Evidence for convergent nucleotide evolution and high allelic turnover rates at the complementary sex determiner (csd) gene of western and Asian honey bees

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 of these evolutionary forces at the complementary sex determiner (csd) gene which 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 to neutral polymorphisms. Here, we report on the analysis of 17, 19 and 15 csd alleles of Apis cerana, Apis dorsata and Apis mellifera honey bees respectively. We observed great heterogeneity of synonymous (pi S) and nonsynonymous (pi N) polymorphisms across the gene, with a consistent peak in exon 6 and 7. We propose that exons 6 and 7 encode the potential specifying domain (csd-PSD) which 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 (pi N/pi S ratios are all < 1, ranging from 0.6 to 0.95). We observed an excess of shared nonsynonymous changes, which suggests 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 a csd-PSD genealogy revealed relatively short average coalescence times (~6 million years), low average synonymous nucleotide diversity (pi S < 0.09) and a lack of trans-specific alleles which 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 turn-over 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. Ne <104), but not high mutation rates, can explain short maintenance times, implicating a strong impact of genetic drift on the molecular evolution of highly social honey bees

Mating flight duration of Apis mellifera queens: As short as possible, as long as necessary

Polyandry in queen honey bees (Apis mellifera) prolongs the duration of nuptial flights which increases costs and risks. Under conditions of limited drone numbers the hypothesis was tested whether or not there is a threshold for successful mating during mating flight. In 29 queens we found a significant negative correlation between mating flight duration and number of spermatozoa in the spermatheca (Pearson r = -0.38, $P$ = 0.04). This negative correlation supports the idea that queens continuously get information on her mating success during flight and return to the colony as soon as they have met a sufficient number of drones. In case of normal availability of drones queens fly from 10 to 30 minutes, so we compared 2 groups of queens (flight duration less than 30 versus more than 30 minutes). Sperm numbers differed significantly between the two groups (3.0 $\pm$ 0.77 and 1.1 $\pm$ 1.04 million, Wilcoxon, $P$ < 0.001). These results further indicate that queens monitor mating success during flight

NEW PHYLOGENETIC ASPECTS OF THE GENUS APIS

International audienc

Variance in spermatozoa number among Apis dorsata drones and among Apis mellifera drones

Published estimates of the mean spermatozoa numbers for Apis dorsata drones vary from 1.2 × 106 and 2.4 × 106; the number of spermatozoa per individual drone vary from 0.22 × 106 to 2.65 × 106. Counts presented here revealed 1.19 × 106 + 0.25 × 106 spermatozoa in drones sampled near a colony and 1.59 × 106 + 0.18 × 106 in drones sampled at a drone congregation area (DCA) in Sabah, Borneo. The difference between the two sites is significant. Further, the degree of variation in sperm numbers among drones near the colonies was higher than at the DCA. Possible reasons are discussed for spermatozoa number variation between drone samples in A. dorsata and in A. mellifera (published estimates). Furthermore, it is discussed if differences in spermatozoa numbers among fathering males may contribute to differences in patriline proportions within colonies

Fight between virgin queens (Apis mellifera) is initiated by contact to the dorsal abdominal surface

To determine the nature of the stimuli involved in queen recognition, we videotaped fighting behaviour between young virgin queens and developed a bioassay. The results of the bioassay were as follows: (1) Under illumination with red light, the queens responded with stinging behaviour (stB.); thus, lack of visual stimuli did not play an essential role in releasing stB. (2) Tethered queens, narcotised queens, and dead queens were stung, demonstrating that movement was not essential for releasing stB. (3) Reduced contact between queens by placing a single screen between them reduced the stinging response, while queens separated by a double screen, blocking direct contact, had no stinging response. (4) StB. was released when queens were in contact with isolated queen abdomens or dorsal abdominal integuments. (5) Workers fitted with queen dorsal abdominal integument released stB. (6) Fifteen day old queen pupae released stB. We hypothesize that the pheromone triggering fighting behaviour is located on the queen's abdominal tergites, which is the location of the tergite glands

Proteins in spermathecal gland secretion and spermathecal fluid and the properties of a 29 kDa protein in queens of Apis mellifera

One and two dimensional SDS-PAGE were used to characterize the protein pattern of the spermathecal gland secretion and spermathecal fluid in Apis mellifera queen pupae and emerged queens of different ages. The concentration of protein varied from 8.5 and 15.3 mg/mL in the spermathecal fluid, and from 5 to 8.5 mg/mL in the secretion. Development of the protein pattern of the gland secretion and spermathecal fluid was identical from pupae until the age of 3 days. In sexually mature queens (10 days or older) the gland secretion and spermathecal fluid each had one additional band at 79 kDa and at 29 kDa respectively. The 29 kDa protein was N-terminal blocked but several peptide fragments were sequenced after digestion with LysC protease. Only 2 of the sequences showed a distinct homology to the N-terminal half of the glycolytic enzyme triosephosphate isomerase (TPI). TPI antibody reacted with the 29 kDa protein, but the enzymatic activity was only 1/100 compared to TPI of hemolymph. The possible function of the protein is discussed