IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella.

The assembled and annotated genome of the masked palm civet (Paguma larvata).

BACKGROUND: The masked palm civet (Paguma larvata) acts as an intermediate host of severe acute respiratory syndrome coronavirus (SARS-CoV), which caused SARS, and transfered this virus from bats to humans. Additionally, P. larvata has the potential to carry a variety of zoonotic viruses that may threaten human health. However, genome resources for P. larvata have not been reported to date. FINDINGS: A chromosome-level genome assembly of P. larvata was generated using PacBio sequencing, Illumina sequencing, and Hi-C technology. The genome assembly was 2.44 Gb in size, of which 95.32% could be grouped into 22 pseudochromosomes, with contig N50 and scaffold N50 values of 12.97 Mb and 111.81 Mb, respectively. A total of 21,582 protein-coding genes were predicted, and 95.20% of the predicted genes were functionally annotated. Phylogenetic analysis of 19 animal species confirmed the close genetic relationship between P. larvata and species belonging to the Felidae family. Gene family clustering revealed 119 unique, 243 significantly expanded, and 58 significantly contracted genes in the P. larvata genome. We identified 971 positively selected genes in P. larvata, and one known human viral receptor gene PDGFRA is positively selected in P. larvata, which is required for human cytomegalovirus infection. CONCLUSIONS: This high-quality genome assembly provides a valuable genomic resource for exploring virus-host interactions. It will also provide a reliable reference for studying the genetic bases of the morphologic characteristics, adaptive evolution, and evolutionary history of this species.

Detection of a novel Pestivirus strain in Java ticks (Amblyomma javanense) and the hosts Malayan pangolin (Manis javanica) and Chinese pangolin (Manis pentadactyla).

Pangolins are endangered animals and are listed in the CITES Appendix I of the Convention International Trade Endangered Species of Wild Fauna and Flora as well as being the national first-level protected wild animal in China. Based on a few reports on pangolins infected with pestiviruses of the Flaviviridae family, Pestivirus infections in pangolins have attracted increasing attention. Pangolin pestivirus is a pathogen that may cause diseases such as acute diarrhea and acute hemorrhagic syndrome. To better understand the epidemiology and genomic characterization of pestiviruses carried by pangolins, we detected pestiviruses in dead Malayan pangolin using metavirome sequencing technology and obtained a Pestivirus sequence of 12,333 nucleotides (named Guangdong pangolin Pestivirus, GDPV). Phylogenetic tree analysis based on the entire coding sequence, NS3 gene or RdRp gene sequences, showed that GDPV was closely related to previously reported pangolin-derived Pestivirus and clustered into a separate branch. Molecular epidemiological investigation revealed that 15 Pestivirus-positive tissues from two pangolins individuals with a positivity rate of 5.56%, and six Amblyomma javanense carried pestiviruses with a positivity rate of 19.35%. Moreover, the RdRp gene of the Pestivirus carried by A. javanense showed a high similarity to that carried by pangolins (93-100%), indicating A. javanense is likely to represent the vector of Pestivirus transmission. This study expands the diversity of viruses carried by pangolins and provides an important reference value for interrupting the transmission route of the virus and protecting the health of pangolins.

Transcriptome analysis reveals the genetic basis underlying the development of skin appendages and immunity in hedgehog (Atelerix albiventris).

The expression of hair features is an evolutionary adaptation resulting from interactions between many organisms and their environment. Elucidation of the mechanisms that underlie the expression of such traits is a topic in evolutionary biology research. Therefore, we assessed the de novo transcriptome of Atelerix albiventris at three developmental stages and compared gene expression profiles between abdomen hair and dorsal spine tissues. We identified 328,576 unigenes in our transcriptome, among which 4,435 were differentially expressed between hair- and spine-type tissues. Dorsal and abdomen skin tissues 5 days after birth were compared and the resulting DEGs were mainly enriched in keratin filament, epithelium cell differentiation, and epidermis development based on GO enrichment analysis, and tight junction, p53, and cell cycle signaling pathways based on KEGG enrichment analysis. MBP8, SFN, Wnt1 and KRT1 gene may involve in the development of hedgehog skin and its appendages. Strikingly, DEGs in hair-type tissues were also significantly enriched in immune-related terms and pathways with hair-type tissues exhibiting more upregulated immune genes than spine-type tissues. Our study provided a list of potential genes involved in skin appendage development and differentiation in A. albiventris, and the candidate genes provided valuable information for further studies of skin appendages.

Combined proteomics and transcriptomics reveal the genetic basis underlying the differentiation of skin appendages and immunity in pangolin.

Pangolin (Mains javanica) is an interesting endangered mammal with special morphological characteristics. Here, we applied proteomics and transcriptomics to explore the differentiation of pangolin skin appendages at two developmental stages and to compare gene expression profiles between abdomen hair and dorsal scale tissues. We identified 4,311 genes and 91 proteins differentially expressed between scale-type and hair-type tissue, of which 6 genes were shared by the transcriptome and proteome. Differentiation altered the abundance of hundreds of proteins and mRNA in the two types of skin appendages, many of which are involved in keratinocyte differentiation, epidermal cell differentiation, and multicellular organism development based on GO enrichment analysis, and FoxO, MAPK, and p53 signalling pathways based on KEGG enrichment analysis. DEGs in scale-type tissues were also significantly enriched in immune-related terms and pathways compared with that in hair-type tissues. Thus, we propose that pangolins have a normal skin innate immune system. Compared with the abdomen, the back skin of pangolins had more genes involved in the regulation of immune function, which may be an adaptive adjustment for the vulnerability of scaly skin to infection and injury. This investigation provides a scientific basis for the study of development and immunity of pangolin skin, which may be helpful in the protection of wild pangolin in China.

SMRT sequencing of the full-length transcriptome of the Sunda pangolin (Manis javanica).

It is widely known that transcriptional diversity contributes greatly to biological regulation in eukaryotes. With the development of next-generation sequencing (NGS) technologies, several studies on RNA sequencing have considerably improved our understanding of transcriptome complexity. However, obtaining full-length (FL) transcripts remains a considerable challenge because of difficulties in short read-based assembly. In the present study, single-molecule real-time (SMRT) sequencing and NGS were combined to generate the complete and FL transcriptome of Manis javanica. The results provide a comprehensive set of reference transcripts and hence contribute to the improved annotation of the M. javanica genome. We obtained 45,530 high-confidence transcripts from 19,109 genic loci, of which 8014 genes have not yet been annotated within the M. javanica genome. Furthermore, we revealed 8824 long-chain noncoding RNAs (lncRNAs). A total of 30,199 alternative splicing (AS) and 11,184 alternative polyadenylation (APA) events were identified in the sequencing data. The structure and expression level of 59 digestive enzyme genes, including 13 carbohydrase genes, 28 lipase genes and 18 protease genes, were analyzed, which might provide original data for further research on M. javanica.

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.

The Fecal Metagenomics of Malayan Pangolins Identifies an Extensive Adaptation to Myrmecophagy.

The characteristics of flora in the intestine of an animal, including the number and abundance of different microbial species and their functions, are closely related to the diets of the animal and affect the physical condition of the host. The Malayan pangolin (Manis javanica) is an endangered species that specializes in myrmecophagy. Analyzing the microbiome in the intestine of the pangolin is imperative to protect this species. By sequencing the metagenomes of the feces of four pangolins, we constructed a non-redundant catalog of 211,868 genes representing 1,811 metagenomic species. Taxonomic annotation revealed that Bacteroidetes (49.9%), Proteobacteria (32.2%), and Firmicutes (12.6%) are the three main phyla. The annotation of gene functions identified 5,044 genes from 88 different glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes database and 114 gene modules related to chitin-degrading enzymes, corresponding to the catalytic domains of GH18 family enzymes, containing chitinase genes of classes III and V in the dataset. Fourteen gene modules corresponded to the catalytic domains of GH19 family enzymes, containing chitinase genes of classes I, II, and IV. These genes were found in 37 species belonging to four phyla: Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria. Moreover, when the metabolic pathways of these genes were summarized, 41,711 genes were associated with 147 unique KEGG metabolic pathways, and these genes were assigned to two Gene Ontology terms: metabolic process and catalytic activity. We also found several species that likely play roles in the digestion of cellulose and may be able to degrade chitin, including Enterobacter cloacae, Lactococcus lactis, Chitinimonas koreensis, and Chitinophaga pinensis. In addition, we identified some intestinal microflora and genes related to diseases in pangolins. Twenty-seven species were identified by STAMP analysis as differentially abundant in healthy and diseased animals: 20 species, including Cellulosilyticum lentocellum and Lactobacillus reuteri, were more abundant in healthy pangolins, while seven species, including Odoribacter splanchnicus, Marinilabilia salmonicolor, Xanthomonas citri, Xanthomonas vasicola, Oxalobacter formigenes, Prolixibacter bellariivorans, and Clostridium bolteae, were more abundant in diseased pangolins. These results will support the efforts to conserve pangolins.

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.