Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity

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

Immune pathways and defence mechanisms in honey bees Apis mellifera

Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens

Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta

Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC50 of 12 mu M, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta

Increased melanizing activity in Anopheles gambiale does not affect development of Plasmodium falciparum

Serpins are central to the modulation of various innate immune responses in insects and are suspected to influence the outcome of malaria parasite infection in mosquito vectors. Three Anopheles gambiae serpins (SRPN1, -2, and -3) were tested for their ability to inhibit the prophenoloxidase cascade, a key regulatory process in the melanization response. Recombinant SPRN1 and -2 can bind and inhibit a heterologous phenoloxiclase-activating protease and inhibit phenoloxidase activation in vitro. Using a reverse genetics approach, we studied the effect of SRPN2 on melanization in An. gambiae adult females in vivo. Depletion of SRPN2 from the mosquito hemolymph increases melanin deposition on foreign surfaces such as negatively charged Sephadex beads. As reported, the knockdown of SRPN2 adversely affects the ability of the rodent malaria parasite Plasmodium berghei to invade the midgut epithelium and develop into oocysts. Importantly, we tested whether the absence of SRPN2 from the hemolymph influences Plasmodium falciparum development. RNAi silencing of SRPN2 in an An. gambiae strain originally established from local populations in Yaounde, Cameroon, did not influence the development of autochthonous field isolates of P. falciparum. This study suggests immune evasion strategies of the human malaria parasite and emphasizes the need to study mosquito innate immune responses toward the pathogens they transmit in natural vector-parasite combinations