Phenotypic and Genetic Analyses of the Varroa Sensitive Hygienic Trait in Russian Honey Bee (Hymenoptera: Apidae) Colonies

April Phenotypic and Genetic Analyses of the Varroa Sensitive Hygienic Trait in Russian Honey Bee (Hymenoptera: Apidae) Colonies Maria J. Kirrane 0 1 Lilia I. de Guzman 0 1 Beth Holloway 0 1 Amanda M. Frake 0 1 Thomas E. Rinderer 0 1 Pdraig M. Whelan 0 1 0 1 School of Biological, Earth and Environmental Sciences (BEES), University College Cork, Cork, Ireland, 2 Environmental Research Institute (ERI), University College Cork , Lee Road, Cork, Ireland, 3 USDA-ARS, Honey Bee Breeding , Genetics and Physiology Laboratory , 1157 Ben Hur Road, Baton Rouge , Louisiana 70820, United States of America, 4 USDA-ARS, Honey Bee Breeding, Genetics and Physiology Laboratory , 1157 Ben Hur Road, Baton Rouge, Louisiana 70820 , United States of America 1 Academic Editor: Nicolas Chaline, Universidade de Sao paulo , BRAZIL Varroa destructorcontinues to threaten colonies of European honey bees. General hygiene, and more specific Varroa Sensitive Hygiene (VSH), provide resistance towards the Varroa mite in a number of stocks. In this study, 32 Russian (RHB) and 14 Italian honey bee colonies were assessed for the VSH trait using two different assays. Firstly, colonies were assessed using the standard VSH behavioural assay of the change in infestation of a highly infested donor comb after a one-week exposure. Secondly, the same colonies were assessed using an actual brood removal assay that measured the removal of brood in a section created within the donor combs as a potential alternative measure of hygiene towards Varroa-infested brood. All colonies were then analysed for the recently discovered VSH quantitative trait locus (QTL) to determine whether the genetic mechanisms were similar across different stocks. Based on the two assays, RHB colonies were consistently more hygienic toward Varroa-infested brood than Italian honey bee colonies. The actual number of brood cells removed in the defined section was negatively correlated with the Varroa infestations of the colonies (r2 = 0.25). Only two (percentages of brood removed and reproductive foundress Varroa) out of nine phenotypic parameters showed significant associations with genotype distributions. However, the allele associated with each parameter was the opposite of that determined by VSH mapping. In this study, RHB colonies showed high levels of hygienic behaviour towards Varroa -infested brood. The genetic mechanisms are similar to those of the VSH stock, though the opposite allele associates in RHB, indicating a stable recombination event before the selection of the VSH stock. The measurement of brood removal is a simple, reliable alternative method of measuring hygienic behaviour towards Varroa mites, at least in RHB stock. - Competing Interests: The authors have declared that no competing interests exist. Hygienic behaviour in honey bees was first studied by Rothenbuhler [1] in relation to the brood disease American Foulbrood (AFB). Hygiene involves three components, namely: the detection, uncapping and removal of diseased or dead brood from the hive [2]. In addition to AFB, it is a means of resistance to a number of in-hive pests including wax moths [3] and small hive beetles [4]. The invasive mite Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae) continues to be regarded as the greatest threat facing honey bees worldwide [5]. In the mites native host, Apis cerana, Varroa only reproduces in drone brood, ensuring populations remain at levels that do not result in significant harm (Rath, 1999). In addition, hygienic removal of Varroa-infested brood contributes to resistance in this bee [6, 7]. Hygienic behaviour has been studied widely in A. mellifera and has been found to be one of a number of traits that are capable of instilling resistance towards the Varroa mite in its invaded host [2, 8]. Hygienic behaviour is a heritable trait [9, 10] and Varroa specific hygiene is the basis of resistance in the Varroa Sensitive Hygienic (VSH) stock from the USDA Laboratory in Baton Rouge, LA [11]. General hygienic behaviour also confers some resistance toward the Varroa mite in the Minnesota hygienic line [11, 12] and may contribute to the resistance of Varroa-surviving colonies in France [13]. A large proportion of Varroa mites in VSH colonies enter cells and either die, produce no progeny, produce only male or only immature progeny [10]. In 2006, Ibrahim and Spivak [14] found that non-reproduction of mites was related to hygienic behaviour and the term Varroa Sensitive Hygiene (VSH) was coined [15]. These bees preferentially remove mite-infested pupae that are between 3 to 5 days post-capping [15]. Varroa mite reproduction is heavily synchronized with the reproductive program of its bee host [16]. Foundress mites must produce at least a mature male and mature female mite within the natal cell in order to produce viable offspring [17]. Mites removed from cells by hygienic behaviour may be forced to reinvade new cells in order to complete reproduction [15]. A break in synchrony between the reproductive cycle of the re-invading mite and the development of its host has previously been shown to negatively impact mite reproduction [18, 19]. Thus, the removal behaviour of VSH stock significantly lowers the population of Varroa mites in these colonies. Another Varroa-resistant stock maintained by the USDA is the Russian honey bee (RHB) [20]. RHB colonies maintain consistently lower levels of Varroa compared with unselected stocks [2123]. No overwhelming resistance mechanism has been identified [23]. However, an increased phoretic period of Varroa on adult bees leads to mites being more susceptible to the bees grooming behaviour [24]. Using the freeze-killed brood (FKB) assay, [25] showed that RHB colonies consistently removed dead brood at levels high enough to be considered hygienic (>95% brood removed within 48 h over two assays). Non-reproduction of Varroa foundresses has also been found to be a contributing factor in the resistance of RHB stocks [26], [23]. This is in part due to the comb built by RHBs but could also be caused, in part, by RHB brood stimulating hygienic behaviour in adult bees [23]. Although originally thought to be controlled by simple Mendelian genetics [1], more recent studies have determined hygienic behaviour to be a more quantitative trait [27, 28] and in fact up to seven loci may be responsible for its expression [29, 30]. Quantitative trait loci (QTL) mapping of genotypes of individual VSH stock that uncapped and removed infested pupae identified candidate genes associated with vision, olfaction, memory and dopamine reception [31]. Those bees that were homozygous for the alleles identified as being associated with the trait were more likely to be observed expressing the behaviour [31]. Differential up-regulation and down-regulation of genes associated with neuronal wiring, olfaction and visual signalling has been shown in VSH and non-VSH stock [32]. In addition, VSH stock and Africanized bee gen (...truncated)