Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (Hymenoptera: Megachilidae).

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000Da. The major component has a monoisotopic molecular mass of 1924.20Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.

Evidence for convergent nucleotide evolution and high allelic turnover rates at the complementary sex determiner gene of Western and Asian honeybees.

Our understanding of the impact of recombination, mutation, genetic drift, and selection on the evolution of a single gene is still limited. Here we investigate the impact of all these evolutionary forces at the complementary sex determiner (csd) gene that evolves under a balancing mode of selection. Females are heterozygous at the csd gene and males are hemizygous; diploid males are lethal and occur when csd is homozygous. Rare alleles thus have a selective advantage, are seldom lost by the effect of genetic drift, and are maintained over extended periods of time when compared with neutral polymorphisms. Here, we report on the analysis of 17, 19, and 15 csd alleles of Apis cerana, Apis dorsata, and Apis mellifera honeybees, respectively. We observed great heterogeneity of synonymous (piS) and nonsynonymous (piN) polymorphisms across the gene, with a consistent peak in exons 6 and 7. We propose that exons 6 and 7 encode the potential specifying domain (csd-PSD) that has accumulated elevated nucleotide polymorphisms over time by balancing selection. We observed no direct evidence that balancing selection favors the accumulation of nonsynonymous changes at csd-PSD (piN/piS ratios are all <1, ranging from 0.6 to 0.95). We observed an excess of shared nonsynonymous changes, which suggest that strong evolutionary constraints are operating at csd-PSD resulting in the independent accumulation of the same nonsynonymous changes in different alleles across species (convergent evolution). Analysis of csd-PSD genealogy revealed relatively short average coalescence times ( approximately 6 Myr), low average synonymous nucleotide diversity (piS < 0.09), and a lack of trans-specific alleles that substantially contrasts with previously analyzed loci under strong balancing selection. We excluded the possibility of a burst of diversification after population bottlenecking and intragenic recombination as explanatory factors, leaving high turnover rates as the explanation for this observation. By comparing observed allele richness and average coalescence times with a simplified model of csd-coalescence, we found that small long-term population sizes (i.e., N(e) < 10(4)), but not high mutation rates, can explain short maintenance times, implicating a strong historical impact of genetic drift on the molecular evolution of highly social honeybees.

Lethal fighting between honeybee queens and parasitic workers (Apis mellifera).

Pheromonal signals associated with queen and worker policing prevent worker reproduction and have been identified as important factors for establishing harmony in the honeybee (Apis mellifera) colony. However, "anarchic workers", which can evade both mechanisms, have been detected at low frequency in several honeybee populations. Worker bees of the Cape honeybee, Apis mellifera capensis, also show this anarchistic trait but to an extreme degree. They can develop into so called "pseudoqueens", which release a pheromonal bouquet very similar to that of queens. They prime and release very similar reactions in sterile workers to those of true queens (e.g. suppress ovary activation; release retinue behavior). Here we show in an experimental bioassay that lethal fights between these parasitic workers and the queen (similar to queen-queen fights) occur, resulting in the death of either queen or worker. Although it is usually the queen that attacks the parasitic workers and kills many of them, in a few cases the workers succeeded in killing the queen. If this also occurs in a parasitized colony where the queen encounters many parasitic workers, she may eventually be killed in one of the repeated fights she engages in.