(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, either 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. All straws were amounts of A aggregata inoculum. Second (and X-rayed to determine live larvae, chalkbrood often early third) instar larvae killed by the fun- and pollen masses. Two of the latter cartons gus are difficult and often impossible to identify were laboratory incubated in March 1979 to re- on the X-ray plate. Thus, any abrupt decrease in move all live bee larvae and parasitoids; and the incidence of 5th instar chalkbrood cadavers the remaining 2 were kept as Adrian 1979 WT which may be accompanied by a comparable in- Controls. crease in pollen masses could reflect a difference in the at which occurred Incubation of the 2 WT cartons (above) re- stage mortality rather than a real diminution of the disease. sulted in emergence from fewer than 30% of the straws, ie the caps were not disrupted on the remaining straws suggesting that they contained only chalkbrood cadavers, pollen masses, and Experiment II dead larvae. As nesting females will only accept tunnels which are uncapped or those in which the cap has been disrupted, and, as our objec- An evaluation of the efficacy of the chalkbrood tive was to expose the renesting selected popu- challenging procedure under field conditions lation to a maximum amount of inoculum, the was undertaken in 1980 on 2 populations of caps of all straws were pierced with a 3 mm x leafcutting bees located north of Nyssa, Oregon. 10 cm reamer and the contents of the straw The Nyssa site was selected because local carefully disrupted along its entire length. Care Californian populations of the size necessary for was taken to minimize the loss of inoculum dur- these tests did not exist, and because we ing the reaming process. Thirty-three and 34 wished to avoid any introductions which would straws containing 220 and 221 live larvae of the jeopardize the isolation of our selected lines. 4th expansion generation (Select Line B) were One population was derived from a 1979 chalk- introduced into these 2 WT cartons and placed brood-free Canadian source; the 2nd from re- in a field south of Gerber, CA. Emergents were gionally trapped bees with a low incidence of thus forced to accept contaminated nesting me- chalkbrood (7.2% at the end of 1979). Each dia. population was housed in different domiciles Caps of all straws in the 2 1979 WT Control and was propagated in drilled wooden boards cartons were pierced and disrupted to a depth from which cells could be removed. of approximately 1.25 cm so as to provide the emerging population with access to an ade- quate supply of tunnels. Such disruption was shallow enough to avoid damage to most if not Inoculum preparation all cells in each straw, yet deep enough to provide females with access to the searching ready Inoculum was prepared using 1977 Adrian wild tunnel. To maintain isolation it was to necessary type (WT) paper soda straw nesting medium. X- locate the WT Controls in a field 24 km west of ray analyses indicated that each carton con- Los Molinos. Controls for Select B 5th expan- tained approximately 700 chalkbrood cadavers sion were re-established at the Los generation (table I). All straws were removed from 1 carton Molinos site. and macerated in a blender. The residual paper Emergence began on July 23 and nesting was then removed, the contents passed through was allowed to continue until August 31 (table a 10-mesh screen to remove pieces of leaf mat- I). Capped straws in each of the 6 cartons were ter and matted pollen, and the screenings thor- painted on August 11 and August 31, permitted oughly pulverized in a mortar. One quarter of to develop for 2 weeks under field conditions, the pulverized contents of each WT carton was and then removed for X-ray analysis. Cells of all dusted into each straw carton prior to field place- capped straws of each population were evaluat- ment, and at weekly intervals thereafter. A ed for live larvae, chalkbrood and pollen mass- hand-operated piston-type duster was used for es. The pollen mass category was included be- spore inoculation. It is estimated that each ca- cause it was suspected (and later confirmed daver produces from 108-109 spores (Stephen, unpublished) and each carton received approxi- from each of the Canadian challenged cartons, mately 175 x 108 spores weekly. and 50 from each carton serving as controls to the 2 to determine the effect of the The experiments were initiated on July 18 populations 1980, approximately 2 wks after each population challenging method on the incidence of chalk- live larvae and masses. Selection had emerged and when the bees were actively brood, pollen nesting. One board from each of 4 field domi- of capped straws was not random for the num- ciles containing active nesting populations of ei- ber of capped straws ranged from 42-177 per carton the 24 cartons used in this test. ther Canadian (2 domiciles) or Nyssa (2 domi- among ciles) females was removed on the morning of July 18 and was replaced with 2 new straw cartons inoculated with 1977 Adrian WT inoculum Experiment III prepared as described above. Two boards were removed from the far side (3-5 m distant) of Chalkbrood resistance in the 6th each of the same domiciles and replaced with 4 expansion gen- straw cartons to serve as controls. Females eration of selected line A was field evaluated by from the displaced boards began provisioning challenging with inoculum. the inoculated straws by late morning. Straws In June 1980, 136 straws containing 791 live were dusted with Adrian WT inoculum at approx- larvae of the 6th expansion generation were di- imately weekly intervals (4 applications) until Au- vided with equal numbers placed in each of 4 gust 10, when flight had ceased. Test straws cartons of new straws prior to emergence. Be- were removed from the field on August 20. Four cause of limited isolation sites, each carton was cartons located approximately 3 m from the test located on different out-buildings of adjacent cartons in each field domicile were removed on farmyards, separated by distances of 150-750 September 3 and served as controls for each m. Inoculum to challenge the selected line was population. Forty capped straws were removed prepared as described above. One-fourth of the from each of the Nyssa challenged cartons, 50 pulverized contents of each 1977 Adrian WT carton was dusted into each of the 4 cartons masses in the calculations would have containing selected bees prior to their emer- highly biased the data. This accounts for This was for gence. procedure repeated weekly the of live larvae and 8 wks with each application of inoculum made in apparent doubling the morning before females began foraging. sharp reduction in chalkbrood incidence in Dusting was terminated on August 8 and car- the Adrian WT 1979 control compared to tons removed for analysis on August 19. An iso- those categories in the Adrian WT 1978 lated population of the selected line served as a Stock. The latter data include all cells con- Select Line A 1980 control (7th expansion gen). structed during both the 1977 and 1978 Four cartons of straws were each supplied with seasons 200 cells from the same susceptible Canadian (table I). bee population used in Exp II. In this experiment Data in table II indicate that the method 2 cartons served as controls "challenge" (Cana- of field inoculation for chalkbrood was ef- da, and were inoculated as described 1980) fective. Chalkbrood in the above. Two cartons were isolated in a field 11 unchallenged endemic increased from 7.6 to km south of Corning as controls. Cartons were population removed from the field between August 19 and 15.7% during 1980, while that of the chal- 21. All capped straws from each treatment were lenged rose to 27.1%. The incidence of X-ray analysed for live larvae, chalkbrood and disease in the challenged Canadian mate- masses. pollen rial was 26.4% whereas that of the unchallenged was 3.8%. Both challenged populations had fewer live larvae and fewer RESULTS pollen masses than the controls (table II). There was little difference between chal- The progeny of selected line B females lenged and control populations on selected forced to nest in heavily contaminated Line A in chalkbrood (5.0 vs 2.8%) or pol- straws (Exp I) showed an increase in len masses (3.2 vs 2.8%) (table III). The chalkbrood from 2.3 to 12.1 %, a decrease proportion of live larvae remained high in in live larvae from 92.3 to 77.7% and no both and both in the of masses. populations (90 93%) higher change proportion pollen than that of the from which Chalkbrood increased 10% in the Select B populations were derived. The line as a result of the but this they spore challenges, challenge, resulted in 27.8% chalkbrood in was more than 21 % less than the disease however, the incidence in the unselected population. chalkbrood-free Canadian material The controls for the selected line (Select B used as the challenge control (table III). Chalkbrood incidence in of the 5th expansion gen) were almost identical subsamples Canadian in to the 1978 populations from which it was population challenged Oregon taken (Select B 4th expansion gen) (table (Exp II) and in California (Exp III) was I). Analysis of the Adrian WT contaminated nearly identical (26.4 vs 27.8%) even the latter was 2 more treat- straws 1978 was made prior to the labora- though given tory emergence of bees and represents ments of inoculum (the population had the status of the material used both as the died off by the 1 st wk in August). The Can- source for inoculum and as the Adrian WT ada (80) challenged population suffered 1979 control for this experiment (table I). bird damage during the 1 st 2 wks of flight Analysis of the Adrian controls was con- and thus reproduction was lower than ex- fined to those straws in which females had pected. The Canadian 1980 controls were nested during the 1979 season, as the in- swamped by inflight of endemic M rotunda- clusion of all previous cadavers and pollen ta beginning during the 2nd wk (table III).

DISCUSSION both lines A and B were derived from the same population, each was expanded from The method used to challenge nesting fe- a limited gene pool, the progeny of 14 and male bees with inoculum appears to the 15 females respectively. It is improbable effective; the incidence of chalkbrood in that factors conferring disease resistance both the Nyssa and Canadian lines, treat- exhibit any form of dominance, for there is ed identically over 4 wks, increased from no evidence of the rapid spread of the trait 7.6 to 27.1% and 0 to 26.4% respectively during the 10 years chalkbrood has been a (table II). The chalkbrood increase from problem. If we assume that resistance in 7.6 to 15.7% in the endemic line is consis- these two lines is recessive and polygenic, tent with the expected rate of disease in- it is probable that allelic components for re- crease in an isolated population (Stephen sistance in the 2 lines differed at the time unpublished), but the 3.8% chalkbrood in of testing. the unchallenged Canadian population af- Chalkbrood in the challenged chalk- ter 1 of is less yr expansion considerably brood-free Canadian population was com- than expected. We assume that inflight of parable (26.4%) to that of the challenged contaminated endemic adults to this site endemic bees (27.1%) (table II). There is a was minimal. The of the method is efficacy widely held belief among leafcutting bee further supported by the increase in chalk- consumers in the USA that Canadian bees brook from 0 to 27.8% as a result of chal- are far more susceptible to chalkbrood lenging a subsample of the same chalk- than endemic US populations. While Exp II brood-free Canadian in population (above) was not directed towards answering that California (table III). question, the data suggest that if differenc- Progeny of Line A females which were es in susceptibility exist, they are minor, at dusted with spores from the equivalent of least in the Canadian population used in 179 cadavers each wk for 8 wks, did not this study. differ in live larvae appreciably chalkbrood, An interesting observation in these ex- or masses from those of the control pollen periments relates to pollen masses. As in- or the 1979 stock from which were they dicated above, pollen masses result from derived This (table III). strongly suggests the absence or death of an egg or early in- that a genetic component for disease re- star larva, leaving the unconsumed provi- sistance is present. sion in the cell. In most areas of the Pacific Chalkbrood was much higher in the Northwest, pollen masses rank second progeny of females renesting in heavily only to chalkbrood as identifiable mortality contaminated soda straws (Exp I) than in in leafcutting bee populations, commonly those of females challenged with chalk- 15 to 35% (see tables I and II). Nest expo- brood spores by dusting (Exp III) (12.1 vs sure to direct sunlight is known to cause 5.0%). This may suggest that : Line A was early instar mortality (Undurraga, 1975) more resistant to the disease than Line B and various other factors such as pesti- as Line A was in its 6th expansion genera- cides and nectar dearth have been sug- tion and Line B in its 4th when challenged; gested as causes. In the original selections or, the 2 methods of challenging differ in from the Adrian WT population, we chose their efficacy — spores were applied at only straws containing 5 or more live lar- weekly intervals in the latter and were vae, and concomitantly, no cells with pol- present continuously from the onset of len masses. During the entire period of se- nesting in the former (Line B). Although lection and expansion, the incidence of pollen masses never exceeded 4.4%. The La seconde moitié des séries sélection- pollen mass trait may also be genetically nées s’est multipliée durant 6 générations mediated, either linked to, or independent puis a été testée avec environ 175 x 108 of, disease resistance. spores chaque semaine pendant 8 se- maines. La différence a été faible entre les populations testées et les témoins (5,0 ACKNOWLEDGMENTS contre 2,8%) (tableau III). Les données suggèrent qu’une composante génétique Technical Paper No. 9065 Oregon Agricultural de résistance au couvain plâtré était pré- Experiment Station. Supported in part by USDA/ sente dans les 2 d’abeilles. La très ARS No 58-9AHZ-3- lignées Cooperative Agreement faible des masses 730. fréquence polliniques dans les 2 lignées tout au long des 4 an- nées d’étude (2,8 et 3,8%) contraste fortement avec celui chez le sau- Résumé — Résistance au couvain plâ- présent type tré (Ascosphaera aggregata) chez la vage (20%), suggérant que cette mégachile (Megachile rotundata). I. Test caractéristique peut être également déter- de lignées sélectionnées. Vingt neuf sé- minée génétiquement, soit en liaison avec, soit de la résistance à la ries de cellules de l’abeille Megachile ro- indépendamment maladie. tundata (Fabr) ont été sélectionnées à par- tir d’une population présentant un taux de rotundata / / couvain plâtré, ou ascosphériose (Ascos- Megachile Ascosphaera phaera aggregata Skou), de 36,1%. couvain plâtré / résistance Seules les séries comprenant 5 larves saines ou plus, pas de couvain plâtré ni de masses polliniques ont été retenues (la Zusammenfassung — Kalkbrut- présence de masses polliniques (Ascos- indique phaera aggregata) Resistenz bei der que l’œuf, ou la est La jeune larve, mort). Blattschneidebiene rotunda- moitié de la population s’est reproduite (Megachile ta). I. Prüfung selektierter Linien unter pendant 4 générations puis nous avons harten Bedingungen. Aus einer Popula- testé sa résistance en forçant les femelles tion der Blattschneidebiene ro- à nidifier dans des matériaux fortement (Megachile tundata mit einem Befall von contaminés. Le taux de couvain Fabr) 36,1% plâtré Kalkbrut parmi la descendance est passé de 2,3 à (Ascosphaera aggregata Skou) 12,1% (tableau I) mais cela représente wurden 29 Zellserien ausgewählt. Es wurden nur Serien die fünf oder une baisse de 60% par rapport à la popu- ausgesucht, mehr Larven und keine Kalkbrut lation sauvage d’origine. Lors d’une série gesunde oder Pollenmassen enthielten de tests menés pour déterminer l’efficacité (Pollenmas- de l’induction du couvain plâtré parmi des sen sind Anzeichen dafür, daß das Ei oder die Larve Die lignées de mégachiles non sélectionnées, junge abgestorben ist). nous avons fait macérer dés pailles à boire Hälfte der Population wurde vier Genera- fortement contaminées. Le produit obtenu tionen hindurch vermehrt und dann auf a été tamisé, pulvérisé et saupoudré dans ihre Resistenz streng geprüft, indem man les matériaux de nidification à intervalles die Weibchen zwang, in schwer verseuch- d’une semaine. Cette méthode s’est mon- tem Material zu nisten. trée efficace : l’incidence du couvain plâtré Die Kalkbrut stieg in diesem Test bei est passée de 7 à 27% dans les popula- der Nachkommenschaft von 2,3 auf 12,1% tions testées (tableau II). an, aber das war um 60% weniger als bei der Wildpopulation, von der sie herstamm- Invertebrate Microbial Diseases (Davidson te. In einer Versuchsserie, in der die Wirk- EW, ed), 511-545 samkeit der künstlichen Kalkbrutauslösung Daly HJ (1952) Records of Palearctic Megachile rotundata in the United States. Entomol bei nicht selektierten Linien geprüft wer- den sollte, wurden schwer kontaminierte News 63, 210-211 Getränke-Halme mazeriert, dann DeJong D (1976) Experimental enhancement of gesiebt chalkbrood infections. Bee World 114- und schließlich wurde das 57, pulverisiert; 115 Nestmaterial mit den Sporen in Intervallen GP, Baker J, Al-Khatib Z, Mellon R, von einer Woche bestäubt. Diese Me- Georghiou je Murray C, Tran H, Vasquez M, Pelsue F, Ha- thode erwies sich als wirksam, da sie den zelrigg J (1983) Insecticide resistance in Kalkbrut-Befall in den Testpopulationen mosquitoes : Research on new chemicals von 7 auf 27% erhöhte. Die zweite Hälfte and techniques for management. Mosquito der Population wurde 6 Generationen hin- Control Research, Annual Report University durch vermehrt und anschließend durch California Bestäuben mit etwa 175 x 108 Sporen Harvey TL, Howell DE (1965) Resistance of the wöchentlich für 8 Wochen geprüft. Es be- housefly to Bacillus thuringiensis. J Invertebr Pathol 7, 92-100 stand ein nur geringer Unterschied zwi- WC Larval hon- schen den Zuchten und den se- Hoage TR, Rothenbuhler (1966) geprüften to various doses of Bacillus lektierten Kontrollen eybee response (5,0 gegen 2,8%). larvae spores. J Econ Entomol 59, 42-45 Diese Daten lassen vermuten, daß in bei- Hurd PD Jr Distributional notes of Eutri- den Linien eine (1954) genetische Komponente charea, a Palearctic subgenus of Megachile, für Krankheitsresistenz bestand. Das auf- which has become established in the United fallend niedrige Vorkommen von Pollen- States. Entomol News 65, 93-95 massen in beiden Linien während der vier Kawakami K (1975) Susceptibility of several va- Jahre der Untersuchung (2,8 bis 3,8%) rieties of the silkworm, Bombyx mori L to As- stand in starkem Gegensatz zu der pergillus disease and germination of fungus Häufigkeit von Pollenmassen in der Wild- spores in larval hemolymph. J Seric Sci (Jpn) population (über 20%). Dies legt die Ver- 44, 39-44 mutung nahe, daß auch dieses Merkmal Kinsinger RA, McGaughey WH (1979) Suscepti- of of Indian meal moth and genetisch übertragen wird, entweder ge- bility populations Almond moth to Bacillus thuringiensis. J oder von der Kalkbrut- koppelt unabhängig Econ Entomol72, 346-349 resistenz. Kish LP, Waters ND, Homan HW (1981) Chalk- brood. University of Idaho. Agric Exp Stn Megachile rotundata / Ascosphaera / Current Info Series No 477 Kalkbrut / Resistenz Klein M, Podoler H (1978) Studies on the application of a nuclear polyhedrosis virus to control populations of the Egyptian cotton worm, REFERENCES Spodoptera littoralis. J Invertebr Pathol 32, 244-248 Aratake Y (1961) Genetic analyses of the infec- Kulincevic JM, Rothenbuhler WC (1975) Selec- tion with muscardines of Bombyx mori L. I. tion for resistance and susceptibility to hair- Infection with the white muscardine. Bull Ser- less-black syndrome in the honeybee. J In- ic Exp Stn (Tokyo) 17, 136-165 vertebr Pathol 25 (3), 289-295 Bailey L (1965) Paralysis of the honey bee, Apis Martignoni M, Schmid P (1961) Studies on the mellifera Linnaeus. J Invertebr Pathol 7, 132- resistance to virus infection in natural popula- 140 tions of Lepidoptera. J Insect Pathol 3, 62-74 Briese DT (1981) Resistance of insect species McManus WR, Youssef NN (1984) Life cycle of to microbial pathogens. In : Pathogenesis of the chalkbrood fungus, Ascosphaera aggregata, in the alfalfa leafcutting bee, Megachile Stephen WP, Vandenberg JD, Fichter BL (1981) rotundata, and its associated symptomatolo- Etiology and epizootiology of chalkbrood in gy. Mycologia 76, 830-842 the leafcutting bee, Megachile rotundata, with Mitchell TB A revision of the notes on other Ascosphaera species. Oregon (1937) genus Meg- State Univ Stn Bull 653 achile in the Nearctic region. Pt. VIII: Taxono- Agric Exp my of the subgenus Chelostomoides, adden- Thomas GM, Poinar GE Jr (1973) Report of di- da and index. Trans Am Entomol Soc 63, agnoses of diseased insects, 1962-1972. Hil- 381-426 gardia 42, 261-359 Rinderer TE, Green TJ (1976) Serological rela- Undurraga JM (1975) Factors affecting the in- tionship between chronic bee paralysis virus crease of the alfalfa leafcutter bee. Proc VI Ann Alfalfa Seed Growers Short Course. and the virus causing hairless-black syndrome in the honeybee. J Invertebr Pathol Oregon State Univ Corvallis, OR, 14-25 27, 403-405 Vandenberg JD, Stephen WP (1982) Etiology Stephen WP (1961) Artificial nesting sites for and symptomatology of chalkbrood in the al- the propagation of the leafcutter bee, Mega- falfa leafcutting bee, Megachile rotundata. J chile (Eutricharea) rotundata (Fabr) for alfalfa Invertebr Pathol 39, 133-137 pollination. J Econ Entomol 54, 989-993 Vandenberg JD, Fichter BL, Stephen WP (1980) load of on WP, Torchio PF Spore Ascosphaera species Stephen (1961) Biological adults of the bee, notes on the leaf-cutter bee, rotun- emerging leafcutting Mega- Megachile chile rotundata. Environ Microbiol 39, data Pan-Pac 85-93 Appl (Fabr). Entomol 37, 650-655 JM X- Stephen WP, Undurraga (1976) Vansulin SA (1974) On the resistance of Culex an tool in radiography, analytical population pipiens molestus Forsk. to Boverin. Parazito- studes of the leafcutter bee, Megachile pacifi- logiya (Leningr) 8, 274-275 ca. J Apic Res 15, 81-87 Watanabe H (1967) Development of resistance Stephen WP, Undurraga JM (1978) Chalkbrood in the silkworm, Bombyx mori, to peroral in- disease in the bee. State leafcutting Oregon fection of a cytoplasmic-polyhedrosis virus. J Univ Agric Exp Stn Bull 630 Invertebr Pathol 9, 474-479