Pathogenesis of varroosis at the level of the honey bee (Apis mellifera) colony.

The parasitic mite Varroa destructor, in interaction with different viruses, is the main cause of honey bee colony mortality in most parts of the world. Here we studied how effects of individual-level parasitization are reflected by the bee colony as a whole. We measured disease progression in an apiary of 24 hives with differing degree of mite infestation, and investigated its relationship to 28 biometrical, physiological and biochemical indicators. In early summer, when the most heavily infested colonies already showed reduced growth, an elevated ratio of brood to bees, as well as a strong presence of phenoloxidase/prophenoloxidase in hive bees were found to be predictors of the time of colony collapse. One month later, the learning performance of worker bees as well as the activity of glucose oxidase measured from head extracts were significantly linked to the timing of colony collapse. Colonies at the brink of collapse were characterized by reduced weight of winter bees and a strong increase in their relative body water content. Our data confirm the importance of the immune system, known from studies of individually-infested bees, for the pathogenesis of varroosis at colony level. However, they also show that single-bee effects cannot always be extrapolated to the colony as a whole. This fact, together with the prominent role of colony-level factors like the ratio between brood and bees for disease progression, stress the importance of the superorganismal dimension of Varroa research.

Development of a 44K SNP assay focussing on the analysis of a varroa-specific defence behaviour in honey bees (Apis mellifera carnica).

Honey bees are exposed to a number of damaging pathogens and parasites. The most destructive among them, affecting mainly the brood, is Varroa destructor. A promising approach to prevent its spread is to breed for Varroa-tolerant honey bees. A trait that has been shown to provide significant resistance against the Varroa mite is hygienic behaviour, a behavioural response of honey bee workers to brood diseases in general. This study reports the development of a 44K SNP assay, specifically designed for the analysis of hygienic behaviour of individual worker bees (Apis mellifera carnica) directed against V. destructor. Initially, 70,000 SNPs chosen from a large set of SNPs published by the Honey Bee Genome Project were validated for their suitability in the analysis of the Varroa resistance trait 'uncapping of Varroa-infested brood'. This was achieved by genotyping of pooled DNA samples of trait bearers and two trait-negative controls using next-generation sequencing. Approximately 36,000 of these validated SNPs and another 8000 SNPs not validated in this study were selected for the construction of a SNP assay. This assay will be employed in following experiments to analyse individualized DNA samples in order to identify quantitative trait loci (QTL) involved in the control of the investigated trait and to evaluate and possibly confirm QTL found in other studies. However, this assay is not just suitable to study Varroa tolerance, it is as well applicable to analyse any other trait in honey bees. In addition, because of its high density, this assay provides access into genomic selection with respect to several traits considered in honey bee breeding. It will become publicly available via AROS Applied Biotechnology AS, Aarhus, Denmark, before the end of the year 2011.

Toxicity of cryoprotectants to honey bee semen and queens.

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.

Characteristics of the spermathecal contents of old and young honeybee queens.

Sperm are often stored, for a long time after mating, in females of various animal species. In case of the queen honeybee (Apis mellifera), sperm remain fertile for several years in the spermatheca. Little information is available regarding the effect of long-term storage of sperm on its fertility. To evaluate this, enzymes and/or sperm have been analysed from the spermatheca of 75 queens of various ages (0 year Y0, n=14; one year Y1, n=14; two years Y2, n=7; virgin queen VQ, n=40) and semen samples have been taken from 46 drones. The sperm from the spermatheca of older queens move more slowly (F=11.45, P < 0.0001) and show different movement patterns (Chi2=90.0, P < 0.0001) from those of the other groups. The spermatheca content of differently aged mated queens differ significantly with respect to the activities of lactate dehydrogenase (F=3.37, P < 0.05), citrate synthase (F=6.24, P < 0.005) and arginine kinase (F=9.44, P < 0.0006). Glyceraldehyde 3-phosphate dehydrogenase (F=0.10, P=0.91) does not differ significantly. The results suggest considerable changes in the energy metabolic profile of the spermatheca tissue, of the sperm or of both during sperm storage.