Breeding honey bees for varroa tolerance

60 SEVEN Breeding honey bees for varroa tolerance Norman L Carreck Introduction Chapters Four, Five and Six have reviewed the current options for the control of varroa, outlining their inherent advantages and disadvantages. From the earliest days of the varroa problem, however, beekeepers have dreamt of breeding bees that would be resistant or tolerant to the mite, so that all of the disadvantages of these treatments could be reduced or avoided. Despite a number of announcements of resistant bees, and commercial strains marketed in various countries and at various times, progress has actually been painfully slow. Survival of bees with varroa - natural varroa tolerance? There have long been anecdotal accounts of European honey bees surviving without treatment in the presence of varroa. A well documented case concerned Italian bees on the island of Fernando de Noronha, off the coast of Brazil (de Jong and Soares, 1997), but details of any explanation for their survival were sketchy, although climate was thought to be an important factor. In Italy, Norberto Milani reported feral colonies living with varroa (Milani et al., 1999) and suggested that the mites had become less virulent. In a study in the Arnot Forest, New York State, USA, Tom Seeley studied feral colonies over three years, in an area where he had previously observed similar numbers of colonies before the arrival of varroa in the late 1980s (Seeley, 2007). He then compared these bees elsewhere with other commercially available strains, and found no difference, concluding that mites in the Forest had become avirulent. In addition, the feral bees swarmed frequently, something already known to reduce mite populations. In Le Mans and Avignon, France, Le Conte et al. (2007) reported feral colonies surviving without treatment, but the mechanism involved was unclear. In probably the most well studied case, in 1999 Ingemar Fries and colleagues from the Swedish University of Agricultural Sciences, Uppsala, placed 150 honey bee colonies on the Island of Sudret, part of Gotland, isolated from other bee colonies, in an experiment known as the Bond Project ( Live and Let Die ). These colonies were left untreated, and monitored for the next six years. As expected, there were heavy losses initially, and few swarms, but by 2005 (Fries et al., 2006) there were 13 colonies remaining, including five survivors from the original group. This demonstrates that untreated colonies in a closed population can survive, but what was the mechanism? For example, did the bees become more resistant, or did the mites become less virulent, or a combination of both? This is difficult to determine, but is of vital importance, when one considers that the life cycle of a varroa mite is only a few weeks, whilst in contrast, that of the honey bee is several years, so one can make much more rapid progress in breeding less virulent mites than in breeding more tolerant bees. It is thus vital to closely study the mechanisms involved. The conclusion was that two mechanisms were involved: firstly, the Bond colonies produced less brood than control colonies, and secondly they had a lower proportion of the mites in the sealed brood compared to control colonies (Fries and Bommarco, 2007). The general rule that in time parasites become less virulent and that their hosts become more resistant, led Tom Rinderer and colleagues at the USDA lab at Baton Rouge, Louisiana, USA, to examine bees from the far east of Russia, where varroa had first been reported to be a problem. Preliminary field studies in the early 1990s led to importations of bees to the USA from the Primorsky region, near Vladivostock in 1997 (Rinderer et al., 1997). After evaluation, these bees were released to commercial breeders in 2000, and studies have shown (Tarpy et al., 2007) that the commercially available stocks are indeed more varroa tolerant than other commercial strains, and that careful crossing has avoided inbreeding, given the

61 limited original gene pool (Bourgeois and Rinderer, 2009). Despite ten years work, however, the precise mechanisms for the varroa tolerance remain somewhat unclear, as does the degree to which these bees will survive without varroa treatment, but it is clear that a number of factors are involved, in particular a reduced number of viable female offspring (de Guzman et al., 2008). Breeding programmes for varroa tolerance As mentioned in Chapter Two, studies of the resistance mechanisms which enable A. cerana and the Africanised bees A. m. scutellata to survive varroa infestation without treatment have not as yet identified traits which have proved useful in breeding programmes. In a review of varroa tolerance that I wrote more than ten years ago (Carreck, 1998), I reported much breeding work from laboratories in central Europe, especially Germany, but progress has been slow, and breeding for a shortened post capping period or for grooming behaviour, for example, seems to have produced little in the way of practical results. Büchler et al. (2010) have recently reviewed the current status of varroa tolerance programmes in Europe. One of the activities of the COLOSS Network, mentioned in Chapter One is to evaluate and compare different strains of bee for a variety of characters, including disease resistance (Bouga et al., 2011), and this includes a bold Genotype Environment Interactions experiment, involving 16 strains of bees being compared at 16 locations throughout Europe. In New Zealand, where varroa was discovered only as recently as 2000, Mark Goodwin and colleagues from HortResearch in Auckland have also reported (Cox et al., 2005; Taylor et al., 2008) promising results in breeding resistant bees, but as yet, the success of these bees in general use is unclear, and little information has been published in refereed scientific journals. Rinderer et al. (2010) have also recently reviewed the current status of varroa breeding programmes in the USA (see Chapter Five), but briefly, a great deal of work by Jeff Harris and John Harbo at the USDA lab at Baton Rouge, Louisiana, USA for many years focussed on a heritable trait which they dubbed Suppressed Mite Reproduction (SMR), which was enhanced in their breeding programmes (Harris and Harbo, 2000). It was believed that the bees were in some way interfering with mite reproduction, leading to a high proportion of infertile mites, although the mechanism was unknown. More recently, however, it has been concluded that the reduced mite numbers were due to the removal of infested pupae (Harris, 2007), in fact the same trait described many years before as hygienic behaviour, and demonstrated to confer resistance to American foulbrood and other brood diseases. This trait has thus been renamed Varroa Sensitive Hygiene (VSH). Hygienic behaviour Hygienic behaviour as a control for varroa had been the focus of attention for other groups, especially Marla Spivak and colleagues at the University of Minnesota. Hygienic behaviour is a trait, whereby worker honey bees are observed to be able to detect sealed brood cells which contain diseased pupae, and then to uncap and remove them. This was first noted many years ago as being effective in limiting the incidence of American foulbrood (Park et al., 1937; Woodrow and Holst, 1942; Rothenbuhler, 1964). Walter Rothenbuhler at Ohio State University concluded (Rothenbuhler, 1964) that hygienic behaviour was controlled by two recessive genes, one for uncapping and one for removal of larvae, and that hygienic behaviour is only effective if both are expressed in a large proportion of workers. More recent work suggests, however, that up to seven gene loci might be involved in what is clearly a complex behaviour (Lapidge et al., 2002). Hygienic behaviour was later found to also have an effect in reducing chalkbrood in colonies (Gilliam et al., 1983). In 1987, Christine Peng and colleagues at the University of Davis, California, reported (Peng et al., 1987a;b) that honey bees also showed hygienic behaviour towards cells artificially infested with varroa. Later, Boecking and Drescher (1992) found a close correlation between the removal of freeze killed brood and the removal of pupae experimentally infested with two mites per cell. By 1996,

62 Fig. 1a. The freeze killed brood test: non-hygienic. Fig. 1b. The freeze killed brood test: hygienic colony. Photos: Norman Carreck. Marla Spivak at the University of Minnesota was (Spivak, 1996) odour. Chalkbrood, caused by a fungus, will produce different investigating the hygienic and non hygienic variants of the commercial odours. Pin killed brood will ooze haemolymph, which may have "Starline" strain of bee. This led to many years of breeding work different odours again. Varroa mites on the other hand, will move improving hygienic behaviour in US honey bees (See Chapter Five). within the cell, and may themselves have a distinctive odour. By A number of tests have been used for hygienic behaviour their feeding on pupae, haemolymph may again be released. Freeze over the years. One involves piercing the cell capping and pupa with killed brood, on the other hand, will be dead but intact. These different a fine pin to kill individual brood cells. The standard test for many cues may therefore elicit differing degrees of hygienic behaviour, so years involved cutting a section of sealed brood from a comb, placing the results of such tests always need careful interpretation. it in a deep freeze overnight to kill it, and then inserting this back in the comb. After 48 hours, the percentage of cells removed was used Intracolony selection as a measure of hygienic behaviour. This technique is, however, Although artificial selection for hygienic honey bees has been conducted time consuming, so Jerry Bromenshenk of the University of Montana for many years, it was only previously carried out at the individual suggested using liquid nitrogen to freeze kill brood. This was further colony level, by making use of queens or males from colonies that developed into the Freeze Killed Brood Assay (FKBA) (Spivak and were demonstrated to be hygienic. Within an individual colony, Downey, 1998), which has been widely used by many laboratories however, although the workers all share the same mother, they as a standard test (Figs. 1a & 1b). consist of a number of groups of half-sisters, known as "patrilines", One needs, however, to carefully consider exactly which corresponding to the various drones with which the queen has cues bees might be using in order to elicit this hygienic behaviour. mated. Even in a colony that has been demonstrated to be hygienic, In the case of American foulbrood, pupae are dead and decomposing, not all of these patrilines may be equally hygienic. In a study at the probably involving secondary bacteria, which produce the characteristic University of Sheffield, Antonio Pérez-Sato, Francis Ratnieks, and

63 colleagues (Pérez-Sato et al., 2009) investigated whether selection of individual patrilines within hygienic colonies ("intracolony selection") could speed up the process of selection. They tested a group of colonies of dark European honey bees A. m. mellifera in Derbyshire for hygienic behaviour using freeze killed brood. Three of the most hygienic colonies were then selected for further study. Two were used to rear queens, the third for producing drones. Within the queen producing colonies, the hygienic behaviour of individually marked workers was recorded in an observation hive, and the workers were then tested for genotype using a polymerase chain reaction (PCR) assay (Châline et al., 2004). Queens were reared from these colonies, genotyped using a small wing tip sample, and then mated in an isolated valley in the Peak District with drones from the third colony. The colonies headed by these queens from hygienic patrilines showed approximately twice the level of hygienic behaviour compared to colonies headed by sister queens from non hygienic patrilines. This showed the potential of the technique in speeding up the process of breeding for hygienic behaviour. This work is now being continued with a programme to use intracolony selection to breed A. m. mellifera for hygienic behaviour at the University of Sussex, by Francis Ratnieks, Karin Alton, Gianluigi Bigio and Norman Carreck. Starting in 2008, queens have been reared from colonies previously identified as hygienic. Some of these have been open mated in our own apiaries, but in order to increase control, instrumental insemination has been used (Figs. 2a to 2i). Tissue samples for DNA extraction from the observation hive studies and from reared queens have been analysed by Annette Jensen at the University of Copenhagen, Denmark (Carreck et al., 2010 a;b;c). Once a suitable strain has been developed at Sussex, this will be passed on to members of the Bee Farmers Association of the UK for testing in the field, and ultimately for multiplication and sale of queens to beekeepers. Inevitably, the process of bee breeding is time consuming and labour intensive, but it is to be hoped that we can significantly improve the hygienic behaviour of our own strain of bee. The degree to which such improved bees will ever be widely used is, however, dependant on the development of a significantly greater queen rearing industry than currently exists in the UK. Bee breeding is of course a continuous process, and one should not underestimate the practical difficulties of maintaining a desired strain of bee. In addition, inbreeding in any organism is in the long term, harmful, and due to their haplodiploid nature, honey bee populations lave less genetic diversity than that of many other groups of insects. Leslie Bailey of Rothamsted pointed out some years ago (Bailey, 1999) that much of the honey bee's inherent resistance to disease stems from its genetic diversity, so that breeding for resistance to one pest or disease may increase susceptibility to others. Fig. 2a. Intracolony selection in practice: Marking newly emerged worker bees. Fig. 2b. Intracolony selection in practice: test comb in observation hive with marked workers.

64 Fig. 2c. Intracolony selection in practice: retrieving marked bees from Fig. 2f. Intracolony selection in practice: cell bar with queen cells. observation hive. Fig. 2d. Intracolony selection in practice: taking wing tip tissue samples. Fig. 2g. Intracolony selection in practice: newly emerged virgin queens held in cages. Fig. 2e. Intracolony selection in practice: grafting larvae for queen rearing. Farm, Shropshire) performing instrumental insemination of selected queens. Fig. 2h. Intracolony selection in practice: Michael Collier (Cornbrook Bee

65 Fig. 2i. Intracolony selection in practice: mated queens in nucleus hives. Acknowledgements The bee breeding project at the University of Sussex is funded by Rowse Honey Ltd., Michael Chowen, The British Beekeepers Association and the Somerset Beekeepers Association. References BAILEY, L (1999) The quest for a super-bee. Bee World 80: 55-60. BOECKING, 0; DRESCHER, W (1992) The removal response of Apis mellifera L colonies to brood in wax and plastic cells after artificial and natural infestation with Varroa jacobsoni Oud and to freeze killed brood. Experimental and Applied Acarology 16: 321-329. BOUGA, M; ALAUX, C; BIENKOWSKA, M; BÜCHLER, R; CARRECK, N L; CAUIA, E; CHLEBO, R; DAHLE, B; DALL'OLIO, R; DE LA RÚA, P; GREGORC, A; IVANOVA, E; KENCE, A; KENCE, M; KEZIC, N; KIPRIJANOVSKA, H; KOZMUS, P; KRYGER, P; LE CONTE, Y; LODESANI, M; MURILHAS, A M (2011) A review of methods for discrimination of honey bee populations as applied to European beekeeping. Journal of Apicultural Research 50: 51-84. DOI: 10.3896/IBRA.1.50.1.06 BOURGEOIS, L; RINDERER, T E (2009) Genetic characterisation of Russian honey bee stock selected for improved resistance to Varroa destructor. Journal of Economic Entomology 102(3): 1233-1238. BÜCHLER, R; BERG, S; CONTE, Y L (2010) Breeding for resistance to Varroa destructor in Europe. Apidologie 41: 393-408. DOI: 10.1051/apido/2010011 CARRECK, N L (1998) Tolerance of honey bees to Varroa jacobsoni. Beekeepers Quarterly, 54: 24-27. CARRECK, N L; ALTON, K L; JENSEN, A B; RATNIEKS, F L W (2010a) Intra-colony selection for hygienic behaviour in the dark European honey bee. In Proceedings of 6th COLOSS Conference - Prevention of Honey bee COLony LOSSes, Hacettepe University, Ankara, Turkey, 5-6 September 2010. 29. CARRECK, N L; ALTON, K L; RATNIEKS, F L W (2010b) Intracolony selection of honey bee colonies for disease resistance. In Abstracts of Royal Entomological Society Annual Meeting Ento 10, Swansea University, UK, 26-28-7/10. 19. CARRECK, N L; ALTON, K L; RATNIEKS, F L W (2010c) Intracolony selection of the Dark European honey bee for disease resistance. In Proceedings of 4 th European Conference of Apidology, METU, Ankara, Turkey, 7-9 September 2010. 52. CHÂLINE, N; RATNIEKS, F L W; RAINE, N E; BADCOCK, N S; BURKE, T (2004) Non-lethal sampling of honey bee, Apis mellifera, DNA using wing tips. Apidologie, 35: 311-318. COX, H; TAYLOR, M; GOODWIN, M; PAGE, N; MCBRYDIE, H (2005) Towards breeding varroa tolerant honey bee stock in New Zealand. Australian Bee Journal 89: 4-6. DE GUZMAN, L I; RINDERER, T E; FRAKE, A M (2008) Comparative reproduction of Varroa destructor in different types of Russian and Italian honey bee combs. Experimental and Applied Acarology 44: 227-238. DE JONG, D; SOARES, A E E (1997) An isolated population of Italian bees that has survived Varroa jacobsoni infestation without treatment for over 12 years. American Bee Journal 137: 742-745. FRIES, I; BOMMARCO, F (2007) Possible host-parasite adaptations in honey bees infested by Varroa destructor mite. Apidologie 38: 525-533. DOI:10.1051/apido:2007039 FRIES, I; IMDORF, A; ROSENKRANZ, P (2006) Survival of mite infested (Varroa destructor) honey bee (Apis mellifera) colonies in a Nordic climate. Apidologie 37: 564-570. DOI: 10.1051/apido.2006031. GILLIAM, M; TABER, S; RICHARDSON, G V (1983) Hygienic behaviour of honey bees in relation to chalkbrood disease. Apidologie 14: 29-39. HARRIS, J W (2007) Bees with Varroa Sensitive Hygiene preferentially remove mite infested pupae aged five days post capping. Journal of Apicultural Research 46: 134-139.

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