(Megachile Rotundata). I

Original article Chalkbrood (Ascosphaera aggregata) resistance in the leafcutting bee (Megachile rotundata). I. Challenge of selected lines WP Stephen * BL Fichter Oregon State University, Department of Entomology,Corvallis, OR 97331-2907, USA (Received 20 April 1989; accepted 13 March 1990) Summary — Twenty-nine cell series of the leafcutting bee, Megachile rotundata (Fabr), each containing 5 or more healthy larvae, no chalkbrood (Ascosphaera aggregata Skou) and no pollen masses were isolated from a population with over 36% chalkbrood. This selected subpopulation was split and isolated for 4 and 6 generations before being challenged by forcing the females to nest in heavily contaminated media, or by weekly dustings with approximately 175 x 108 A aggregata spores. The incidence of chalkbrood in both challenged lines was markedly lower than that of the wildtype, and comparable to that of the selected lines, suggesting a genetic component for resistance in both lines. The very low incidence of pollen masses (dead eggs or early instar larvae) in the selected lines throughout the 4 yrs of the study indicates that this trait may also be genetically mediated, ei- ther linked to, or independent of disease resistance. chalkbrood / Ascosphaera / Ieafcutting bee / resistance / Megachile INTRODUCTION mori L (Watanabe, 1967; Briese, 1981), and 3 reports describe resistance to the Few studies have been conducted on re- "hairless-black syndrome" in honey bees sistance in endemic insects to naturally oc- (Bailey, 1965; Kulincevic and Rothenbuh- curring pathogens. Most data deal with ler, 1975; Rinderer and Green, 1976). Re- susceptibility differences among laboratory sistance to chalkbrood, Ascosphaera apis, populations of insect pest species to viral among colonies of honey bees was sug- (Martignoni and Schmid, 1961; Klein and gested by DeJong (1976), and large differ- Podoler, 1978) and bacterial pathogens ences in susceptibility among silkworm (Harvey and Howell, 1965; Hoage and Ro- populations to Beauveria bassiana (Ara- thenbuhler, 1966; Kinsinger and McGau- take, 1961) and Aspergillus flavus (Kawa- ghey, 1979; Georghiou et al, 1983). kami, 1975) have been noted. The only di- Among non-pest species, extensive stud- rect selection for resistance to an ies have been conducted on resistance to entomogenous fungal pathogen cited in viral pathogens among strains of Bombyx current literature is the study by Vansulin * Correspondence and reprints (1974) in which the mortality in Culex pipi- production, accounting for losses up to ens molestus is halved after 8 generations 75% in some areas of the Pacific North- of exposure to constant doses of B bassia- west (Stephen et al, 1981). Chalkbrood is na. spread by spores in contaminated nesting media and carried on the Megachile rotundata (Fabr) is a gregari- by spores body ous Eurasian bee first recorded from the surface of adult bees (Vandenberg et al, eastern United States in the mid 1930’s 1980). Spores deposited in cell provisions are a larva and (Mitchell, 1937). It spread rapidly across ingested by developing temperate North America reaching the germinate in the gut. The mycelia pene- western states by the late 1950’s (Daly, trate the gut wall and rapidly fill the hemo- 1952; Hurd, 1954). In 1959, when the bee coel (Vandenberg and Stephen, 1982; McManus and At was first recognized as a domesticable Youssef, 1984). present, there is no effective control for the disease oligolege on alfalfa (Stephen, 1961; Ste- phen and Torchio, 1961), it was apparently other then prophylactic measures directed ubiquitous throughout the Pacific North- at reduction of inoculum (Stephen and Un- west with its numbers limited by the availa- durraga, 1978; Kish et al, 1981). bility of suitable nesting sites. Populations Although various materials are used as increased rapidly through trap nesting and, nesting media for the bee, paper soda by 1962, large commercial operations in straws are the medium of choice for popu- bee sales were established. lation monitoring. Selected soda straws The leafcutting bee propagation indus- can be removed from a box without disrup- try has been centered in western Canada tion of the cell series and their contents for more than a decade with annual com- can be evaluated through X-ray analysis mercial sales of 200 to 300 million cells, (Stephen and Undurraga, 1976). The cells principally to alfalfa seed producers in the in a nesting tunnel (from 1-12) are usually western United States. We suggest that those of a single female. We examined these importations, extensive local and re- thousands of straws while monitoring bee gional commerce, as well as the tendency populations in the western United States of the species to disperse widely have pre- for chalkbrood. In heavily diseased popula- vented the development of locally adapted tions, cadavers occur in a near-random populations of the bee. The result is a near distribution within and among straws, but continuous array of highly adaptable, poor- occasionally an entire series of from 5-10 ly adapted local populations throughout cells in a single straw will be found to be western America. The rate at which chalk- disease-free. As only 500 to 1000 spores brood has spread tends to support this constitute an LD50 (Fichter and Stephen, idea. unpublished), and nearly 108 spores are carried by a single female (Vandenberg et Chalkbrood is a fungal disease of leaf- al, it is that an en- cutting bee larvae (Megachile rotundata 1980), highly improbable tire tunnel in a diseased (Fabr) caused by Ascosphaera aggregata nesting heavily could escape inoculation. Skou, and was first identified from Nevada population populations in 1973 (Thomas and Poinar, Thus, the premise of these studies was 1973). It spread rapidly throughout all are- that, if all the progeny of a single female as of the western United States in which were chalkbrood-free in a population with the bee was propagated and is now cos- more than 35% diseased larvae, genetical- mopolitan (Stephen et al, 1981). This dis- ly-mediated factors for disease resistance ease is the principal limiting factor to bee may be present in the population. Our ob- jectives were to demonstrate that intrapop- bees. A 2nd location near Nyssa, OR was uti- ulation differences in pathogen susceptibil- lized to test the protocol for the chalkbrood chal- ity exist and that through selection and lenge studies. challenge, disease resistance lines may be established. Expansion and selection MATERIALS All bees were reared in 9 cm long x 5.5 mm di- ameter brown, paper soda straws packed tightly in cartons with approximately 250 straws each. Oregon stock Tight packing permitted the removal of capped straws and replacement with new without alter- ing the position of other straws. Each spring, A population of leafcutting bees near Adrian, bees were incubated at 30 °C until emergence Oregon with 36.1 % chalkbrood was chosen as of the first males after which they were placed the wild type (WT) from which selections were into the field. Every 10 d after field placement, were and left in situ made. Approximately 10 000 straws were X- capped straws color-coded rayed at the end of the 1977 season. Only for an additional 20 d to mature. They were then straws containing 5 or more healthy larvae and removed from the carton and returned to the la- no chalkbrood or pollen masses were selected: boratory for X-ray analysis. Live larvae, chalk- a total of 29 straws with from 5-9 live cells each brood, dead larvae, pollen masses (= provi- formed the base population from which expan- sioned cells with no eggs or with dead eggs or sion was made. early instar larvae), and parasitism were recorded. Any straw containing a chalkbrood cadaver was removed and destroyed during the expansion phase. No spore challenges were made Study sites during the 1st 4 generations of expansion. Cells parasitized by Tetrastichus spp were removed from each straw to avert a of The straws were incubated and placed out for build-up parasites the season. The straws were increase in 1978 at 2 sites near Los Molinos, during remaining returned to their sites for CA. The sites were 7 km original emergence approximately apart and of the with walnut and almond orchards intervening. renesting subsequent generation. Both sites were carefully surveyed and trap- nested in 1977 and no M rotundata were found. Approximately half of the selected material was METHODS placed at each site with new, brown paper soda straws provided for renesting. Cells from the 2 sites were Lines A and B. Los Moli- designated Experiment I nos was selected as the principal experimental area because of its long, dry summer season during which at least 3 generations of the leaf- To ascertain whether the selected lines were re- cutting bee could be reared and because of the sistant to chalkbrood, the 4th expansion genera- relatively low endemic bee populations. All but 2 tion was forced to nest in soda straws heavily (original) replication sites had to be abandoned contaminated with chalkbrood. Six straw car- during the 4-year study period because our se- tons, used for propagation of the Adrian wild lected lines were swamped by in-flight from en- type (= WT) population in 1977 and 1978 and demic bee populations. Thus, replications were heavily contaminated with chalkbrood, were se- confined to data gathered from individual car- lected for this test. Each carton contained ap- tons placed at different locations at the same proximately 220 straws, all of which were site. Outbuildings at each site were sprayed with capped indicating that each had been occupied Dibrom in late May and/or early June of each (and presumably provisioned) at some time dur- year to eliminate any residual population of ing the 2 yr period. Two cartons were destruc- tively sampled for 150 straws each and 30 [Fichter and Stephen, unpublished]) that early capped straws were selected haphazardly from instar larvae could be killed after ingesting large each of the remaining 4 cartons.

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