmful metabolite production, biofilm formation ability, and bacterial adhesion to gastrointestinal mucosa. RESULTS: The AKK-D strain has a probiotic function similar to that of the standard strain in humans (AKK-H). An in vivo study found that AKK-D significantly ameliorated symptoms in the enterotoxigenic Escherichia coli (ETEC)-induced murine diarrhea model. AKK-D improved organ damage, inhibited inflammatory responses, and improved intestinal barrier permeability. Additionally, AKK-D promoted the reconstitution and maintenance of the homeostasis of gut microflora, as indicated by the fact that AKK-D-treated mice showed a decrease in Bacteroidetes and an increase in the proportion of other beneficial bacteria like Muribaculaceae, Muribaculum, and unclassified f_Lachnospiaceae compared with the diarrhea model mice. CONCLUSION: Taken together, our data show that this novel AKK-D strain might be a potential probiotic for use in musk deer breeding, although further extensive systematic research is still needed.

Single-nucleus transcriptomics and chromatin accessibility analysis of musk gland development in Chinese forest musk deer (Moschus berezovskii).

Musk secreted by male forest musk deer (Moschus berezovskii) musk glands is an invaluable component of medicine and perfume. Musk secretion depends on musk gland maturation; however, the mechanism of its development remains elusive. Herein, using single cell multiome ATAC + gene expression coupled with several bioinformatic analyses, a dynamic transcriptional cell atlas of musk gland development was revealed, and key genes and transcription factors affecting its development were determined. Twelve cell types, including two different types of acinar cells (Clusters 0 and 10) were identified. Single-nucleus RNA and single-nucleus ATAC sequencing analyses revealed that seven core target genes associated with musk secretion (Hsd17b2, Acacb, Lss, Vapa, Aldh16a1, Aldh7a1, and Sqle) were regulated by 12 core transcription factors (FOXO1, CUX2, RORA, RUNX1, KLF6, MGA, NFIC, FOXO3, ETV5, NR3C1, HSF4, and MITF) during the development of Cluster 0 acinar cells. Kyoto Encyclopedia of Genes and Genomes enrichment showed significant changes in the pathways associated with musk secretion during acinar cell development. Gene set variation analysis also revealed that certain pathways associated with musk secretion were enriched in 6-year-old acinar cells. A gene co-expression network was constructed during acinar cell development to provide a precise understanding of the connections between transcription factors, genes, and pathways. Finally, intercellular communication analysis showed that intercellular communication is involved in musk gland development. This study provides crucial insights into the changes and key factors underlying musk gland development, which serve as valuable resources for studying musk secretion mechanisms and promoting the protection of this endangered species.

Maslinic acid supplementation prevents di(2-ethylhexyl) phthalate-induced apoptosis via PRDX6 in peritubular myoid cells of Chinese forest musk deer.

Chinese forest musk deer (FMD), an endangered species, have exhibited low reproductive rates even in captivity due to stress conditions. Investigation revealed the presence of di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, in the serum and skin of captive FMDs. Feeding FMDs with maslinic acid (MA) has been observed to alleviate the stress response and improve reproductive rates, although the precise molecular mechanisms remain unclear. Therefore, this study aims to investigate the molecular mechanisms underlying the alleviation of DEHP-induced oxidative stress and cell apoptosis in primary peritubular myoid cells (PMCs) through MA intake. Primary PMCs were isolated and exposed to DEHP in vitro. The results demonstrated that DEHP significantly suppressed antioxidant levels and promoted cell apoptosis in primary PMCs. Moreover, interfering with the expression of PRDX6 was found to induce excessive reactive oxygen species (ROS) production and cell apoptosis in primary PMCs. Supplementation with MA significantly upregulated the expression of PRDX6, thereby attenuating DEHP-induced oxidative stress and cell apoptosis in primary PMCs. These findings provide a theoretical foundation for mitigating stress levels and enhancing reproductive capacity of in captive FMDs.

Chemical composition and microbiota changes across musk secretion stages of forest musk deer.

Forest musk deer is the most important animal for natural musk production, and the musk composition changes periodically during musk secretion, accompanied by variation in the com-position of deer-symbiotic bacteria. GC-MS and 16S rRNA sequencing were conducted in this study, the dynamic changes to correlated chemical composition and the microbiota across musk secretion periods (prime musk secretion period, vigorous musk secretion period and late musk secretion period) were investigated by integrating its serum testosterone level in different mating states. Results showed that the testosterone level, musk composition and microbiota changed with annual cycle of musk secretion and affected by its mating state. Muscone and the testosterone level peaked at vigorous musk secretion period, and the microbiota of this stage was distinct from the other 2 periods. Actinobacteria, Firmicutes and Proteobacteria were dominant bacteria across musk secretion period. PICRUSt analysis demonstrated that bacteria were ubiquitous in musk pod and involved in the metabolism of antibiotics and terpenoids in musk. "Carbohydrates and amino acids," "fatty acids and CoA" and "secretion of metabolites" were enriched at 3 periods, respectively. Pseudomonas, Corynebacterium, Clostridium, Sulfuricurvum were potential biomarkers across musk secretion. This study provides a more comprehensive understanding of genetic mechanism during musk secretion, emphasizing the importance of Actinobacteria and Corynebacterium in the synthesis of muscone and etiocholanone during musk secretion, which required further validation.

SSR Marker Acquisition and Application from Transcriptome of Captive Chinese Forest Musk Deer (Moschus berezovskii).

Forest musk deer (Moschus berezovskii) is one of the most endangered medicinally important wild animals in the world. Forest musk deer farming is the main way of production of musk. However, the single provenance and lack of genetic information lead to reduced genetic diversity of forest musk deer. Therefore, more SSR markers need to be developed to identify forest musk deer germplasm. In this study, bone marrow derived mesenchymal cells were used to construct cDNA library for transcriptome sequencing. The datasets were de novo assembled and annotated. 9 polymorphic simple sequence repeat (SSR) markers were finally identified and used to detect population genetic diversity. 6.07 Gb clean data were generated using Illumina sequencing technology, and de novo assembled into 138,591 transcripts and 81,553 unigenes. 5,777 simple sequence repeats (SSRs) were identified, in which there were 578 repeating motif types, with mono-nucleotide and tri-nucleotides comprising 55.88% and 25.60%, respectively. 100 primer pairs were designed to validate amplification and polymorphism using DNA from fecal samples. 9 polymorphic SSRs were developed and used to detect population genetic diversity of 122 forest musk deer in 2 farms. The average number of alleles per locus varied from 4 to 15 (average = 8.3). The observed heterozygosity (HO) per locus ranged from 0.102 to 0.941, while the expected heterozygosity (HE) per locus was from 0.111 to 0.651. All loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.001). The polymorphism information content (PIC) of these loci varied from 0.108 to 0.619. 9 polymorphic SSR markers were developed in this research. These sites can be used for breeding planning and conservation of germplasm resources.

Identification of Potential miRNA-mRNA Regulatory Network Associated with Growth and Development of Hair Follicles in Forest Musk Deer.

In this study, sRNA libraries and mRNA libraries of HFs of FMD were constructed and sequenced using an Illumina HiSeq 2500, and the expression profiles of miRNAs and genes in the HFs of FMD were obtained at the anagen and catagen stages. In total, 565 differentially expressed unigenes (DEGs) were identified, 90 of which were upregulated and 475 of which were downregulated. In the BP category of GO enrichment, the DEGs were enriched in the processes related to HF development and differentiation, including the hair cycle regulation and processes, HF development, skin epidermis development, regulation of HF development, skin development, the Wnt signaling pathway, and the BMP signaling pathway. Through KEGG analysis it was found that DEGs were significantly enriched in pathways associated with HF development and growth. A total of 186 differentially expressed miRNAs (DEmiRNAs) were screened (p < 0.05) in the HFs of FMD at the anagen stage vs. the catagen stage, 33 of which were upregulated and 153 of which were downregulated. Through DEmiRNA-mRNA association analysis, we found DEmiRNAs and target genes that mainly play regulatory roles in HF development and growth. The enrichment analysis of DEmiRNA target genes revealed similarities with the enrichment results of DEGs associated with HF development. Notably, both sets of genes were enriched in key pathways such as the Notch signaling pathway, melanogenesis, the cAMP signaling pathway, and cGMP-PKG. To validate our findings, we selected 11 DEGs and 11 DEmiRNAs for experimental verification using RT-qPCR. The results of the experimental validation were consistent with the RNA-Seq results.

Quercetin promotes the secretion of musk by regulating the hormone level and microbial structure of forest musk deer.

Musk is a scarce and precious medical resource secreted by male forest musk deer (FMD). Current research to promote musk secretion in FMD has used almost exclusively hormone injections, but this approach can be detrimental to the health of FMD. In order to conserve this endangered species as much as possible while increasing the production of musk, this study first used bioinformatics methods to predict the function of quercetin, a flavonoid that promotes testosterone (T) production and prevents late-onset male hypogonadism. On the basis of good prediction effect, different concentrations of quercetin were added to the diet of FMD. The results showed that quercetin could change the levels of T, luteinizing hormone releasing hormone, luteinizing hormone, and estradiol, and regulate the structure of intestinal microorganisms and musk microorganisms of FMD. Moreover, there is a correlation among musk components, hormones, intestinal microorganisms, and musk microorganisms, which indicates that the production of musk may be regulated by these three at the same time, and the addition of quercetin with 800 mg per kg diet could significantly increase the yield of muscone (P < 0.05), the most effective ingredient in musk. In addition, quercetin decreased the high level of cortisol during musk secretion, which may relieve the stress on FMD in this process. This may help to protect the health of FMD. Combined with the results of software prediction, we finally proposed a possible mechanism for the complex process of musk secretion in FMD with a view to providing ideas for further studies.

Detecting Forest Musk Deer Abscess Disease Pathogens Using 16S rRNA High-Throughput Sequencing Technology.

Currently, researchers use bacterial culture and targeted PCR methods to classify, culture, and identify the pathogens causing abscess diseases. However, this method is limited by factors such as the type of culture medium and culture conditions, making it challenging to screen and proliferate many bacteria effectively. Fortunately, with the development of high-throughput sequencing technology, pathogen identification at the genetic level has become possible. Not only can this approach overcome the limitations of bacterial culture, but it can also accurately identify the types and relative abundance of pathogens. In this study, we used high-throughput sequencing of 16S rRNA to identify the pathogens in purulent fluid samples. Our results not only confirmed the presence of the main pathogen reported by previous researchers, Trueperella pyogenes, but also other obligate anaerobes, Fusobacterium necrophorum and Bacteroides fragilis as the dominant pathogens causing abscess diseases for the first time. Therefore, our findings suggest that high-throughput sequencing technology has the potential to replace traditional bacterial culture and targeted PCR methods.

Genetic and histological relationship between pheromone-secreting tissues of the musk gland and skin of juvenile Chinese forest musk deer (Moschus berezovskii Flerov, 1929). / 中国幼年林麝（Moschus berezovskii Flerov, 1929ï¼‰é¦™è ºå’Œçš®è‚¤åˆ†æ³Œä¿¡æ¯ç´ çš„é—ä¼ å’Œç»„ç»‡å­¦å ³ç³».

BACKGROUND: The musk glands of adult male Chinese forest musk deer (Moschus berezovskii Flerov, 1929) (FMD), which are considered as special skin glands, secrete a mixture of sebum, lipids, and proteins into the musk pod. Together, these components form musk, which plays an important role in attracting females during the breeding season. However, the relationship between the musk glands and skin of Chinese FMD remains undiscovered. Here, the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing (RNA-seq), and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin. METHODS: A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted. Then, based on the anatomical structure of the musk gland, skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD. These tissues were used for RNA-seq, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod, while juvenile FMD did not have any well-developed musk pods. Transcriptomic data revealed that 88.24% of genes were co-expressed in the skin and musk gland tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis found that the genes co-expressed in the abdomen skin, back skin, and musk gland were enriched in biological development, endocrine system, lipid metabolism, and other pathways. Gene Ontology (GO) enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division. Moreover, the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific. qRT-PCR and WB revealed that sex-determining region Y-box protein 9 (Sox9),Caveolin-1 (Cav-1), andandrogen receptor (AR) were expressed in all three tissues, although the expression levels differed among the tissues. According to the IHC results, Sox9 and AR were expressed in the nuclei of sebaceous gland, hair follicle, and musk gland cells, whereas Cav-1 was expressed in the cell membrane. CONCLUSIONS: The musk gland of Chinese FMD may be a derivative of skin tissue, and Sox9, Cav-1, and AR may play significant roles in musk gland development.

mtDNA CR Evidence Indicates High Genetic Diversity of Captive Forest Musk Deer in Shaanxi Province, China.

Forest musk deer (Moschus berezovskii) are endangered ruminants whose adult males secrete musk. China has been breeding forest musk deer artificially since the 1950s in an effort to restore wild populations, with Shaanxi and Sichuan provinces as the two main sites for captive breeding. Genetic diversity is a significant indicator that determines the long-term viability and status of a population, particularly for species at risk of extinction. In this study, we analyzed the current genetic makeup of seven captive forest musk deer populations in the Shaanxi province, using the mitochondrial DNA (mtDNA) control region (CR) as the molecular marker. We sequenced 604 bp of mtDNA CR, with an average content of A+T higher than G+C. We observed 111 variable sites and 39 different haplotypes from 338 sequences. The nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.02887 and 0.908, respectively. Genetic differentiation between these populations was not significant, and the populations might not have experienced rapid growth. By combining our sequences with previous ones, we identified 65 unique haplotypes with 26 rare haplotypes and estimated a total of 90 haplotypes in Shaanxi province captive populations. The Shaanxi province and Sichuan province obtained 88 haplotypes, the haplotypes from the two populations were mixed together, and the two populations showed moderate genetic differentiation. Our findings suggested that captive forest musk deer populations in the Shaanxi province had high genetic diversity, with a rich founder population of about 90 maternal lines. Additionally, managers could develop genetic management plans for forest musk deer based on the haplotype database. Overall, our study will provide insights and guidelines for the conservation of genetic diversity in captive forest musk deer populations in the Shaanxi province.

Single-nucleus RNA and ATAC sequencing uncovers the molecular and cellular characteristics in the musk gland of Chinese forest musk deer (Moschus berezovskii).

The Chinese forest musk deer (FMD; Moschus berezovskii) is an endangered artiodactyl mammal. Musk secreted by the musk gland of male has extremely high economic and medicinal value. However, the molecular and cellular characteristics of the musk gland have not been studied. Here, we investigated the diversity and transcriptional composition of musk gland cell types and the effect of cell type-specific chromatin accessibility on gene expression using single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) association analysis. Based on uniform manifold approximation and projection (UMAP) analysis, we identified 13 cell types from the musk gland, which included two different acinar cells (cluster 0 and cluster 10). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that many pathways related to musk secretion were enriched in acinar cells. Our analysis also revealed acinar cell core transcription factors and core target genes, and further constructed acinar cell-specific regulatory networks. In cluster 0, 11 core target genes (Nedd4l, Adcy9, Akr1c1, Vapb, Me1, Acsl1, Acss3, Srd5a1, Scnn1a, Acadm, and Nceh1) possibly related to musk secretion were regulated by 24 core transcription factors (SP3, NFIC, NR6A1, EHF, RUNX1, TFAP2A, RREB1, GRHL2, NFIB, ELF1, MAX, KLF5, REL, HES1, POU2F3, TFDP1, NR2C1, ATF7, MEIS1, NR4A2, NFIA, PBX1, ZNF652, and NFKB1). In cluster 10, four core target genes (Akr1c1, Pcca, Atp1b1, and Sgk1) possibly related to musk secretion were regulated by 10 core transcription factors (BARX2, EHF, PBX1, RUNX1, NFIB, FOXP1, KLF3, KLF6, ETV6, and NR3C2). Moreover, the credibility of snRNA-seq and snATAC-seq data was verified by fluorescence in situ hybridization and immunohistochemistry. Finally, cell communication analysis demonstrated that the two types of acinar cells mainly have communications in musk secretion-related processes. In conclusion, we provided important insights and invaluable resources for the molecular and cellular characteristics of the musk gland, which will lay a foundation for the study of musk secretion mechanism in the future.

ITRAQ-based quantitative proteomics analysis of forest musk deer with pneumonia.

Pneumonia can seriously threaten the life of forest musk deer (FMD, an endangered species). To gain a comprehensive understanding of pneumonia pathogenesis in FMD, iTRAQ-based proteomics analysis was performed in diseased (Pne group) lung tissues of FMD that died of pneumonia and normal lung tissues (Ctrl group) of FMD that died from fighting against each other. Results showed that 355 proteins were differentially expressed (fold change ≥ 1.2 and adjusted P-value < 0.05) in Pne vs. Ctrl. GO/KEGG annotation and enrichment analyses showed that dysregulated proteins might play vital roles in bacterial infection and immunity. Given the close association between bacterial infection and pneumonia, 32 dysregulated proteins related to Staphylococcus aureus infection, bacterial invasion of epithelial cells, and pathogenic Escherichia coli infection were screened out. Among these 32 proteins, 13 proteins were mapped to the bovine genome. Given the close phylogenetic relationships of FMD and bovine, the protein-protein interaction networks of the above-mentioned 13 proteins were constructed by the String database. Based on the node degree analysis, 5 potential key proteins related to pneumonia-related bacterial infection in FMD were filtered out. Moreover, 85 dysregulated proteins related to the immune system process were identified given the tight connection between immune dysregulation and pneumonia pathogenesis. Additionally, 12 proteins that might function as crucial players in pneumonia-related immune response in FMD were screened out using the same experimental strategies described above. In conclusion, some vital proteins, biological processes, and pathways in pneumonia development were identified in FMD.

Transcriptome profiling of lung immune responses potentially related to acute respiratory distress syndrome in forest musk deer.

BACKGROUND: Forest musk deer is an endangered species globally. The death of captive forest musk deer can be caused by certain respiratory system diseases. Acute respiratory distress syndrome (ARDS) is a huge threat to the life of forest muck deer that breed in our department. METHODS: Lung histopathologic analysis was conducted by hematoxylin and eosin (HE) staining. The lung gene changes triggered by ARDS were examined by RNA sequencing and related bioinformatics analysis in forest musk deer. The potential functions of unigenes were investigated by NR, SwissProt KOG, GO, and KEGG annotation analyses. Vital biological processes or pathways in ARDS were examined by GO and KEGG enrichment analyses. RESULTS: A total of 3265 unigenes were differentially expressed (|log2fold-change|> 2 and adjusted P value < 0.01) in lung tissues of 3 forest musk deer with ARDS compared with normal lung tissues of the non-ARDS group. These differentially expressed unigenes (DEGs) played crucial roles in immunity and defense responses to pathogens. Moreover, we identified the DEGs related to one or more of the following biological processes: lung development, immunity, and bacterial/viral/fungal infection. And six DEGs that might be involved in lung injury caused by immune dysregulation or viral/fungal infection were identified. CONCLUSION: ARDS-mediated lung gene alterations were identified in forest musk deer. Moreover, multiple genes involved in lung development and lung defense responses to bacteria/viruses/fungi in ARDS were filtered out in forest musk deer.

MicroRNA let-7f-5p regulates PI3K/AKT/COX2 signaling pathway in bacteria-induced pulmonary fibrosis via targeting of PIK3CA in forest musk deer.

Background: Recent studies have characterized that microRNA (miRNA) is a suitable candidate for the study of bleomycin/LPS-induced pulmonary fibrosis, but the knowledge on miRNA in bacteria-induced pulmonary fibrosis (BIPF) is limited. Forest musk deer (Moschus berezovskii, FMD) is an important endangered species that has been seriously affected by BIPF. We sought to determine whether miRNA exist that modulates the pathogenesis of BIPF in FMD. Methods: High-throughput sequencing and RT-qPCR were used to determine the differentially expressed miRNAs (DEmiRNAs) in the blood of BIPF FMD. The DEmiRNAs were further detected in the blood and lung of BIPF model rat by RT-qPCR, and the targeting relationship between candidate miRNA and its potential target gene was verified by dual-luciferase reporter activity assay. Furthermore, the function of the candidate miRNA was verified in the FMD lung fibroblast cells (FMD-C1). Results: Here we found that five dead FMD were suffered from BIPF, and six circulating miRNAs (miR-30g, let-7f-5p, miR-27-3p, miR-25-3p, miR-9-5p and miR-652) were differentially expressed in the blood of the BIPF FMD. Of these, let-7f-5p showed reproducibly lower level in the blood and lung of the BIPF model rat, and the expression levels of PI3K/AKT/COX2 signaling pathway genes (PIK3CA, PDK1, Akt1, IKBKA, NF-κB1 and COX2) were increased in the lung of BIPF model rats, suggesting that there is a potential correlation between BIPF and the PI3K/AKT/COX2 signaling pathway. Notably, using bioinformatic prediction and experimental verification, we demonstrated that let-7f-5p is conserved across mammals, and the seed sequence of let-7f-5p displays perfect complementarity with the 3' UTR of PIK3CA gene and the expression of the PIK3CA gene was regulated by let-7f-5p. In order to determine the regulatory relationship between let-7f-5p and the PI3K/AKT/COX2 signaling pathway in FMD, we successfully cultured FMD-C1, and found that let-7f-5p could act as a negative regulator for the PI3K/Akt/COX2 signaling pathway in FMD-C1. Collectively, this study not only provided a study strategy for non-invasive research in pulmonary disease in rare animals, but also laid a foundation for further research in BIPF.

Changes in the gut microbiota of forest musk deer (Moschus berezovskii) during ex situ conservation.

Ex situ conservation is an important technique for protecting rare and endangered wildlife, and maintaining stable individual health is crucial to its success. Gut microbiota composition is a critical indicator of animal health and should therefore be closely monitored during ex situ conservation to track impacts on animal health. Forest musk deer (Moschus berezovskii) were historically distributed in Hebei Province, China, however, they are now extinct in the region. Thus, ex situ conservation efforts were conducted in 2016 whereby approximately 50 individuals were artificially migrated from Weinan, Shaanxi to Huailai, Hebei. To monitor gut health of these migrated individuals, we used 16S rRNA high-throughput sequencing technology to examine the microbiota differences between Huailai juvenile and Weinan juvenile groups, and between Huailai adult and Weinan adult groups. Alpha diversity analysis indicated that the richness of microbiota significantly decreased after migration to the Huailai area, and the beta diversity results also showed significant dissimilarity in gut microbial communities, demonstrating the distinct microbial structure differences in the forest musk deer population from the two areas, for both juvenile and adult groups, respectively. In addition, PICRUSt functional profile prediction indicated that the functions of gut digestion and absorption, and degradation of toxic substances were significantly weakened after ex situ conservation. Differences in diet composition between the individuals of the two sites were also observed and the impact of food on gut microbiota compositions within forest musk deer during ex situ conservation was investigated. This study provides a theoretical basis for developing ex situ conservation measures, especially for the protection of forest musk deer.

Comparative study of the function and structure of the gut microbiota in Siberian musk deer and Forest musk deer.

Musk deer are famous for their secretion of musk; however, the scale of artificial breeding of musk deer is limited. Considering the lack of a comprehensive understanding of the gut microbiota, there is a need to study the gut microbiota of Siberian musk deer (SMD). Quantitative PCR analysis and high-throughput sequencing were used to show the differences in gut microbial communities and functions between SMD and forest musk deer (FMD). The relative abundance of Firmicutes was significantly higher in SMD than in FMD, with a corresponding decrease in Bacteroidetes, and showed significant seasonal variation. The gut microbiome of FMD has enriched activity for carbohydrate metabolism, while in SMD, amino acids and energy metabolism was higher. The key enzymatic reactions were related to pyruvate metabolism in SMD; however, in FMD, enzymes that digest cellulose (EC:3.2.1.21, EC:3.2.1.4.) were more abundant, and these were related to the living environment and feeding habits. This indicates that FMD and SMD have significant differences in their microbial communities and functions. Furthermore, antibiotic resistances were identified and significantly different in gut microbes of SMD and FMD. For SMD, seasonal variations alter microbial communities and function. The key enzymes of the short-chain fatty acids (EC:1.3.1.44, EC:6.4.1.2) were significantly different, with higher relative abundance in winter-a mechanism of natural selection and environmental adaptation. This study is the first to analyze the composition of the gut microbiota of SMD and can be used to develop or modify conservation and husbandry strategies for musk deer, to improve their productivity. KEY POINTS: • Significant differences in microbial communities and their function between FMD and SMD. • The energy metabolism and the relative abundance of Firmicutes were significantly higher in SMD. • Seasonal variations alter microbial function in SMD, carbohydrate metabolism was higher in summer.

Population genomics reveals moderate genetic differentiation between populations of endangered Forest Musk Deer located in Shaanxi and Sichuan.

BACKGROUND: Many endangered species exist in small, genetically depauperate, or inbred populations, hence promoting genetic differentiation and reducing long-term population viability. Forest Musk Deer (Moschus berezovskii) has been subject to illegal hunting for hundreds of years due to the medical and commercial values of musk, resulting in a significant decline in population size. However, it is still unclear to what extent the genetic exchange and inbreeding levels are between geographically isolated populations. By using whole-genome data, we reconstructed the demographic history, evaluated genetic diversity, and characterized the population genetic structure of Forest Musk Deer from one wild population in Sichuan Province and two captive populations from two ex-situ centers in Shaanxi Province. RESULTS: SNP calling by GATK resulted in a total of 44,008,662 SNPs. Principal component analysis (PCA), phylogenetic tree (NJ tree), ancestral component analysis (ADMIXTURE) and the ABBA-BABA test separated Sichuan and Shaanxi Forest Musk Deer as two genetic clusters, but no obvious genetic differentiation was observed between the two captive populations. The average pairwise FST value between the populations in Sichuan and Shaanxi ranged from 0.05-0.07, suggesting a low to moderate genetic differentiation. The mean heterozygous SNPs rate was 0.14% (0.11%-0.15%) for Forest Musk Deer at the genomic scale, and varied significantly among three populations (Chi-square = 1.22, p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest (0.11%). The nucleotide diversity of three populations varied significantly (p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest genetic θπ (1.69 × 10-3). CONCLUSIONS: Genetic diversity of Forest Musk Deer was moderate at the genomic scale compared with other endangered species. Genetic differentiation between populations in Sichuan and Shaanxi may not only result from historical biogeographical factors but also be associated with contemporary human disturbances. Our findings provide scientific aid for the conservation and management of Forest Musk Deer. They can extend the proposed measures at the genomic level to apply to other musk deer species worldwide.

Occurrence, genetic diversity and zoonotic potential of Blastocystis sp. in forest musk deer (Moschus berezovskii) in Southwest China.

Blastocystis sp. is a common anaerobic protist with controversial pathogenicity that can infect various animals and humans. However, there are no reports of Blastocystis sp. infections in forest musk deer (Moschus berezovskii). The present study was designed to examine the occurrence, subtype distribution and genetic characterization of Blastocystis sp. in forest musk deer in southwestern China, and to assess the potential for zoonotic transmission. A total of 504 fresh stool samples were collected from captive forest musk deer in four distinct areas of southwestern China. Overall, 14.7% of the forest musk deer (74/504) were found to be infected with Blastocystis sp. The highest occurrence of Blastocystis sp. was observed in Dujiangyan (27.5%), followed by Maerkang (23.3%). The occurrence of Blastocystis sp. was 7.9% and 4.1% in Shimian and Hanyuan, respectively. Significant differences in the occurrence of Blastocystis sp. among different areas were observed (p < 0.05), while we did not observe significant differences among animals of different age and sex (p > 0.05). Two known zoonotic subtypes (ST1 and ST5) and three animal-predominant subtypes (ST10, ST13, and ST14) were identified, of which ST10 was the most common (36/74, 48.6%). Our findings highlight that forest musk deer may be potential reservoirs of zoonotic human Blastocystis sp. infections.

Title: Présence, diversité génétique et potentiel zoonotique de Blastocystis sp. chez le cerf porte-musc (Moschus berezovskii) dans le sud-ouest de la Chine. Abstract: Blastocystis sp. est un protiste anaérobie commun, de pathogénicité controversée, et qui peut infecter divers animaux et les humains. Cependant, aucun cas d'infection par Blastocystis sp. n'a été rapporté chez le cerf porte-musc (Moschus berezovskii). La présente étude a été conçue pour examiner la présence, la distribution des sous-types et la caractérisation génétique de Blastocystis sp. chez le cerf porte-musc du sud-ouest de la Chine et pour évaluer son potentiel de transmission zoonotique. Au total, 504 échantillons de selles fraîches ont été prélevés sur des cerfs porte-musc captifs dans quatre régions distinctes du sud-ouest de la Chine. Dans l'ensemble, 14,7 % (74/504) des cerfs porte-musc se sont avérés infectés par Blastocystis sp. La plus forte occurrence de Blastocystis sp. a été observée à Dujiangyan (27,5 %), suivi de Maerkang (23,3 %). La présence de Blastocystis sp. était respectivement de 7,9 % et 4,1 % à Shimian et Hanyuan. Des différences significatives dans la présence de Blastocystis sp. entre les différentes zones ont été observées (p < 0,05), alors que nous n'avons pas observé de différences significatives entre les animaux d'âge et de sexe différents (p > 0,05). Deux sous-types zoonotiques connus (ST1 et ST5) et trois sous-types à prédominance animale (ST10, ST13 et ST14) ont été identifiés, dont ST10 était le sous-type le plus courant (36/74, 48,6 %). Nos découvertes mettent en évidence que le cerf porte-musc forestier peut être un réservoir potentiel d'infections à Blastocystis sp.

Changes in Gut Microbiota Composition Associated with the Presence of Enteric Protist Blastocystis in Captive Forest Musk Deer (Moschus Berezovskii).

Blastocystis is a common protistan parasite inhabiting the gastrointestinal tract of a wide range of hosts including humans and domestic and wild animals. Many studies have revealed the associations between Blastocystis and gut microbiome in humans. However, only a few studies have focused on the associations between Blastocystis and gut microbiome of animals, especially in forest musk deer (Moschus berezovskii). We investigated the effects of the Blastocystis colonization on the intestinal bacterial community compositions using amplicon sequencing targeting the V4 variable region of the 16S rRNA. Two subtypes of Blastocystis (ST5 and ST10) and Blastocystis-free (control) were included in this study. We found that compared with the forest musk deer without Blastocystis, ST10-colonized forest musk deer had higher bacterial richness and diversity, while ST5-colonized forest musk deer showed a comparable bacterial diversity. Likewise, beta diversity revealed significant differences in bacterial community structure between ST10-colonized and Blastocystis-free forest musk deer. The proportion of Bacteroidetes were significantly enriched in ST10-colonized forest musk deer. Bacterial community structure between ST5-colonized and Blastocystis-free forest musk deer did not differ significantly. The present study explored the associations between Blastocystis and gut microbial community of forest musk deer for the first time, and revealed ST10 colonization, instead of ST5, is associated with higher bacterial diversity and shifted microbial structure. Our data provides valuable insights into the associations between gut microbiomes and parasites. IMPORTANCE Forest musk deer is listed as an endangered species by International Union for Conservation of Nature Red List, and the Chinese government has introduced captivity breeding measures to curb the rapid decline of the musk deer population since the 1950s. It has been suggested that Blastocystis colonization can modulate the composition of the host's intestinal microbiota, thereby affecting the host health. The present study investigated the effects of the Blastocystis colonization on the gut microbiota in the feces of forest musk deer in Sichuan Province, China. Two subtypes (ST5 and ST10) have differential effects on the bacterial diversity and community composition, suggesting that the study of Blastocystis should be distinguished at the subtype level. Because the pathogenicity of Blastocystis is controversial, pathogenic, or commensal, continuous monitoring of the impact of Blastocystis colonization on the intestinal microbiota is of great significance to assess its health effects on forest musk deer.