Apis mellifera Sampling and Experimental Conditions

2.5.1 Adult A. mellifera sampling conditions

All adult A. mellifera for proteomic analysis were sampled from apiaries in Counties Meath, Galway or Carlow. Samples for viral analysis were from apiaries throughout the Republic of Ireland (Figure 2.1a). Adult bees were sampled from the same area of the hive near the centre brood boards to ensure the same age of bee was taken (Figure 2.1b). A container of bees was sampled and kept frozen until further use. Adults workers were sampled from colonies where the level of parasitization by V. destructor was known. This was determined by examining the sticky inserts placed in the floor of the hive for the level of natural mite drop per daBees were chosen for analysis from very mildly parasitized and very heavily parasitized colonies for comparison. Individual bees were subsequently examined for the presence of Varroa mites before use also. Samples were also chosen from mildly parasitized and heavily parasitized Winter colonies and Summer colonies for comparison.

Figure 2.1a: Example of an apiary showing the hives. Figure 2.1b: Image shows the inside of hive. Each section is a brood board, and adults were sampled from the centre of these.

2.5.2 Worker A. mellifera sampling conditions at defined pupal age

Worker A. mellifera were collected from an apiary in Kilmessan, County Meath, using a queen trapping method. A queen excluder box was placed around an empty brood comb and the queen was placed inside. This was carried out in three different hives. The queen excluder comb allows the worker bees to travel through to tend to the queen but due to her larger size, she remains in place on the brood comb (Figure 2.2). This ensures that all of the eggs that are laid in the hive are contained on this piece of brood comb, helping to ensure that all of the pupae are of a similar age. The containment of all brood onto a single piece of brood comb means that any Varroa present in the colony will all move into this area, increasing natural infestation levels for experimental use of the pupae. Each cell was uncapped and the number of adult female Varroa present upon uncapping was recorded, along with the number of progeny present. A pupa was considered as unparsitized and used as a control sample if there were no female Varroa observed upon uncapping of the cell.

a) b)

Figure 2.2: Example of a queen excluder (metal) being placed over a brood board (wood).

The size of the holes in the excluder means that workers can pass freely but the larger queen is secured in place.

To correctly match the age of the pupae taken for experiments, only pupae in the purple eye phase with no darkening of the cuticle were used.

During development from larvae to adult, eye colour changes from white to purple as the pupae develops and so this can be used as an accurate guide to the age of the pupae (Figure 2.3).

Figure 2.3: Image shows the distinct eye type for the various developmental stages of the A. mellifera pupae.

At the bottom of the image, the headless larval stage can be seen (1) followed on top by the white eye stage of the pupae (2), then the pink eye stage (3), finally followed by the purple eye stage (4).

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2.5.3 Drone A. mellifera sampling conditions at determined pupal age

Drone pupae were received from apiaries in Counties Meath, Carlow and GalwaBrood comb containing drone cells was removed and frozen until use. All pupae used for proteomic and molecular analyses were chosen by age, based on the eye colour and cuticle darkness (See section 2.3.3). As for the worker pupae, each cell was uncapped prior to use and the number of adult and young Varroa inside each cell was enumerated. A pupa was considered to be unparasitized and used as a control sample if no adult female Varroa was observed upon uncapping.

2.5.4 Artificial infestation of A. mellifera pupae by adult female V. destructor mites

A brood comb was obtained from a heavily infested colony in County Carlow and maintained at optimum temperature in an insulated box to be brought back to the laboratory. Upon arrival, the lids were removed from the brood cells piece by piece and the Varroa adult females that ran from each cell were captured and secured in a petri dish using a fine paintbrush. Four worker and two drone pupae were removed from the brood cells using a blunt tweezers to avoid damage, and each was placed in a sterile 1.5ml eppendorf containing a small piece of moist cotton wool to maintain humidity (Figure 2.4). Between eight and twelve adult Varroa were then placed into each tube using the fine paintbrush and the lid closed to secure. The eppendorf tubes were placed in an incubator at 35^oC for 48hours. Pupae were inspected following removal from the incubator to ensure that they were still in a viable condition following 48 hours and only those that looked healthy and had no signs of melanisation were used for subsequent analysis.

Figure 2.4: Method used for artificial infestation of A. mellifera.

Image shows an eppendorf tube containing purple eye stage worker pupa, a number of adult female Varroa, and a piece of moist cotton wool for natural humidity replication.