Fig. 4. Pedigree experimental design.

A three-generation Varroa pedigree (F0-in red, F1-in orange, and F2-in yellow) of 30 families was constructed via the following steps: a Collecting a naturally infested family: foundress female mite (F0), and her two adult offspring: male and female F1 mites (F0 male’s genotype was not possible to study because the F0 female was previously mated, typically by a single male); b “Incubation phase”: Transferring the young F1 female onto a bee pupa for 1-4 days, till cuticle sclerotized and darkened; c “Dispersal phase”: Transferring F1 female onto a nurse adult bee for another 3 days; d Introducing the matured F1 female to a freshly capped 5th instar larva cell; e F1 female reproducing inside the bee cell; f 10 days post introduction, collecting the artificially infested family: F1 female and her F2 offspring. The F2 generation included a son and one or more daughter nymphs. This design provided known parents and genotypes for the F1 and F2 generations, allowing a detailed examination of inheritance.