Infestation experiments

Mite collections and honey bee colonies

Adult V. destructor female mites were collected from drone and worker brood of A. mellifera colonies. We used the same colonies as mite sources to infest both species at each location. Batches of 10–20 collected mites were kept on 15–20 A. mellifera adult workers in plastic cages for at least two days, thereby mimicking the wandering stage of mites prior to reproduction in honey bee brood cells6. The invasive K1 (Korean) V. destructor haplotype was confirmed on a mite subsample (N = 146) using standard methods20 by comparing mitochondrial DNA sequences to references deposited in GenBank (V. destructor Cox-1 gene 458 bp fragment; Korean haplotype, accession number AF106899.1)18. Infestation experiments were performed in six colonies of A. mellifera in Chiang Mai (Northern Thailand; N 18°48′13″ and E 98°57′22″, A. mellifera can only survive in the northern part of the country) and on five, five and four colonies of A. cerana in Chiang Mai, Phatthalung (Southern Peninsular Thailand; Khuan Khanun, N 7°44′57″ and E 100°00′03″) and Samui island (N 9°32′05″ and E 99°58′28″), respectively.

Brood comb preparation and infestations

V. destructor mites reproduce on honey bee brood developing within the wax cells of host nests. They enter these cells just before they are sealed with a wax cap by workers6. In A. mellifera combs, we mapped the positions of cells containing uncapped larvae at the appropriate stage (L5 larvae) on transparent sheets and reintroduced the comb into its colony of origin for natural capping by the workers to occur. Experimental cells were then selected among those that were capped within 6 hours and subsequently used for infestations20. Such infestations do not limit mite reproduction33,34. We partially opened the capping of selected cells using a razor blade edge and assigned one of two treatments: (1) infested cells: a single adult V. destructor female mite was introduced into the cell by using a fine paint brush and (2) control cells: the opening was only lightly brushed without introducing a mite. In both cases, the cell capping was then resealed.

In A. cerana, natural wax combs are not built on wooden frames so they cannot be placed back into their colony of origin to be capped naturally. Thus, the cells used for these experiments were identified based on a visual assessment of larval developmental stage and when possible of partial capping of the cells. Infestation (1) or sham treatment (2) was performed as above for A. mellifera, but the cells were capped with a layer of paper tissue. This layer of paper was sealed to the cell walls with the use of a pre-heated nail head.

For each colony, we used approximately the same number of infested and control cells, which varied from 8–45 and 9–46 respectively, depending on the number of L5 larvae available at the beginning of each infestation experiment. Once the infestations were performed, we cut out the portion of comb with the infested cells and suspended it in an incubator at developing brood conditions, i.e. 34.5 °C and 70–90% RH. Whenever field conditions did not allow us an access to a laboratory incubator, we used a portable incubator connected to a 12 V battery and yielding identical temperature and humidity conditions in order to store the combs. Apis cerana and A. mellifera workers display the same developmental periods before capping: 3 days from egg to larva followed by 6 days of molting larval instars (L1 to L5). After capping occurs, however, their preimaginal stages differ by one day, as A. mellifera workers need 12 days of pupation to fully develop into an adult, while A. cerana workers only require 11 days until bee emergence35. We thus opened the experimental cells one day before the expected emergence date and reported the developmental stage of the opened brood as well as its survival status. We recorded the developmental data as follows: larva, pre-pupa, white-eyed, pink-eyed and purple-eyed pupae were considered to be immature stages of development (stages 1–5), whereas yellow-thorax, grey-winged, grey-thorax, grey-abdomen pupae and adults were defined as mature stage (stage 6) based on the developmental pattern observed in the control at the time of cell opening.

Data and statistical analyses

We used data from cells containing brood that was not affected by chalkbrood infection, by wax moths or by involuntary crushing of the comb. In addition, we only considered artificially infested cells that showed a clear sign of infestation, i.e. the occurrence of feces or the presence of a V. destructor mite in the cell and that were not naturally infested by either Tropilaelaps spp. or local Varroa spp. mites, prior to the experiment.

We ranked each individual from 1 (larva) to 6 (mature), depending on its developmental stage at cell opening. Within each replicate, we calculated an average developmental stage for infested and control cells separately and subtracted the former from the latter, in order to obtain a mean developmental delay per colony. All residuals were normally distributed within populations and the variance was homogeneous between populations. We then compared the average developmental delays within each species and each location by using a one-way ANOVA on log-transformed values combined with a Dunnett’s post hoc test and A. mellifera as the control group. Statistical calculations were performed using Systat software (version 13).

Further analyses were performed with generalized linear models (GLMs) applied to the same developmental stage dataset with colony as random factor and using the lmer function from the package lme436. Response variables were the frequencies of control and infested individuals at each stage in each colony. They were modelled with Poisson error distributions. Statistical tests were performed in R software (R version 3.2.2)37 and are summarized in the Supplementary Table S1.

Larvae wounding experiments

Honey bee colonies and brood comb preparations

We used 5 colonies of Apis mellifera and 5 of Apis cerana kept in Langstroth hives in an apiary at Zhejiang University, Hangzhou (China) for the larval wounding experiments. On the combs of both species, we identified cells by mapping brood at the L5 stage, as described above. We then replaced the comb into the colony for capping by the workers to occur and identified the capped cells 6 hours after the mapping. We used a fine pulled-glass capillary with a diameter similar to that of the V. destructor mite chelicerae (Ø = 50 μm) to wound the larvae by pricking, simulating the wound induced by V. destructor mites feeding on the bee larvae38,39. We inflicted wounding by pressing the tip of the capillary against the cuticle until it gave way. We sterilized the needle with ethanol before each use and replaced it anew for each colony tested. Two experiments were conducted in each species in order to quantify the susceptibility of larvae towards wounding, firstly in the absence of workers and secondly in their presence to compare the removal of wounded brood by workers, i.e. hygienic behaviour.

(1) To measure the susceptibility of wounded larvae, we pricked 30 freshly capped larvae and instead of resealing the wax capping, we removed it and closed the cell with a transparent gelatin cap, allowing for the observation of brood development40. As a control, we opened 15 freshly capped cells and sealed them with a gelatin cap without pricking the larvae (controls for the effect of pricking) and we left 15 cells non-manipulated (controls for the effect of gelatin cap on larva survival). We then placed the comb into an incubator at developing brood conditions, i.e. 34.5 °C and 70% RH and checked the experimental cells every 12 hours during the next three days. At every check, we reported the survival status of the brood in the cells. We considered a larva dead when it turned black and deflated or when it did not stretch in the cell as is typical for the transition to pre-pupal stage. We opened the non-manipulated cells after 72 hours to identify the final state of larvae/pre-pupae.

(2) To measure the hygienic removal of wounded larvae, we opened 30 freshly capped cells and gently pricked the larvae inside of them. As a control, we opened 15 cells without pricking the larvae (positive controls). Wax caps of cells with pricked and non-pricked larvae were then resealed. As a control for the effect of cell opening on removal by workers, we mapped an additional 15 cells that we did not manipulate (negative controls). The comb was then replaced into its original colony and the experimental cells were checked every 12 hours during the next 3 days. At every comb check, we reported the number of mapped brood cells that had been cleaned out by the workers of each species during an interval, i.e. brood removal.

Data and statistical analyses

(1) To assess susceptibility to wounding, we counted the dead and surviving larvae in each species and at each time interval. (2) To compare hygienic removal of wounded brood, we counted the number of wounded brood removed by the workers of each species at each time interval. As the respective mortality and removal rates of the different controls were not significantly different for either experiment (log-rank Mantel test; susceptibility: A. mellifera, df = 1, Chi2 = 0.34, p = 0.56; A. cerana, df = 1, Chi2 = 0.0, p = 0.99; hygienic removal: A. mellifera, df = 1, Chi2 = 2.0, p = 0.16; A. cerana, df = 1, Chi2 = 1.0, p = 0.32), these data were pooled. The validity of our assays was confirmed by the control mortality being below a 15% threshold41. We then compared the mortality rates and wounded brood removal rates between bee species by means of log-rank Mantel tests.