Final Report for GS09-086

Project Type: Graduate Student
Funds awarded in 2009: $9,963.00
Projected End Date: 12/31/2011
Grant Recipient: Univ. Tennessee
Region: Southern
State: Tennessee
Graduate Student:
Major Professor:
Dr. John Skinner
Univ. Tennessee
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Project Information

Summary:

The purpose of this study was to evaluate the effect of fumagillin, thymol, Nozevit and Honey-B-Healthy on Honey bees infected with either Nosema ceranae or Nosema apis. Prospective treatments for Nosema disease were evaluated by examining both mortality and spore production in caged honey bees. Bees used in the study were shaken from frames of uncapped brood taken from hives visibly free of Nosema infection. These bees were chilled with ice water, placed in cages (180 bees/cage) and immediately infected with either N. apis or N. ceranae through gravity feeders at a rate of 40,000 spores/bee. Treatment began 3 days after inoculation. Dead bees were removed and counted every subsequent day. Twenty-three days after inoculation, 10-15 bees were removed, abdomens were crushed individually in water, and spores were counted using a hemocytometer.

Although treatment greatly affected mortality, no difference was noted with respect to the species of Nosema. Bees treated with thymol and Honey-B-Healthy were found to have rates of mortality similar to uninfected bees. Bees treated with Nozevit had rates of mortality similar to untreated bees. Fumagillin improved mortality, but not to levels of uninfected bees (Table 2).

Honey-B-Healthy had no effect on spore production, therefore the reduction in mortality displayed under treatment must be attributed to positive effects on the bee itself rather than to an antagonistic effect on the pathogen. Fumagillin was the only compound to significantly and consistently reduce spore production. The failure of fumagillin to reduce mortality congruently is unexplained. Thymol and Nozevit had some effect on spore production but it was unclear how significant this effect was.

Introduction

The purpose of this project is to determine the best way to treat the honeybee disease Nosema in Tennessee by comparing the relative efficacy of fumagilin, thymol, Nozevit and Honey-B-Healthy on two species of Nosema through experimental infection and treatment.

Nosema infection (nosemosis) has been a longstanding problem in beekeeping. Queens infected with Nosema may be more prone to superscedure. Infected individual bees have a shortened lifespan and collect less pollen. Overall, nosemosis can lead to reduced productivity and colony death. Light infections are often asymptomatic.

The diagnosis of nosemosis has become more complex recently with the emergence of a new species of Nosema. Nosema apis has been a recognized (microsproridian) parasite of honey bees for a century. Until recently all nosemosis in honey bees was attributed to this species. Nosema ceranae, isolated in 1996, was initially thought to be an exclusive parasite of the Asian honey bee (Apis cerana). Nosema ceranae has recently been found infecting Apis melifera worldwide and throughout the United States. The exact distribution of each species is unknown. There is evidence that Nosema cerenae has greater pathogenicity and is more likely to cause hive collapse than N. apis.

Currently, the most commonly used treatment for nosemosis is Fumigillin. There is evidence it controls both species of Nosema quite well. Though it is currently very effective, there are concerns exclusive use of Fumagillin will foster resistance in Nosema. Furthermore Fumigillin is a teratogen and its use has been banned in the EU, in Great Britain and elsewhere.

Nozevit is an oak bark extract produced in Croatia. It has been found to be as effective as fumigillin in controlling Nosema ceranae.

Honey-B-Healthy is made form lemongrass and spearmint oil. It’s marketed as a feeding stimulant and is has not been tested for Nosema control.

Several experiments have been done examining the usefulness of thymol in the treatment of nosemosis but the results are conflicting. In some cases, with rigorous control, thymol was found to work better than fumigillin for treatment of both Nosema ceranae and N. apis. Yet in other cases it was found to have no effect.

Project Objectives:

• Determine effect of treatment with thymol solution, Nozevit, Honey-B-Healthy, on honey bees infected with either Nosema apis or N. ceranae.
– Does treatment have an effect on longeveity of caged honey bees?
– Does treatment have an effect on Nosema spore production in caged honey bees?
– How do the prospective treatments listed above compare to Fumagillin, the standard for adequate treatment of Nosema in honey bees?
Determine if infection with Nosema apis is more or less virulent than infection with Nosema ceranae in terms of accelerated honey bee mortality.

Cooperators

Click linked name(s) to expand
  • Paul Rhoades
  • Dr. John Skinner

Research

Materials and methods:

Hives visibly free of Nosema were found by collecting 50 bees from the entrance and crushing the abdomens in 5ml of water. The suspension was then viewed with a light microscope for any presence of Nosema. Bees were gathered from Nosema free hives by shaking frames of uncapped brood into a plastic tub ringed with Vaseline to prevent crawling bees from escaping. Bees that could fly were allowed to escape leaving only young workers. These bees were brought back to the laboratory and chilled in ice water before being counted and divided into cages (180 bees/cage). Twelve total cages were produced in this way (Table 1). Bees to be infected were immediately fed spores of either N. apis or N. ceranae suspended in sugar syrup (1:1 sucrose : distilled water by weight) through a gravity feeder at a rate of 40,000 spores/bee.

Bees were then placed in an incubator at 27°C temp and were fed with a 1:1 by weight sucrose : distilled water solution and mixture of sugar solution and Megabee (Tuscon, AZ) for protein. Treatment began on day three. All treatments were mixed by label instructions except thymol. Thymol was dissolved into ethanol and mixed at a rate of 0.6?l ethanol containing 0.1mg thymol per ml of sugar solution. Dead bees were counted and removed every subsequent day. At the end of the trial, guts 10-15 bees were removed for spore counting.

ANOVA was used to determine the significance of final spore count differences. Survival analysis was used to determine the significance of differences in mortality among treatments. All statistics were run using JMP.

Research results and discussion:

An original goal of this project was to characterize the response of the vegetative (living) stage of Nosema apis and N. ceranae to various treatments. This was to be accomplished by viewing thin sections of fixed, paraffin embedded gut tissue under a light microscope. By evaluating relative rates of vegetative reproduction under different treatment regimens I hoped to gather data on the efficacy of different treatments as well as determine how the treatment may work. For example, does the treatment attack the vegetative state directly by inhibiting reproduction within the cell or does the treatment prevent cell colonization, spore germination or final spore formation? Unfortunately, the individual response to the initial stages of infection was highly variable even when factors such as inoculation dose and honey bee age are held constant. Making and analyzing thin sections was very time consuming and processing the amount of specimens necessary was not feasible. Therefore, determining treatment effects on intracellular Nosema performance were not possible with these methods.

I instead chose to evaluate efficacy of treatment with two metrics: Survivorship and spore production in caged honey bees. This method had the benefit of large sample size to obscure the effect of variability of infection.

First, there was no difference in mortality between bees infected with N. ceranae and N. apis. This confirms results concurrently obtained by Zachary Huang at Michigan State University (personal communication). The similarity in mortality persisted under all treatments.

The results for mortality and spore production under various treatments were more complex. The results are summarized in Table 2.

Differences in mortality between infected and uninfected bees became apparent immediately. Differences in mortality were statisticly significant at p=0.05 by the 13th day. Mortality of infected bees continued to increase. 60% of infected bees were dead after 23 days, while only 10% of uninfected bees had died. (Table 2; Figure 1)
Among bees treated with thymol, Honey-B-Healthy and fumagillin there was no difference in mortality until day 18 (Figure 2). After this time bees treated with fumagillin began experiencing greater rates of mortality, while bees treated with Honey-B-Healthy or thymol had statistically similar rates of mortality to uninfected bees.

Conversely infected bees treated with Nozevit began experiencing greater mortality by day 13(Figure 3). The differences in mortality between uninfected bees and those treated with Nozevit became statistically significant by day 15. Mortality of bees treated with Nozevit continued to increase until final mortality was statistically similar to untreated infected bees.

Spore count data is difficult to interpret. Although the data seems to suggest thymol and Nozevit slow spore production in the gut, the wide range of susceptibility to infection means more experiments will need to be run to determine the consistency of this trend (Figure 4).

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

These results were presented at the North American Bee Research Conference in Galveston, Texas in February, 2011. An abstract of those proceedings will be published in the American Bee Journal. I’ve also discussed these results with many individual beekeepers through extension work.

Project Outcomes

Project outcomes:

These results demonstrate the relationship between disease severity and mortality are not necessarily linear. The deleterious effects of Nosema infection may be mitigated by feeding stimulants such as Honey-B-Healthy, effectively raising the economic threshold for treatments with harder chemicals such as Fumagillin. Similarly the stress Fumagillin places on honey bees is illustrated here.

Economic Analysis

Thymol is a commonly used by beekeepers to prevent fermentation of sugar syrup fed to bees. Honey-B-Healthy and Nozevit are commonly used spring feeding stimulants. These compounds are widely available and cheaper than Fumagillin. The cost of beekeeping and its associated products (pollination, honey) may be reduced if treatment with fumagillin can be eliminated in some years by use of alternative compounds.

Farmer Adoption

Beekeeper adoption of alternate treatments for Nosema is unknown at this point although some have expressed interest in trying it. The data produced by these experiments aren’t enough to make any firm recommendations at this time. However, the possibility that it might work is enough for some beekeepers to begin experimenting on their own.

Recommendations:

Areas needing additional study

Although thymol and Honey-B-Healthy reduce mortality of infected bees it is unknown if treatments with these compounds will delay rapid reoccurrence of disease in the real world or just obscure the symptoms. In order to really determine the efficacy of these compounds for treatment of Nosema disease a number of questions will need to be resolved:
1. Is it necessary to lower spore production in the gut or, if symptoms are treated, will the disease fade from the colony as environmental conditions change? Fumagillin, while effective, does not eliminate disease from the hive entirely, it just lowers infection in treated bees. Treatment with fumagillin generally reduces Nosema presence in a hive for a full year. This is may be because it’s stored in honey, and consumed slowly for many months. Or it may be because treatment allows bees to get through more difficult times of the year.
2. Nosema infection causes afflicted bees to defecate inside a hive instead of outdoors. This spreads the pathogen within the hive through feces. Treatment with fumagillin may break the infection cycle in a hive by limiting this symptom. Judging by the appearance of the cages after a few weeks of confinement Honey-b-Healthy and thymol may also limit inadvertent defecation. If this is true Nosema presence may be reduced for a time frame similar to fumagillin.
3. Will consumption of feed in a working hive have similar effects to what was observed in lab cages?

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.