Background: Tribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species. Results: Around 300 candidate defense proteins are identified based on sequence similarity to homologs known to participate in immune responses. In most cases, paralog counts are lower than those of Drosophila melanogaster or Anopheles gambiae but are substantially higher than those of Apis mellifera. The genome contains probable orthologs for nearly all members of the Toll, IMD, and JAK/STAT pathways. While total numbers of the clip-domain serine proteinases are approximately equal in the fly (29), mosquito (32) and beetle (30), lineage-specific expansion of the family is discovered in all three species. Sixteen of the thirty-one serpin genes form a large cluster in a 50 kb region that resulted from extensive gene duplications. Among the nine Toll-like proteins, four are orthologous to Drosophila Toll. The presence of scavenger receptors and other related proteins indicates a role of cellular responses in the entire system. The structures of some antimicrobial peptides drastically differ from those in other orders of insects. Conclusion: A framework of information on Tribolium immunity is established, which may serve as a stepping stone for future genetic analyses of defense responses in a nondrosophiline genetic model insect.

Background: In eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (Bombyx moriL.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.Results: We generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults ofB. moriand obtained ~2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.Conclusions: We identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.

We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera . Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non‐catalytic structural units in these SP‐like proteins −12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein–protein interactions, including disulphide‐stabilized structures (LDL _r A, SRCR, frizzled, kringle, Sushi, Wonton and Pan/apple), carbohydrate‐recognition domains (C‐type lectin and chitin‐binding), and other modules (such as zinc finger, CUB, coiled coil and Sina). Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos. Multiple sequence alignments revealed evolutionary relationships of honey bee SPs and SPHs with those in Drosophila melanogaster , Anopheles gambiae , and Manduca sexta . We identified homologs of D. melanogaster persephone, M. sexta HP14, PAP‐1 and SPH‐1. A. mellifera genome includes at least five genes for potential SP inhibitors (serpin‐1 through ‐5) and three genes of SP putative substrates (prophenoloxidase, spätzle‐1 and spätzle‐2). Quantitative RT‐PCR analyses showed an elevation in the mRNA levels of SP2, SP3, SP9, SP10, SPH41, SPH42, SP49, serpin‐2, serpin‐4, serpin‐5, and spätzle‐2 in adults after a microbial challenge. The SP41 and SP6 transcripts significantly increased after an injection of Paenibacillus larva , but there was no such increase after injection of saline or Escherichia coli . mRNA levels of most SPs and serpins significantly increased by 48 h after the pathogen infection in 1st instar larvae. On the contrary, SP1, SP3, SP19 and serpin‐5 transcript levels reduced. These results, taken together, provide a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera .

Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 Å, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.