The Effect of Disease and Season to Hepatopancreas and Intestinal Mycobiota of Litopenaeus vannamei

Increasing evidence has manifested that the gut bacterial microbiota of shrimps is closely related to the environmental factors, host developmental stage and health status like that of humans and animals does. These studies have provided an important guidance for improving shrimp culture benefits. In practice, aside from bacteria, eukaryotic microorganisms dominated by fungal microbiota (mycobiota), also play a key role in host growth, metabolism and homeostasis. However, little so far is known about the mycobiota in the digestive tract of shrimp. In this study, we used high-throughput sequencing of internal transcribed spacer 1 region to characterize the hepatopancreas and intestinal mycobiota of Pacific white shrimp and their connections with disease incidence and seasonal variation. The results showed that the hepatopancreas and intestinal mycobiota of Litopenaeus vannamei are dominated by the phyla Ascomycota and Basidiomycota, and the genera Alternaria, Tuber, Hortaea, Sarocladium, and Stagonospora. The fungal microbiota significantly varies under the influence of disease and seasonal variation. Sick shrimps had a higher level of potential pathogenic fungus, Candida in the intestine. Healthy shrimps had a higher abundance of the genera Didymella and Filobasidium in the gut, and Pyrenochaetopsis in the hepatopancreas. Of note, most of the fungi carried by Pacific white shrimps were pathogens to humans. This study has revealed the intestinal and hepatopancreas mycobiota of L. vannamei and the effects of diseases and seasonal variation to the mycobiota. Our study provides important guidance for Pacific white shrimp farming and sheds further insight on the fungal microbiota

Identification and differential expression of piRNAs in the gonads of Amur sturgeon (Acipenser schrenckii)

Objective Sturgeons are considered living fossils, and have a very high conservation and economic value. Studies on the molecular mechanism of sturgeon gonadal development and sex differentiation would not only aid in understanding vertebrate sex determination but also benefit sturgeon aquaculture. Piwi-interacting RNAs (piRNAs) have been shown to function in germline or gonadal development. In this study, we performed small RNA deep sequencing and microarray hybridization to identify potential sturgeon piRNAs. Methods Male and female sturgeon gonads were collected and used for small RNA sequencing on an Illumina HiSeq platform with the validation of piRNA expression by microarray chip. The program Bowtie and k-mer scheme were performed to filter small RNA reads and discover potential sturgeon piRNAs. A known piRNA database, the coding sequence (CDS), 5′ and 3′ untranslated region (UTR) database of the A. Schrenckii transcriptome, Gene Ontology (GO) database and KEGG pathway database were searched subsequently to analyze the potential bio-function of sturgeon piRNAs. Results A total of 875,679 putative sturgeon piRNAs were obtained, including 93 homologous to known piRNAs and hundreds showing sex-specific and sex-biased expression. Further analysis showed that they are predominant in both the ovaries and testes and those with a sex-specific expression pattern are nearly equally distribution between sexes. This may imply a relevant role in sturgeon gonadal development. KEGG pathway and GO annotation analyses indicated that they may be related to sturgeon reproductive processes. Conclusion Our study provides the first insights into the gonadal piRNAs in a sturgeon species and should serve as a useful resource for further elucidation of the gene regulation involved in the sex differentiation of vertebrates. These results should also facilitate the technological development of early sex identification in sturgeon aquaculture

Identification of Austwickia chelonae as cause of cutaneous granuloma in endangered crocodile lizards using metataxonomics

The crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) is an endangered reptile species, and in recent years many have died from diseases, especially the rescued and breeding individuals. However, pathogens underlying these diseases are unclear. In this study, we report our effort in rapidly identifying and isolating the pathogen that causes high mortality in crocodile lizards from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve. The typical symptom is cutaneous granuloma in the infected crocodile lizards. Metagenomic next-generation sequencing (mNGS) is a comprehensive approach for sequence-based identification of pathogenic microbes. In this study, 16S rDNA based mNGS was used for rapid identification of pathogens, and microscopy and microbe isolation were used to confirm the results. Austwickia chelonae was identified to be the dominant pathogen in the granuloma using 16S rDNA based mNGS. Chinese skinks were used as an animal model to verify the pathogenicity of A. chelonae to fulfill Koch’s postulates. As expected, subcutaneous inoculation of A. chelonae induced granulomas in the healthy Chinese skinks and the A. chelonae was re-isolated from the induced granulomas. Therefore, A. chelonae was the primary pathogen that caused this high mortality disease, cutaneous granuloma, in crocodile lizards from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve. Antibiotics analysis demonstrated that A. chelonae was sensitive to cephalothin, minocycline and ampicillin, but not to kanamycin, gentamicin, streptomycin and clarithromycin, suggesting a possible treatment for the infected crocodile lizards. However, surgical resection of the nodules as early as possible was recommended. This study is the first report of pathogenic analysis in crocodile lizards and provides a reference for disease control and conservations of the endangered crocodile lizards and other reptiles. In addition, this study indicated that mNGS of lesions could be used to detect the pathogens in animals with benefits in speed and convenient

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

Diverse single-stranded DNA viruses from viral metagenomics on a cynopterus bat in China

Bats serve as reservoirs for many emerging viruses. Cressdnaviruses can infect a wide range of animals, including agricultural species, such as pigs, in which porcine circoviruses cause severe gastroenteritis. New cressdnaviruses have also attracted considerable attention recently, due to their involvement with infectious diseases. However, little is known about their host range and many cressdnaviruses remain poorly characterized. We identified and characterized 11 contigs consisting of previously unknown cressdnaviruses from a rectal swab sample of a Cynopterus bat collected in Yunnan Province, China, in 2011. Full genomes of two cressdnaviruses (OQ267680, 2069 nt; OQ351951, 2382 nt), and a nearly complete genome for a third (OQ267683, 2361 nt) were obtained. Phylogenetic analyses and the characteristics of these viral genomes suggest a high degree of ssDNA virus diversity. These results shed light on cressdnavirus diversity and the probable role of Cynopterus bats as their hosts

Top 20 highest expressed miRNAs of <i>Acipenser schrenckii</i> identified by Illumina sequencing.

<p>ASY_id, <i>A. schrenckii</i> mature miRNAs ID annotated as described in the text. For detailed information about sturgeon miRNAs please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115251#pone.0115251.s005" target="_blank">S2 Table</a>.</p><p>Top 20 highest expressed miRNAs of <i>Acipenser schrenckii</i> identified by Illumina sequencing.</p

Classification of small RNA reads of <i>Acipenser schrenckii</i>.

a<p>miRNA reads identified by searching against the Sanger miRBase (Release 19).</p>b<p>Screening against the Rfam database to identify the noncoding RNAs using the program Bowtie (-v 0 -k 1).</p>c<p>Screening against the RepeatMasker database to identify the repeat sequences using the program Bowtie (-v 0 -k 1).</p>d<p>Novel miRNAs were identified by miREvo and mirdeep2 (-i -r -M -m -k -p 10 -g 50000; quantifier.pl -p -m -r -y -g 0 -T 10).</p><p>Classification of small RNA reads of <i>Acipenser schrenckii</i>.</p

Hierarchical analysis of molecular variance (AMOVA) apportioned among geographical regions, ponds, and individuals.

<p>*p<0.05; **p<0.01; ***p<0.001.</p