Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor.

Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and α-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.

Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor.

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and ß-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-ß-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea.

Cytochrome P450 monooxygenases (CYPs) are present in almost all areas of the tree of life. As one of the largest and most diverse superfamilies of multifunctional enzymes, they play important roles in the metabolism of xenobiotics and biosynthesis of endogenous compounds, shaping the success of insects. In this study, the CYPome (an omics term for all the CYP genes in a genome) diversification was examined in the four Tenebrionidea species through genome-wide analysis. A total of 483 CYP genes were identified, of which 103, 157, 122, and 101 were respectively deciphered from the genomes of Tebebrio molitor, Asbolus verucosus, Hycleus cichorii and Hycleus phaleratus. These CYPs were classified into four major clans (mitochondrial, CYP2, CYP3, and CYP4), and clans CYP3 and CYP4 are most diverse. Phylogenetic analysis showed that most CYPs of these Tenebrionidea beetles from each clan had a very close 1:1 orthology to each other, suggesting that they originate closely and have evolutionally conserved function. Expression analysis at different developmental stages and in various tissues showed the life stage-, gut-, salivary gland-, fat body-, Malpighian tubule-, antennae-, ovary- and testis-specific expression patterns of T. molitor CYP genes, implying their various potential roles in development, detoxification, immune response, digestion, olfaction, and reproduction. Our studies provide a platform to understand the evolution of Tenebrionidea CYP gene superfamily, and a basis for further functional investigation of the T. molitor CYPs involved in various biological processes.

The Wnt gene family in Tenebrio molitor and other coleopterans.

The Wnt gene family is involved in a wide range of developmental processes. Despite its significance, the evolution and function of Wnt genes remain largely unclear. Here, an exhaustive survey of Wnt genes was conducted in Tenebrio molitor and 17 other beetle genomes. A total of 146 Wnt genes were identified, creating a comprehensive coleopteran Wnt gene catalog. Comparative genomics indicates that dynamic evolutionary patterns of Wnt gene loss and duplication occurred in Coleoptera, leading to the diverse Wnt gene repertoire in various beetles. A striking loss of particular Wnt gene subfamilies occurs in Coleoptera. Remarkably, Wnt gene duplication was discovered for the first time in insects. Further analysis of Wnt gene expression in T. molitor indicates that each Wnt gene, including the duplicated ones, has a unique spatial or temporal expression pattern. The current study provides valuable insight into the evolution and functional validation of Wnt genes in Coleoptera.

A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.