nmiao Li

High-throughput sequencing of microRNA transcriptome and expression assay in the sturgeon, Acipenser schrenckii.

Sturgeons are considered as living fossils and have very high evolutionary, economical and conservation values. The multiploidy of sturgeon that has been caused by chromosome duplication may lead to the emergence of new microRNAs (miRNAs) involved in the ploidy and physiological processes. In the present study, we performed the first sturgeon miRNAs analysis by RNA-seq high-throughput sequencing combined with expression assay of microarray and real-time PCR, and aimed to discover the sturgeon-specific miRNAs, confirm the expressed pattern of miRNAs and illustrate the potential role of miRNAs-targets on sturgeon biological processes. A total of 103 miRNAs were identified, including 58 miRNAs with strongly detected signals (signal >500 and P≤0.01), which were detected by microarray. Real-time PCR assay supported the expression pattern obtained by microarray. Moreover, co-expression of 21 miRNAs in all five tissues and tissue-specific expression of 16 miRNAs implied the crucial and particular function of them in sturgeon physiological processes. Target gene prediction, especially the enriched functional gene groups (369 GO terms) and pathways (37 KEGG) regulated by 58 miRNAs (P<0.05), illustrated the interaction of miRNAs and putative mRNAs, and also the potential mechanism involved in these biological processes. Our new findings of sturgeon miRNAs expand the public database of transcriptome information for this species, contribute to our understanding of sturgeon biology, and also provide invaluable data that may be applied in sturgeon breeding.

Evidence for Retrovirus and Paramyxovirus Infection of Multiple Bat Species in China

Bats are recognized reservoirs for many emerging zoonotic viruses of public health importance. Identifying and cataloguing the viruses of bats is a logical approach to evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats comprising 20 common species, which were sampled in three locations of Yunnan province, by combining reverse transcription polymerase chain reaction (RT-PCR) assays and next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR using degenerate primers, and these paramyxoviruses were mainly classified into three genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of 7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus), 17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the 103 vertebrate viral contigs, 79 displayed low identity (<70%) to known viruses including human viruses at the amino acid level, suggesting that these belong to novel and genetically divergent viruses. Overall, the most frequently identified viruses, particularly in bats from the family Hipposideridae, were retroviruses. The present study expands our understanding of the bat virome in species commonly found in Yunnan, China, and provides insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into humans.

Selective evolution of Toll-like receptors 3, 7, 8, and 9 in bats

Previous studies have shown that bats are reservoirs of a large number of viruses, many of which cause illness and mortality in humans and other animals. However, these bat-associated pathogens cause little, if any, clinicopathology in bats. This long-term adaptation should be reflected somewhat in the immune system. Toll-like receptors (TLRs) are the first line of immune defense against pathogens in vertebrates. Therefore, this study focuses on the selection of TLRs involved in virus recognition. The coding sequences of TLR3, TLR7, TLR8, and TLR9 were sequenced in ten bats. The selection pressure acting on each gene was also detected using branch- and site-specific methods. The results showed that the ancestor of bats and certain other bat sublineages evolved under positive selection for TLR7, TLR8, and TLR9. The highest proportion of positive selection occurred in TLR9, followed by TLR8 and TLR7. All of the positively selected sites were located in the leucine-rich repeat (LRR) domain, which implied their important roles in pathogen recognition. However, TLR3 evolved under negative selection. Our results are not in line with previous studies which identified more positively selected sites in TLR8 in mammalian species. In this study, the most positively selected sites were found in TLR9. This study encompassed more species that were considered natural reservoirs of viruses. The positive selection for TLR7, TLR8, and TLR9 might contribute to the adaptation of pathogen-host interaction in bats, especially in bat TLR9.

Phylogeny and dating of divergences within the genus Thymallus (Salmonidae: Thymallinae) using complete mitochondrial genomes

Abstract The genus Thymallus has attracted increasing attention in recent years because of its sharp demographic decline. In this study, we reported four complete mitochondrial genomes in the Thymallus genus: Baikal-Lena grayling (T. arcticus baicalolenensis), lower Amur grayling (T. tugarinae), Yalu grayling (T. a. yaluensis), and Mongolian grayling (T. brevirostris). The total length of the four new grayling mtDNAs ranged from 16 658 to 16 663 bp, all of which contained 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. The results suggested that mitochondrial genomes could be a powerful marker for resolving the phylogeny within Thymallinae. Our study validated that the Yalu grayling should be a synonym of the Amur grayling (T. grubii) at the whole mitogenome level. The phylogenetic and dating analyses placed the Amur grayling at the deepest divergence node within Thymallus, diverging at ∼14.95 Ma. The lower Amur grayling diverged at the next deepest node (∼12.14 Ma). This was followed by T. thymallus, which diverged at ∼9.27 Ma. The Mongolian grayling and the ancestor of the sister species, T. arcticus and T. arcticus baicalolenensis, diverged at ∼7.79 Ma, with T. arcticus and T. arcticus baicalolenensis separating at ∼6.64 Ma. Our study provides far better resolution of the phylogenetic relationships and divergence dates of graylings than previous studies.

Genetic diversity and population demography of the Chinese crocodile lizard (Shinisaurus crocodilurus) in China.

The Chinese crocodile lizard Shinisaurus crocodilurus is a critically endangered species, listed in Appendix II of CITES. Its populations and habitat in China have undergone significant changes in recent years. Understanding the genetic variability and phylogeography of this species is very important for successful conservation. In this study, samples were taken from 11 wild ponds and two captive populations in China. We sequenced mitochondrial CYTB, partial ND6, and partial tRNA-Glu and genotyped 10 microsatellite loci. Our analyses of these data showed low genetic variability, no strong isolation caused by distance, and a lack of a phylogeographic structure in this species. Based on our results, the basal divergence between two clades of S. crocodilurus in China may have been caused by the formation of the Pearl River system. We found a population expansion in one of these clades. Microsatellite analysis indicated the presence of three clusters, separated by significant genetic differences. We found that most individuals in the two captive populations were from the Luokeng (Guangdong) and Guangxi wild source populations, respectively.

Conservation, sex-biased expression and functional annotation of microRNAs in the gonad of Amur sturgeon (Acipenser schrenckii)

MicroRNAs (miRNAs) are involved in post-transcriptional gene regulation and have crucial roles in regulating the expression of gametogenesis-related genes in animals. However, the mechanism of sex determination and differentiation in the sturgeon has remained unclear. Identifying miRNAs and characterizing sex-biased miRNA expression is therefore critical for understanding the role of miRNAs during sexual differentiation in sturgeon. In this study, five different tissues from sturgeon before sex differentiation and the gonads were used for miRNA expression profiling. We screened 1037 miRNAs in miRBase 20.0 and an additional 103 sturgeon miRNAs using microarray and real-time PCR. We found that the sequences of 477 miRNAs out of a total of 1140 miRNAs were highly conserved (100%) among different fish species. From a total of 663 non-redundant miRNA probes, 481 miRNAs were detected in the gonads of both sexes. Of the 148 miRNAs that were identified to have sex-biased expression patterns between the testis and the ovary (P<0.01), 21 miRNAs (14.19%) were relatively highly expressed in the testis or the ovary with fold-changes >2. The microarray expression patterns of 13 randomly selected sex-biased miRNAs were validated using real-time PCR. Target gene prediction revealed a significant enrichment of functional groups (88 GO terms) and 18 KEGG pathways (P<0.05) and suggested that there are interactions between sex-biased miRNAs and 25 putative gametogenesis-related targets. Therefore, our miRNA expression analysis in juvenile A. schrenckii establishes a foundation for understanding and further investigating the role of miRNAs in sturgeon sex differentiation.

The complete mitochondrial genome of the Acipenser schrenckii (Acipenseridae: Acipenser)

Abstract The Acipenser schrenckii is an ancient freshwater Chondrostei species, which is mainly distributed in Amur River in China and Russian. The complete mitochondrial genome of A. schrenckii was obtained for the first time in this study. The circular genome (16,548 bp in length) consists of 37 typical animal mitochondrial genes (13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes) and 1 control region. Overall base composition of the complete mitochondrial DNA was 30% A, 24% T, 29.4% C and 16.5% G with AT (54%).

Transcriptomic analysis identifies genes and pathways related to myrmecophagy in the Malayan pangolin (Manis javanica)

The Malayan pangolin (Manis javanica) is an unusual, scale-covered, toothless mammal that specializes in myrmecophagy. Due to their threatened status and continuing decline in the wild, concerted efforts have been made to conserve and rescue this species in captivity in China. Maintaining this species in captivity is a significant challenge, partly because little is known of the molecular mechanisms of its digestive system. Here, the first large-scale sequencing analyses of the salivary gland, liver and small intestine transcriptomes of an adult M. javanica genome were performed, and the results were compared with published liver transcriptome profiles for a pregnant M. javanica female. A total of 24,452 transcripts were obtained, among which 22,538 were annotated on the basis of seven databases. In addition, 3,373 new genes were predicted, of which 1,459 were annotated. Several pathways were found to be involved in myrmecophagy, including olfactory transduction, amino sugar and nucleotide sugar metabolism, lipid metabolism, and terpenoid and polyketide metabolism pathways. Many of the annotated transcripts were involved in digestive functions: 997 transcripts were related to sensory perception, 129 were related to digestive enzyme gene families, and 199 were related to molecular transporters. One transcript for an acidic mammalian chitinase was found in the annotated data, and this might be closely related to the unique digestive function of pangolins. These pathways and transcripts are involved in specialization processes related to myrmecophagy (a form of insectivory) and carbohydrate, protein and lipid digestive pathways, probably reflecting adaptations to myrmecophagy. Our study is the first to investigate the molecular mechanisms underlying myrmecophagy in M. javanica, and we hope that our results may play a role in the conservation of this species.

Diets Alter the Gut Microbiome of Crocodile Lizards

The crocodile lizard is a critically endangered reptile, and serious diseases have been found in this species in recent years, especially in captive lizards. Whether these diseases are caused by changes in the gut microbiota and the effect of captivity on disease remains to be determined. Here, we examined the relationship between the gut microbiota and diet and disease by comparing the fecal microbiota of wild lizards with those of sick and healthy lizards in captivity. The gut microbiota in wild crocodile lizards was consistently dominated by Proteobacteria (∼56.4%) and Bacteroidetes (∼19.1%). However, the abundance of Firmicutes (∼2.6%) in the intestine of the wild crocodile lizards was distinctly lower than that in other vertebrates. In addition, the wild samples from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve also had a high abundance of Deinococcus–Thermus while the wild samples from Guangxi Daguishan Crocodile Lizard National Nature Reserve had a high abundance of Tenericutes. The gut microbial community in loach-fed crocodile lizards was significantly different from the gut microbial community in the earthworm-fed and wild lizards. In addition, significant differences in specific bacteria were detected among groups. Notably, in the gut microbiota, the captive lizards fed earthworms resulted in enrichment of Fusobacterium, and the captive lizards fed loaches had higher abundances of Elizabethkingia, Halomonas, Morganella, and Salmonella, all of which are pathogens or opportunistic pathogens in human or other animals. However, there is no sufficient evidence that the gut microbiota contributes to either disease A or disease B. These results provide a reference for the conservation of endangered crocodile lizards and the first insight into the relationship between disease and the gut microbiota in lizards.

Acidic mammalian chitinase gene is highly expressed in the special oxyntic glands of Manis javanica

The Malayan pangolin (Manis javanica) is a mammal that feeds primarily on ants and termites, which contain the energy‐rich carbohydrate chitin. Chitin is digestible by endogenous enzymes of the typical mammalian gastrointestinal tract, especially the acidic mammalian chitinase (AMCase). The objective of this research was to determine whether AMCase activity is expressed in the stomach of M. javanica. The stomach tissues were divided into three parts: the gastric sack, the oxyntic glands, and the pyloric musculature, which were assayed by conventional RT‐PCR, quantitative reverse transcriptase‐coupled PCR (qPCR) and western blot. Information regarding 3D structural models of AMCase was also obtained. In conclusion, acidic mammalian chitinase is highly expressed in the oxyntic glands of the M. javanica species.