Final Report for GNC09-102
Honey bees are the major providers of pollination services for cultivated and native flowering plants. The output derived predominantly from honey bee pollination ensures a diverse and nutrient-rich food source for human and animal consumption, as well as provides billions of dollars in domestic and global revenue. The condition of the pollinating capacity and overall health of honey bees in the U.S. has eroded in recent years. Beekeepers have bared the brunt of this health crisis through heavy annual losses of their colonies starting in 2006. Contributing to these losses are mite pests, pathogens, pesticides, and nutritional deficiencies that can interact and jeopardize honey bee colony productivity and survival. One pathogen, Nosema ceranae, has caused reason for concern as it is often found at high levels in both healthy and dying colonies.
An age-based division of labor maintains social organization in honey bee colonies, such that young bees tend to remain in the nest as nurse bees, feeding larvae and the queen, and older bees tend to forage for nectar and pollen outside the nest. Many factors such as the environment, nutrition, or pathogens may act on honey bee physiology to disrupt the timing of the transition from nursing to foraging. This research used cage and field studies to examine the effects of N. ceranae infection on levels of vitellogenin and juvenile hormone, endocrine signaling factors that are fundamental to regulating foraging onset and lifespan in honey bees.
Many beekeepers are finding that it is increasingly difficult to keep their honey bee colonies alive. Exposure to mite pests, pathogens, pesticides, and nutritional deficiencies create a ‘perfect storm’ of factors that can interact and jeopardize colony productivity and survival. The outcome of this health crisis has been losses of nearly one-third of U.S. honey bee colonies annually. Our current rate of colony death is unsustainable and will ultimately result in fewer bees available to pollinate the fruits, vegetables, and native plants that we and other animals depend on for food and shelter.
No one factor can be singled out as an explanation for widespread colony losses; however, the emerging pathogen, Nosema ceranae, has caused reason for concern as it is often found at high levels in both healthy and dying colonies. N. ceranae is a spore-forming fungus that infects and reproduces inside an adult bee’s gut. When a bee ingests N. ceranae spores, they germinate and undergo reproduction inside cells that line the area of the bee gut where food is absorbed. N. ceranae may limit an infected bee’s access to nutrients as the bee and fungus compete for resources, ultimately shortening a bee’s lifespan. During the course of infection, millions of spores are produced and released back into the environment when the bee defecates. These spores serve as a source of infection for other bees. For a honey bee colony that contains tens of thousands of workers, infection and premature death of one bee would be an insignificant loss; however, as infection spreads to a large number of bees a cascade effect can ensue as the foraging population dies and the queen is not able to lay enough eggs to keep up with the loss.
There are gaps in our understanding of how N. ceranae may cause colony death. This research focuses on finding answers to the following fundamental questions to help fill some of these gaps: How does infection by N. ceranae affect bee behavior? What is the underlying physiological mechanism responsible for these behavioral changes?
A honey bee colony is a family comprised of a queen mother, up to 40,000 daughters (workers) and a small percentage of sons (drones). Workers typically live 6 weeks during the summer. Young workers remain in the nest as nurses, feeding the larvae and queen, and at about 3 weeks of age the workers switch from nursing duties to foraging for nectar and pollen outside the nest for their remaining 2-3 weeks of life. Underlying this age-based division of labor is a protein called vitellogenin (Vg) and juvenile hormone (JH). Nurse bees have high levels of Vg and low levels of JH, whereas at the onset of foraging, they have low levels of Vg and high levels of JH. The interaction of Vg and JH functions as a pacemaker that drives the sequence of behaviors that workers perform throughout their lives. Factors such as the environment or pathogens may act on honey bee physiology to disrupt this developmental pacemaker.
N. ceranae could provide a mechanism that promotes premature and pathological onset of foraging in workers, ultimately leading to a shortened life span at the individual level and an absence of adult bees at the colony level. Moreover, N. ceranae infection could exert behavioral effects on its host through disruption of Vg/JH endocrine signaling.
Objective 1. Determine the life span and identify the onset of foraging behavior in newly emerged worker bees infected with N. ceranae.
Objective 2. Determine how N. ceranae infection affects expression of vitellogenin and juvenile hormone, which are physiological regulators of adult development in honey bees.
Objective 1: Nosema ceranae spores were purified from bees from an infected colony and confirmed to species using conventional PCR. Newly emerged bees were infected by individually feeding 10,000 spores in sugar solution or sugar solution without spores as a control. Bees were paint-marked by treatment and released into small nucleus colonies, each containing a queen and equal amounts of workers, brood, pollen, and nectar. Starting at day 7 after infection until day 21 (i.e., precocious window of foraging) the entrance to the nucleus colonies was blocked and the number of marked bees returning to the colonies was recorded during a 30-min observation period. Marked bees were recorded as foragers if they were seen carrying a pollen load, and these bees were collected for later analyses. The age of foraging onset in N. ceranae-infected bees was compared to non-infected bees.
Objective 2: Newly emerged bees were infected by individually feeding 10,000 spores in sugar solution or sugar solution without spores as a control. Bees were caged by treatment and placed in a controlled environment. A sample of bees was taken from each cage every 4 days during a 16-day period for a total of 4 collection time points after infection (day 4, 8, 12, and 16). A small volume of haemolymph was collected from each bee in acetonitrile at -20 C and whole bee samples were stored at -80 C and until analysis.
The presence and spore load of N. ceranae was determined by microscopic inspection of the gut of each bee. Total RNA was isolated from tissues of each bee to quantify Vg mRNA by rt-PCR. Juvenile hormone titer in haemolymph was determined by standard radioimmunoassay. Gene expression (i.e., levels of mRNA transcript) of Vg in tissues and JH titer in haemolymph from bees infected with N. ceranae was compared to samples from age-matched, non-infected bees.
Foraging behavior was observed daily from day 7 through 21 in several nucleus colonies containing both infected and uninfected bees. For a typical honey bee, engaging in foraging behavior at less than 21 days old would likely be considered precocious. During this precocious window of foraging, significantly more marked bees from the infected group were observed returning to colonies than marked bees from the uninfected group. In addition, there were significantly more marked pollen foragers from the Nosema-infected group compared to the uninfected group that were collected at the entrances to the colonies during the observation period. These findings would suggest that Nosema ceranae affects the behavior of infected bees leading them to transition prematurely from nursing to foraging tasks.
Quantitative rt-PCR results from cage studies showed that levels of Vg followed a normal trajectory in uninfected control bees. Levels of Vg peaked between 4 and 8 days after emergence and then decreased to low levels from day 12 through day 16 in control bees. Bees infected with N. ceranae showed a different pattern of Vg expression. Levels of Vg were significantly lower at 4 and 8 days after infection in bees infected with N. ceranae compared to controls. Interestingly, Vg levels in infected bees remained static from day 12 through day 16 and were significantly higher compared to uninfected bees. Since nurse bees typically consume large amounts of pollen that they use to acquire amino acids for the synthesis of proteins, it is possible that low levels of Vg in infected bees is the result of inhibited nutrient processing.
Results from the radioimmunoassay showed that levels of JH peaked between 4 and 8 days after infection and then decreased to low levels from day 12 through day 16 in infected bees. This trend corresponds nicely to the opposing pattern seen in Vg levels. Levels of JH in uninfected bees remained relatively static throughout the 16-day observation period.
The data are still being analyzed and the complete set of data will be made available at a later date.
Educational & Outreach Activities
Results from this study were disseminated to the broader beekeeping and research community in 2011 as presentations at the American Bee Research Conference, Beltsville, MD; Canadian Beekeeping Research Symposium, Winnipeg, MB; Eastern Apiculture Society Summer Convention, Warwick, RI; Empire State Honey Producers Association Fall Meeting, Syracuse, NY; Wisconsin Honey Producers Association Fall Convention, Wisconsin Rapids, WI; Minnesota Honey Producers Association Annual Convetion, Detroit Lakes, MN; and several hobby beekeeping groups throughout Minnesota.
A manuscript is in prepration for publication in a scientific journal.
Establishing a link between infection with N. ceranae and disruption of a fundamental endocrine signaling pathway that regulates development and lifespan in adult honey bees is a novel finding that has important consequences for a honey bee colony. During the course of infection, millions of N. ceranae spores are produced and released back into the environment when an infected bee defecates. These spores serve as a source of infection for other bees. For a honey bee colony that contains tens of thousands of workers, infection and premature death of one bee may be an insignificant loss; however, as infection spreads to a large number of bees a cascade effect can ensue as the foraging population dies and the queen is not able to lay enough eggs to keep up with the loss.
It is important to regularly monitor honey bee colonies for the presence and level of Nosema spp. infection before a treatment regimen is initiated. Monitoring can be inexpensive but time consuming; it requires taking a sample of preferably older bees, homogenizing the sample in water, and inspecting the homogenate for spores by microscopy. While medications such as Fumagillin are available to treat honey bee colonies for Nosema spp, these treatments may only be effective against the vegetative stage of infection and may not kill mature spores, which are the vehicles of infection. A proactive approach may be to ensure honey bees have access to diverse and abundant sources of pollen. This may require movement of colonies to suitable sites of forage or the purchase of protein supplements.
The long-term goal of this research was to help North Central Region and U.S. beekeepers develop management techniques and sustainable methods to control for the effects of N. ceranae infection. Findings from this research show that honey bees infected with N. ceranae had lower levels of Vg earlier in life compared to controls. Although a firm recommendation for implementation of a best management practice cannot be made at this time, this research would support the need for honey bees to have access to forage that contains both diverse and abundant sources of pollen to compensate for deficiencies in Vg and overall proteins as a result of infection.
Areas needing additional study
An important question that remains unanswered is how N. ceranae contributes to the death of a honey bee colony? This research examined the impact of N. ceranae infection on individual bees. Individual bees infected with N. ceranae were found to engage in foraging behavior at earlier times during their adult development than uninfected bees of the same age. Corroborating this finding is the suppression of Vg levels and an increase in JH titer seen in infected nurse-aged bees. If infection by N. ceranae disrupts Vg/JH endocrine signaling, which is thought to regulate the division of labor within the worker caste, then is it possible to determine a threshold or proportion of infected bees that would trigger a cascade effect leading to premature death of the foraging population and queen unable to lay enough eggs to keep up with the loss?