Transcriptomics and differential gene expression in Whitmania pigra (Annelida: Clitellata: Hirudinida: Hirudinidae): Contrasting feeding and fasting modes.

The medicinal utility of leeches has been demonstrated through decades of use in modern hospital settings, mainly as relievers of venous congestion following flap or digit replantation surgery. In the present study, we sequence and annotate (through BLAST- and Gene Ontology-based approaches) the salivary transcriptome of the nonblood feeding hirudinid Whitmania pigra and assess the differential gene expression of anticoagulation factors (through both quantitative real-time PCR [qRT-PCR] and in silico-based methods) during feeding and fasting conditions. This was done in order to evince the diversity of putative anticoagulation factors, as well as estimate the levels of upregulation of genes immediately after feeding. In total, we found sequences with demonstrated orthology (via both phylogenetic analyses and BLAST-based approaches) to seven different proteins that have previously been linked to anticoagulatory capabilities-eglin C, bdellin, granulin, guamerin, hyaluronidase, destabilase I, and lipocalin. All of these were recovered from leeches both in the fasting and in the feeding conditions, but all show signs of upregulation in the feeding leeches. Interestingly, our RNA-seq effort, coupled with a hypergeometric test, indicated that the differentially expressed genes were disproportionately involved in three main immunological pathways (endocytosis, peroxisome regulation, and lysosome regulation). The results and implications of the finding of anticoagulants in this nonblood feeding leech and the putative upregulation of anticoagulation factors after feeding are briefly discussed in an evolutionary context.

Analysis of synonymous codon usage pattern of genes in unique non-blood-sucking leech Whitmania pigra.

Whitmania pigra is a unique, fluid-sucking ectoparasite and an anticoagulant medical leech. The codon usage bias (CUB) is the nonuniform usage of synonymous codons in which some codons are more preferred than others. Here, we performed a comprehensive analysis of CUB of genes in W. pigra, analyzing 140 780 transcripts, 59 553 unigenes, and 20 304 qualified coding sequences (CDSs) from the transcriptomic data of W. pigra. The effective number of codons values suggested that the CUB was low in these genes. We recognized profoundly favored codons in W. pigra that have a G/C-ending. Parity rule two-bias plots suggested that both mutation pressure and natural selection might have influenced the CUB. However, neutrality plots revealed that natural selection might have played a major role while mutation pressure might have played a minor role in shaping the CUB. We applied principal component analysis to relative synonymous codon usage values for divided CDSs based on GC content and codon-ending bases. Codon usage in W. pigra had a general inclination toward C-ending codons and natural selection rather than mutation pressure is the dominant force in the genetic evolution of W. pigra. To our knowledge, this is the first study to describe a complete codon usage analysis of W. pigra; this will increase the understanding of CUB and evolution in W. pigra. The analysis of codon usage patterns in W. pigra aids in understanding its evolution and genetic architecture.

Draft Genome of a Blister Beetle Mylabris aulica.

Mylabris aulica is a widely distributed blister beetle of the Meloidae family. It has the ability to synthesize a potent defensive secretion that includes cantharidin, a toxic compound used to treat many major illnesses. However, owing to the lack of genetic studies on cantharidin biosynthesis in M. aulica, the commercial use of this species is less extensive than that of other blister beetle species in China. This study reports a draft assembly and possible genes and pathways related to cantharidin biosynthesis for the M. aulica blister beetle using nanopore sequencing data. The draft genome assembly size was 288.5 Mb with a 467.8 Kb N50, and a repeat content of 50.62%. An integrated gene finding pipeline performed for assembly obtained 16,500 protein coding genes. Benchmarking universal single-copy orthologs assessment showed that this gene set included 94.4% complete Insecta universal single-copy orthologs. Over 99% of these genes were assigned functional annotations in the gene ontology, Kyoto Encyclopedia of Genes and Genomes, or Genbank non-redundant databases. Comparative genomic analysis showed that the completeness and continuity of our assembly was better than those of Hycleus cichorii and Hycleus phaleratus blister beetle genomes. The analysis of homologous orthologous genes and inference from evolutionary history imply that the Mylabris and Hycleus genera are genetically close, have a similar genetic background, and have differentiated within one million years. This M. aulica genome assembly provides a valuable resource for future blister beetle studies and will contribute to cantharidin biosynthesis.

Analysis of codon usage patterns in Hirudinaria manillensis reveals a preference for GC-ending codons caused by dominant selection constraints.

BACKGROUND: Hirudinaria manillensis is an ephemeral, blood-sucking ectoparasite, possessing anticoagulant capacities with potential medical applications. Analysis of codon usage patterns would contribute to our understanding of the evolutionary mechanisms and genetic architecture of H. manillensis, which in turn would provide insight into the characteristics of other leeches. We analysed codon usage and related indices using 18,000 coding sequences (CDSs) retrieved from H. manillensis RNA-Seq data. RESULTS: We identified four highly preferred codons in H. manillensis that have G/C-endings. Points generated in an effective number of codons (ENC) plot distributed below the standard curve and the slope of a neutrality plot was less than 1. Highly expressed CDSs had lower ENC content and higher GC content than weakly expressed CDSs. Principal component analysis conducted on relative synonymous codon usage (RSCU) values divided CDSs according to GC content and divided codons according to ending bases. Moreover, by determining codon usage, we found that the majority of blood-diet related genes have undergone less adaptive evolution in H. manillensis, except for those with homologous sequences in the host species. CONCLUSIONS: Codon usage in H. manillensis had an overall preference toward C-endings and indicated that codon usage patterns are mediated by differential expression, GC content, and biological function. Although mutation pressure effects were also notable, the majority of genetic evolution in H. manillensis was driven by natural selection.

Geographic variation in wing size and shape of the grasshopper Trilophidia annulata (Orthoptera: Oedipodidae): morphological trait variations follow an ecogeographical rule.

A quantitative analysis of wing variation in grasshoppers can help us to understand how environmental heterogeneity affects the phenotypic patterns of insects. In this study, geometric morphometric methods were used to measure the differences in wing shape and size of Trilophidia annulata among 39 geographical populations in China, and a regression analysis was applied to identify the major environmental factors contributing to the observed morphological variations. The results showed that the size of the forewing and hindwing were significantly different among populations; the shape of the forewing among populations can be divided into geographical groups, however hindwing shape are geographical overlapped, and populations cannot be divided into geographical groups. Environmental PCA and thin-plate spline analysis suggested that smaller individuals with shorter and blunter-tip forewings were mainly distributed in the lower latitudes and mountainous areas, where they have higher temperatures and more precipitation. Correspondingly, the larger-bodied grasshoppers, those that have longer forewings with a longer radial sector, are distributed in contrary circumstances. We conclude that the size variations in body, forewing and hindwing of T. annulata apparently follow the Bergmann clines. The importance of climatic variables in influencing morphological variation among populations, forewing shape of T. annulata varies along an environmental gradient.