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Project description

The fungus, Hirsutella thompsonii, for the biological control of the Varroa mite, a pest of honey bees. (01BC006)
Program UC IPM competitive research grants program
Principal
investigators 		C.Y. Peng, Entomology, UC Davis
E.C. Mussen, Entomology, UC Davis
Host/habitat Honey Bee
Pest Varroa Mite Varroa jacobsoni
Discipline Entomology
Beneficial
organism 		Hirsutella thompsonii
Review
panel 		Biological Control
 Start year (duration)  2001 (Three Years)
Objectives Develop the fungus, Hirsutella thompsonii, as a biological control agent of the honey bee ectoparasitic mite, Varroa jacobsoni.

Establish a standard protocol for infecting Varroa with various isolates of H. thompsonii.

Examine and evaluate the infectivity of various isolates of H. thompsonii under different temperature and humidity regimes that simulate the fluctuations within a bee hive against Varroa mite.

Assess the nontarget effects of the most virulent isolate(s) to the Varroa mite against honey bee larvae, pupae, and adults of worker, drone, and queen castes.

Conduct field trials of the most virulent isolate(s) in mite-infested bee colonies.

Final report Because the widespread infestation of the extoparasitic mite, Varroa destructor, have decimated numerous feral and managed honeybee colonies and have developed resistance to fluvalinate acaracide, which also has caused the contamination of beeswax, the growers who depend on honeybee for crop pollination and beekeepers share grave concerns about the mites. Clearly, there is a compelling need to find alternative control methods for this devastating mite. The acaricidal fungus, Hirsutella thompsonii, is a strong candidate for bicontrol of Varroa because it infects a range of mite species with no known adverse effects to other taxa. In addition, we have preliminary results indicating that H. thompsonii can infect Varroa and increases mite mortality under laboratory conditions. We proposed to develop a standard protocol for effectively infecting the Varroa mite with H. thompsonii, conducting laboratory and field tests of ineffective isolates of H. thompsonii, and developing H. thompsonii as a control agent for Varroa. A total of four experiments were conducted from summer 2001 to fall 2003 as planned in the proposal.

Experiment I. Among the nine isolates of H. thompsonii obtained from the University of Florida and the USDA, only the three USDA isolates (ARSEF 257, 1947, and 3323) were infectious to the Varroa mite in laboratory tests. The mite became infected when it was allowed to walk on a sporulating H. thompsonii culture for five minutes. SEM study revealed that thee membranous arolium of the mite leg sucker is the focus of infection where the fungal conidia adhered and germinated. The infected mites died from mycosis with LT50s ranging from 52.7 to 96.7 h depending on the isolates. The fungus did not infect the honeybee in all developmental stages. The results concluded that some isolates of H. thompsonii have the potential to be developed as a biocontrol agent for Varroa mite.

Experiment II. The laboratory rearing results and SEM observations clearly showed that H. thompsonii only infected Varroa mites and did not infect honeybee in all developmental stages. It is concluded that the fungus is safe for honeybees.

Experiment III. The mortality data of Varroa mite, subsequently inoculated with H. thompsonii and maintained on host honeybees in the cage experiments, showed that H. thompsonii can infect mites at 30, 50, 70, and 90% relative humidity (RH) at 34 to 35 degrees C and subsequently kill the mites, although higher RH also cause higher mite mortality. Varroamite mortality also increases with the increase of RH. The data indicated that H. thompsonii could cause mycosis of Varroa mites while parasitizing host honeybees in laboratory rearing cages in RH simulating the beehive condition.

Experiment IV. Experiment IV was modified to test variously formulated H. thompsonii in cage rearing experiments instead of testing the fungus in field bee colonies, because of limitation of available quantity of the fungus, Varroa mites, and manpower to manage field bee colonies. The results showed a minimal infection of Varroa mites using these tested formulations. Significant improvement on the formulation of H. thompsonii is needed before further tests should be conducted.

Third-year
progress 		During 2003, several caged experiments were conducted. Worker bees infested with mites were treated with Hirsutella conidia formulated in bran. The viability of the conidia was high at the time of treatment (>90%) and moderate one day later at 34 °C (>60%). Some mites died from Hirsutella infection (range 0 to 33%), but mite mortality in the control treatment was also high. The cause of death in the control treatment was unknown. Mortality of the workers bees occurred at equal levels in both treated and control cages. No Hirsutella infection was observed in the dead workers.

Second-year
progress 		Varroa destructor, an ectoparasitic mite of the honey bee (Apis mellifera), is the major culprit causing a worldwide decline of honey bee populations. It feeds on the blood of immature and adult bees that weaken them or contribute to their death. Heavy mite infestations decrease the bee populations for pollinating plants and adversely affect production of hive products such as honey and beeswax. We are evaluating a biological control alternative, the mite fungus Hirsutella thompsonii, to suppress the Varroa mite.

In 2001, we obtained nine H. thompsonii isolates and bioassayed them against adult female mites. In our bioassay, we found that three of the nine fungal isolates were pathogenic and killed the mites with a lethal time to kill 50% (LT50s) averaging 52.7, 77.2, and 96.7 hours post-fungal inoculation. Total mortality of the treated mites ranged from 80-100%, whereas mortality of the control mites ranged from 0-6.7% at seven days post-treatment. Using a scanning electron microscope (SEM), we demonstrated that the mites' membranous suckers or arolia (= bottom of the legs) served as the primary site of fungal infection. The conidia germinated about 12 hours post-fungal inoculation causing high mite mortality by 100 hours. During the 7-day period, none of the bee pupae were adversely affected by H. thompsonii. The above information has been published (Peng, C. Y., X. Zhou, and H. K. Kaya. 2002. J. Invertebr. Pathol. 81: 185-195), and a copy is attached to our report.

In 2002, we determined the infectivity of the three virulent isolates of H. thompsonii against the Varroa mite at humidity regimes that may occur within a beehive. Mites exposed to H. thompsonii conidia were placed on worker honey bees which were then held in rearing cages at 90, 70, 50 and 30% relative humidity (RH) at 34 oC for 2 weeks. The mite mortality by the fungus isolate 257 14 days post-treatment was >75% for the 90 and 70% RH, >50% for the 50% RH, and <15% for the 30% RH. Although there was some variation, the other two fungal isolates showed similar trends. Control mortality ranged from 13 to 21% at the various RH. The data suggest that higher humidity is needed for the fungus to be effective against the Varroa mite, but this higher humidity occurs in the beehive. In preparation for field tests, we examined survival of fungal conidia in various formulations that could potentially be used in the beehive. We examined 13 different formulations and so far, two formulations, 10% maltose and 10% skim milk, appear to provide the best conidial survival. Further work on formulation is being conducted to optimize conidial survival.

The nontarget effects of H. thompsonii were evaluated against various life stages of the honey bee. Adult worker bees were exposed to conidia, held in cages with 50% sucrose and water and incubated at 34 oC and 93% RH. Late larvae, prepupae, and pupae were exposed to conidia, held in wells of tissue culture plates, and checked for mortality daily. Adults that emerged were held for seven days and checked for mortality daily. H. thompsonii had no effect on larvae, prepupae, pupae, and adults.

First-year
progress 		Varroa destructor is an ectoparasitic mite of the honey bee Apis mellifera and is the major culprit causing a worldwide decline of honey bee populations. It feeds on the blood of the immature and adult bees which weaken them or contribute to their death. Heavy mite infestations significantly decrease their populations for pollinating plants, and adversely affect production of hive products such as honey and beeswax. Control of the mite is difficult because of its resistance to chemical pesticides. Moreover, the presence of pesticides in beeswax and concerns of pesticide use, in general, have necessitated that an alternative control measure be pursued. We are evaluating a biological control alternative that is examining the mite fungus, Hirsutella thompsonii, to suppress the Varroa mite.

We obtained six H. thompsonii isolates and bioassayed them for activity against adult female mites. The mites were allowed to walk on the fungal culture isolates for 5 minutes, collected immediately and observed with a Hitachi S3500N scanning electron microscope (SEM) or placed individually on a white-body, white-eyed worker pupa from a mite-free colony, and maintained in a clear rearing cup. Control mites were handled as above except that they were allowed to walk for 5 minutes on the culture medium without fungus. The cups were incubated at 34-35°C and 93% RH. Mites were collected at 6-hour intervals thereafter over a 96-hour period and examined under SEM to determine the site of fungal infection. In separate mortality experiments, treated and control mites were handled as above except that all six fungal isolates were tested and checked for mite and host pupal mortality daily at 6-hour intervals for 7 days post-treatment.

SEM studies revealed that the mites membranous suckers served as the primary site of fungal infection. Mites, taken immediately after walking on the culture, had many fungal conidia attached on the sucker. The conidia germinated about 12 hours post-fungal inoculation, causing high mite mortality by 100 hours. In our bioassay study, we found that three of the six fungal isolates were pathogenic and killed the mites with a lethal time to kill 50% (LT50s) averaging 51, 73, and 95 hours post-fungal inoculation. Total mortality of the treated mites ranged from 80-100%, whereas mortality of the control was 2% at 7 days post-treatment. During the 7-day period, none of the bee pupae were adversely affected by H. thompsonii.

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