JOURNAL OF INVERTEBRATE PATHOLOGY 43, 11-20 (1984)

In Vivo Development and Pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the Alfalfa Leafcutting Bee,

Megachile rotunda ta

NABIL N. YOUSSEF

Department of Biology, Utah State University. Logan, Utah 84322

CARL E ROUSH

Lower Columbia College, Longview, Washington 98632

AND

WILLIAM R. MCMANUS

Department of Biology, Utah State University. Logan, Utah 84322

Received August 30, 1982; accepted March 16, 1983

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 x 10’ to 3 x 106) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.

KEY WORDS: Ascosphaera proliperda; Megachile rotundata; fungus.

INTRODUCTION

Skou (1972) identified an undescribed fungus that caused a chalkbrood-like dis- ease in the larvae of the leafcutting bee Me- gachile centuncularis and named it Ascos- phaera proliperda. In addition, he studied its life cycle in vitro and compared its bi- ology with other known Ascophaeraceae. His studies were based on isolates from a total of 10 diseased larvae collected during a 4-year period (1967-1970) from a popu- lation of M. centuncularis maintained in greenhouses at Copenhagen, Denmark. Unfortunately, little attention, has been given to the study of A. proliperda in the United States, due primarily to the belief (Dr. Leslie Kish, oral presentation, Chalk-

brood Steering Committee, 1980; Vanden- berg and Stephen, 1983; Rose, Christensen, and Wilson, unpubl.) that this fungus is not authentically known from the United States.

In 1980, we discovered this fungus in field-collected larvae of Megachile rotun- data that showed the typical symptoms of chalkbrood disease induced by A. aggre- gata. Because M. rotundata is a commer- cially important pollinator of alfalfa, it was apparent that this discovered association could potentially implicate A. proliperda as the source for current increase in mortality of young bee larvae of M. rotundata. Therefore, an in vitro study of A. proli- perda in M. rotundata, with emphasis on development, symptoms, and pathogen-

11 0022-201 l/84 $1 so Copyright 63 1984 by Academic Press, Inc.

12 YOUSSEF, ROUSH, AND MC MANUS

icity was completed and results obtained are reported herein.

MATERIALS AND METHODS

Source of inoculum. Spores of A. proli- perdu used in this investigation were ob- tained from in vitro isolates originating in Utah and from another isolate obtained from Dr. Leslie Kish (University of Idaho, Moscow, Idaho). According to Kish, the European isolate originated from in vitro cultures from CBS, Baarn, The Nether- lands; the original culture (provided by Skou) was entered as Accession No. CBS 687.71. The Utah isolate was recovered ser- endipitously in 1980 from an in vitro culture that was inoculated by what we thought to be pure spores of A. aggregata. These spores were isolated from alfalfa leafcutting bee larvae that showed the typical charac- teristics of chalkbrood disease caused by this species. These larvae were obtained from a commercial nesting site in northern Utah.

Znoculation of natural provision. Cells containing natural provisions of the alfalfa leafcutting bee were collected from nests placed in a shelter within an isolated alfalfa seed field at Clarkston, Utah. Nesting holes were prepared by placing pieces of paper soda straw (each 10 mm long x 5 mm di- ameter) in predrilled wood blocks. Straws were examined daily and those with com- pleted cells were transferred to the labora- tory. Cells were then removed from straws, cell caps were removed, and each cell was placed into a No. 00 Beem capsule. A small drop of spore inoculum, made by squashing several ascocytes in small amounts of honey, was placed in close proximity to the egg with a syringe fitted with an l&gauge needle. Spore counts were adjusted to about 250,00O/drop by adding either more spores or more honey to the inoculum. Beem capsules containing provisions and eggs were incubated at 30°C and 80% rela- tive humidity.

Preparation and inoculation of artificial provision. Frozen honey bee-collected

pollen pellets, were used as the pollen source for this study. These pellets were soaked in cool water for several hours, strained repeatedly through several layers of cheese cloth, and concentrated to a paste by centrifugation. The resulting paste was transferred to cans and sterilized under fractional steam sterilization (Tyndalliza- tion) to avoid dissolution of its consistency. Sterilized cans were kept intact until usage.

To prepare the artificial provision, a por- tion of the sterilized pollen paste was mixed thoroughly with “pure alfalfa honey” (Miller Co., Salt Lake City, Utah) to a con- sistency that closely resembled a “typical” leafcutting bee provision in both taste and texture. The pollen/honey mixture was then drawn into sterile syringes and kept at 4°C until usage.

To inoculate the artificial provision, sev- eral ascocysts were removed asceptically from culture dishes and physically crushed and stirred into 0.20 ml honey in a Petri dish until the spores were completely dispersed. The spore/honey mix was then blended thoroughly and gradually with sufficient pollen/honey mix (artificial provision) to yield 5 ml of mixture. Spore count/provi- sion was adjusted to that required for each treatment following the method described in the previous section. The artificial pro- vision/spore mix was drawn into a syringe, and kept at 4°C until usage.

Artificial provision mix or spore/artificial provision mix was then dispensed from the syringe (in aliquots equal to that of natural provision) into No. 00 Beem capsules, which were placed vertically in specially designed trays. Eggs were removed from naturally provisioned cells and transferred onto the artificial media. Trays with their holdings were maintained under the same environmental conditions as capsules con- taining naturally provisioned cells.

To estimate the number of spores used per provision, an aliquot equal to provision was suspended in 5 ml of water. The sus- pension was vigorously agitated and passed through a filter to remove pollen grains. A

DEVELOPMENT AND PATHOGENICITY OF A. proliperda 13

spore count of the filtrate was then made with the aid of a Neubauer hemocytometer.

An alternative to the artificial cell tech- nique was also used (Figs. 7, 8). Three mil- liliters of provision mix was dispensed in small Petri dishes (35 mm diameter) and al- lowed to spread evenly to a thickness of about 3 mm. Eggs were then transferred onto the surface of the provision at 1 cm apart. A small piece of sterilized soda straw (10 mm long, 6 mm diameter) was placed upright over each egg and pressed into the provision surface, thus forming a separate cell for each egg. Petri dishes were covered and placed under the same environmental conditions as those described for the indi- vidual cells.

RESULTS

Symptoms of Infection

Larvae reared individually in Beem cap- sule cells containing the artificial provision/ spore mix were overcome by the infection and died irrespective of the spore concen- trations tested. Figures 1-6 demonstrate how a typical progressive sequence of in- fection and fungal development occurred under laboratory conditions. Typically, the larvae died within 1 week after eclosion, before reaching one-half the size of healthy fifth instar larvae. Dying larvae frequently defecated a small number of soft pellets. Dead larvae became flaccid and their color changed to cream or light brown. During the week following death, corpses gradu- ally dessicated and shriveled slightly prior to developing a light dimpling of the integ- ument. The dimpling was apparently due to the pressure of growing hyphae that even- tually penetrated the integument (Figs. 12, 13). Approximately a week after death, the corpses developed a “fuzzy” appearance (to the naked eye) due to hyphal fdaments protruding through the integument (Figs. 2, 3). Soon after “fuzzing” occurred, each corpse became obscured as the cell filled with a profusion of hyphae (Fig. 4). Scat- tered nutriocytes in the vegetative growth

were visible 5 to 8 days after the hyphae first protruded above larval cuticle (Fig. 4). Opaque white nutriocytes darkened as they developed and became the characteristi- cally black ascocyst structures 5 to 7 days (at 30°C) after their formation (Fig. 6).

Only 1 of the 143 larvae reared individ- ually in Beem capsules provided with arti- ficial provision laden with A. proliperda spores survived to pupate, while all the eggs of the control sample that hatched (15) produced healthy larvae. Typically, larvae feeding upon the lower concentrations of spores (1500-3300) lived 3 to 4 days longer than larvae on the more concentrated spore/provision mixes. These larvae often attained near normal fifth instar size before succumbing with chalkbrood symptoms. Hyphal protrusion following death was also sometimes delayed.

M. rotundata larvae that had been reared in groups on the artificial provision/spore mix in Petri dishes (Table 3, Figs. 7, 8) de- veloped symptoms of infection similar to those described for the Beem capsule trials. Expression of symptoms was also delayed at lower concentrations.

Course of Infection

Histological and scanning electron mi- croscope examinations of sectioned treated larvae revealed that infection began with the germination of fungal spores that had been introduced orally into the lumen of the gut (Fig. 9). No evidence was found to sug- gest that infection took place via penetra- tion of larval integument or via the respi- ratory system. Developing hyphae imme- diately penetrated the gut wall regardless of location (Fig. 10) and invaded all larval tis- sues including muscles, ganglia, glands, and fat bodies (Fig. 11). It is of interest to note that hyphae of this fungus were ca- pable of penetrating all cuticular elements regardless of system (foregut, the respira- tory system, larval integument, etc.). Prior to consuming all tissues, the fungus pene- trated the larval integument randomly and began its aerial development (Fig. 12). In

YOUSSEF, ROUSH, AND MC MANUS

FIGS. 14 Leafcutting bee larvae fed on artificial provision that was inoculated with spores of As- cosphnera proliperdu showing the sequential development of the fungus. Note beginning of aerial growth of fungus and its lack of growth on provision (Fig. 2). 5 x

FIG. 7. Different method of raising bee larvae on artificial provision. FIG. 8. Group of leafcutting bee larvae similar to those in Fig. 7 two months after inoculation of the

provision with fungus. Note prolific aerial growth of fungus with many mature ascocysts.

DEVELOPMENT AND PATHOGENICITY OF A. prdiperda

FIG. 9. An interference light micrograph of a transverse section of a portion of midgut (MG) of leafcutting bee larva three days after inoculation. Note germinating spore (arrow) surrounded by pollen grains (P). 788 x

FIG. 10. An interference light micrograph of a similar section to that of Fig. 9 showing a later stage of fungal development. Note penetrating hyphae (arrow heads) from gut lumen and the destruction of gut wall. Also note the apparent sparsity of hyphae in gut lumen. 281 x

FIG. 11. An interference light micrograph of a transverse section of a portion of an infected larvae. Note prolific growth of hyphae in the haemoceol and the complete destruction of larval tissues. 281 x

YOUSSEF, ROUSH, AND MC MANUS

FIG. 12. A scanning electron micrograph of infected larvae. Note protruding hyphae (arrow) on surface of integument identified by the presence of spicules (SP) and setae (ST). 248 x

FIG. 13. Scanning electron micrograph of groups of hyphae (double arrow) that appeared to penetrate the larval integument as a bundle. 500 x

FIG. 14. A scanning electron micrograph of aerial growth of A. proliperda. Note the formation of nutriocyte (N) and trycogen cells (T) necessary for sexual reproduction of the fungus. 300 x

FIG. 15. A scanning electron micrograph of a group of spore balls of Ascosphaera protiperda that were isolated from ascocysts developed from artificially inoculated leafcutting bee larvae. Note the lack of membrane surrounding the spore balls (SB). 1120 x

DEVELOPMENT AND PATHOGENICITY OF A. proliperda 17

many instances, a group of hyphae (Fig. 13) were observed to have pushed through the integument collectively and penetrated it as a bundle. Aerial hyphae grew rapidly, forming a white mat that eventually encap- sulated the larvae. Within a few days of penetration, the reproductive phase of the fungus was observed aerially (Fig. 14) and viable spores were obtainable within a month from infection (Fig. 15).

Pathogenicity

Mortality rate induced by A. proliperda to leafcutting bee larvae was constant ir- respective of source of inoculum, type of provision, and method of rearing (Tables l- 3). In these experiments, all treated larvae (except three) died prior to pupation even at the low dose of 1500 spore/provision. However, there was an apparent delay in symptoms associated with low doses.

Delaying the onset of infection to leaf- cutting bee larvae seemed to have had no effect on the final results in one experi- ment. In an additional experiment when in- fection was delayed by “sandwiching” the spore inoculum between the two layers of clean provision, thus allowing the larvae to reach the fourth instar before feeding on the spore inoculum, symptoms of chalkbrood did not appear until the infected larvae reached maturity. By the time larvae began to defecate, however, they were overcome by the fungus in a manner similar to those larvae allowed to feed on provision spore mix immediately after hatching.

Larvae of the control group, which fed upon clean provision only, developed nor- mally, with no incidence of infection.

DISCUSSION

A. proliperda is pathogenic to the larvae of M. rotundata, at least under laboratory conditions. This statement is supported by the fact that of 405 larvae fed on either nat- ural or artificial provisions that were inoc- ulated with spores of either isolate, only three larvae survived the treatment even at the low dose level of 1500 spore/bee. It should be noted, however, that the num- bers of spores given in the study are by no means a true reflection of the number of spores ingested by the larvae; in almost all instances, larvae did not consume more than half the provided provisions. In a sim- ilar experiment, but on a rather small scale, Stephen et al. (1981), using a Denmark iso- late obtained from J. Rose (University of Wyoming, Laramie), reported that from a total of nine treated larvae, two succumbed with chalkbrood-like symptoms, six died without being infected while the fungus grew on the pollen provision, and one died from undiagnosed reasons. Their data could be used to argue the point that A. proliperda is merely an opportunist as is its relative A. apis. Indeed, Stephen (pers. commun.) has since referred to this possi- bility. Bailey (1967) characterized A. apis as an opportunistic pathogen that kills honey bee larvae only when larvae are sub- jected to environmental stress. This view was also shared by Gochnauer and Mar-

TABLE 1 COMPARATIVE PATHOGENICITY OF Two ISOLATES OF Ascosphaera proliperda OF DIFFERENT SOURCES TO THE

LARVAE OF THE LEAFCUTTING BEE, Megachile rotundata RAISED ON NATURAL PROVISION, EACH CONTAINING

ABOUT 250,000 SPORES

Source of inoculation

No. of eggs

No. larvae with CB”

Nonfungal death Healthy

Isolates from Utah 200

Isolate L. Kish 16

0 CB = chalkbrood symptoms.

175 25

12 3

None

I --

18 YOUSSEF, ROUSH, AND MC MANUS

TABLE 2 PATHOGENICITY OF DIFFERENT SPORE DOSE OF Ascosphaera proliperda TO LARVAE OF THE LEAFCUI-HNG BEE,

Megachile rotundataa

Treatment Results

Spore/ No. larvae No. healthy provision No. eggs Egg deathsb with CB larvae

3 x 106 22 2 20 0 3 x 106 83 6 77 0 3 x 105 15 2 13 0 3 x 104 13 1 11 1 3 x 103 10 1 9 0

0 19 4 0 15

0 Each larvae was reared on artificial provision in a separate Beem capsule. b Deaths due to mechanical damage.

getts (1979), who stated that A. upis is a “relatively non-invasive pathogen” that kills the honey bee larvae by competing for primary nutrients. This is not the case for A. proliperdu. In our study, the chalkbrood disease was expressed at high levels be- tween the different experimental groups compared to the control. Under identical conditions we also proved that A. proli- perdu spores will not germinate when in- oculated onto provision only.

Our study shows that infection with A. proliperda occurred only when larvae of the leafcutting bee, M. rotunduta, ingested the spores. At no time have we seen infec- tion take place by fungal penetration of the larval integument. McManus (1982) re- ported similar results for A. aggregata, an- other pathogen of the leafcutting bee. Bar- the1 (1971) and Matus and Sarbak (1974) al- leged, however, that natural infection of the honey bee larvae by A. upis occurs either by ingestion of spores with food or by pen- etration of the larval integument.

Symptoms of the chalkbrood-like disease induced by oral inoculation with spores of A. proliperda are quite different from those induced by the other recorded pathogenic species of the Ascosphaeraceae. Symptoms of the disease in larvae infected with A. proliperdu are recognized by the random protrusion of the hyphae through the integ- ument at early stages of larval development (Figs. 12, 13). However, Vandenberg and Stephen (1983) claim that the growing hy- phae of A. proliperda, “first erupted through the intersegmental membrane,” thus implying that the intersegmental mem- brane of the larvae has different morpho- logical characteristics than the rest of the larval integument (except the head). In bees, as in many holometabolous insects, larval cuticle remains undifferentiated (Dennell, 1946; Locke, 1960; Way, 1950) (Figs. 12, 13). Within a day or two, asco- cysts were formed aerially on many of these mycelia (Fig. 14). Ascocysts were never observed beneath the integument of

TABLE 3 PATHOGENICITY OF DIFFERENT SWRE DOSES OF Ascosphaera proliperda TO THE LARVAE OF THE LEAFCUI-~ING

BEE, M. rotundata

No. of Spore/

es.3 provision

15 1.5 x 105 16 1.5 x 104

15 1.5 x 103

EB deaths

0 3 1

No. larvae with CB

15 13 14

No. healthy larvae

0 0 0

0 Each larvae was reared in a separate compartment in a petri dish on artificial provision (Figs. 7, 8).

DEVELOPMENT AND PATHOGENICITY OF A. proliperda 19

the infected larvae. Consequently, our ob- servation regarding the formation of cysts are in full agreement with the original de- scription of the fungus (Skou, 1972). On the other hand, A. aggregata, which also in- fects larvae of M. rotundata, seldom induce any disease symptoms prior to the larvae becoming mature (McManus , 1982). During the late fourth stadium the color of infected larvae usually changes to creamy, then to a light brown, and finally to brown black. This color change is actually a reflection of changes in the color of the developing as- cocysts, which develop beneath the almost translucent integument. Contrary to claims by Vandenberg and Stephen (1982), at no time have we seen A. aggregata grow out- side the host. These authors state, “but oc- casionally the fungus erupted and grew to a limited extent on the residual pollen diet.”

Symptoms induced by A. apis, in honey bee larvae are quite different from those of A. proliperda in the leafcutting bee. Ac- cording to Bailey (1967), the infected honey bee larvae swell greatly following death, thus filling their occupied cells with hyphae penetrating through the integument. At later stages, these larvae dried to a chalky, hard lump that was gray and black. Exam- ination of similar larvae (in our laboratory) revealed that ascocysts were produced both under and above the integument.

Our results suggest that additional atten- tion should be given to studies of etiology, cross infectivity, and natural distribution of A. proliperda among wild bee taxa. These studies would serve as a foundation for the development of control measures of the fungus. Unfortunately, control measures may become necessary because in a recent field survey (July, 1982) designed to esti- mate levels of infection with A. aggregata in a commercial alfalfa leafcutting bee op- eration at Clarkston, Utah, we found 4 of 127 larvae with similar symptoms to those produced in the laboratory by A. proli- perdu. Although these data suggest that the incidence of the disease is currently low,

one is reminded how rapidly the chalk- brood disease caused by A. aggregata has spread in commercial populations of the al- falfa leafcutting bee in the United States and other regions (Stephen and Undurraga, 1978).

ACKNOWLEDGMENTS

The Multistate Chalkbrood Project is supported by contributions from grower groups and industry rep- resentatives in Idaho, Nevada, Oregon, and Wash- ington; the Idaho, Nevada, and Washington Alfalfa Seed Commission; Universities of Idaho and Nevada- Reno; Oregon, Utah, and Washington State Universi- ties; and by a cooperative agreement with the USDA/ SEA Federal Bee Laboratory, Logan, Utah. Many thanks are due to William P. Nye, Collaborator, U.S. Department of Agriculture, ARS, Logan, Utah, for providing honey bee-collected pollen, V. J. Tepedino and Philip Torchio, USDA, ARS, Logan, Utah, for critically reviewing the manuscript, and Nancy So- rensen for typing it. This paper is a contribution from Utah State Agriculture Experiment Station, Journal Paper No. 2768, and the Department of Biology, Col- lege of Science, Utah State University.

REFERENCES

BAILEY, L. 1967. The effect of temperature on the pathogenicity of the fungus, Ascosphaera upis, for larvae of the honeybee, Apis mellifera. In “Insect Pathology and Microbial Control” (P. A. van der Laan, ed.), pp. 162-167. North-Holland, Am- sterdam .

BARTHEL, B. 1971. Der Kalkbrut auf der Sput. Carten Kleintierzucht C. Imker., 10, 12-13.

DENNELL, R. 1946. A study of an insect cuticle: The larval cuticle of Sarcophaga fulculata Pand. (Dip- tera) Proc. Roy. Sot. Ser. B, 133, 348-373.

GOCHNAUER, T. A., AND MARGETTS, V. J. 1979. Prop- erties of honeybee larvae killed by chalkbrood dis- ease. J. Aqic. Res., 18, 212-216.

LOCKE, M. 1960. The cuticle and wax secretion in Cal- podes ethlius (Lepidoptera, Hesperidae). Quart. J. Microsc. Sci., 101, 333-338.

MAASSEN, A. 1916. Uber Bienenkrankheiten. Mitt. Kaiserl. Biol. Amt. Land.-Forstw., 16, 51-58.

MATUS, F., AND SARBAK, I. 1974. Occurrence of chalk- brood disease in Hungary. Mayar Allatorvosk Lapis, 29, 250-255.

MCMANUS, W. R. 1982. “Zn vivo Development of Ascosphaeru aggregata Skou in the Alfalfa Leaf- cutting Bee, Megachile rotundata (Fabricius): .4 Light and Electron Microscope Study.” M.S. Thesis, Utah State University.

SKOU, J. P 1972. Ascosphaerales. Friesia, 10, 1-24

20 YOUSSEF, ROUSH, AND MC MANUS

SKOU, J. P. 1975. Two new species of Ascosphaera and notes on the conidial state of Bettsia alvei. Friesia, 11, 62-74.

STEPHEN, W. P., AND UNDURRAGA, J. M. 1978. “Chalk- brood Disease in the Leafcutting Bee.” Oregon State Univ. Agric. Exp. Sta. Bull. 630.

STEPHEN, W. P., VANDENBERG, J. D., AND FICHTER,

B. L. 1981. “Etiology and Epizootiology of Chalk- brood in the Leafcutting Bee, Megachile rotundata (Fabricius), with Notes on Ascosphaera Species.” Oregon State Univ. Agric. Exp. Sta. Bull. 653.

VANDENBERG, J. D., AND STEPHEN, W. P. 1982. Etiology and symptomatology of chalkbrood in the alfalfa leafcutting bee, Megachile rotundata. J. In- vertebr. Patho/., 39, 133-137.

VANDENBERG, J. D., AND STEPHEN, W. D. 1983. Patho- genicity of Ascosphaera species for larvae of Me- gachile rotundata, J. Apic. Res. 22, 57-63.

WAY, M. J. 1950. The structure and development of the larval cuticle of Diataraxia oleracea (Lepidop- tera). Quart. J. Microsc. Sci., 91, 145-182.