Intestinally indisposed: Nosema infection in honey bees

Nosema apis, N. ceranae and N. neummani are intestinal parasites of honey bees.

Microsporidia are a highly specialized group of fungal parasites that are transmitted through infectious spores.1 Three microsporidian species infect European honey bees (Apis mellifera). Spores of Nosema apis were discovered in European honey bees in the mid-19th century, and the parasite was officially described in 1909.2 In contrast, N. ceranae was recently introduced to European honey bee populations from Asian honey bees (A. cerana). Though introductions may have occurred earlier, the first N. ceranae infections in European honey bees were reported in Spain and Taiwan in 2006 and 2007, respectively. Subsequent reports found that N. ceranae has a worldwide distribution.3 A third microsporidian species, N. neumanni, was recently discovered in Ugandan honey bees.4 N. neumanni's distribution and associated colony morbidity and mortality have not been determined. The remainder of this report will focus on N. apis and N. ceranae.

Early reports from Spain linked N. ceranae infection to high levels of colony mortality. Since European honey bees and N. ceranae did not coevolve, bees were suspected to lack natural defenses against this new microsporidian parasite. Indeed, subsequent experiments using caged workers generally found that N. ceranae was more virulent than N. apis. However, studies from the last decade have painted a more nuanced picture of N. ceranae infection and its costs. Worldwide, colony loss associated with N. apis, N. ceranae, or mixed infections is highly variable. This variability is likely explained by several factors: 1) genetic variability in Nosema strains linked to virulence, 2) genetic variability in bee populations (and within colonies) linked to Nosema resistance, 3) regional differences in climate, 4) the presence of other stressors including pathogens, parasites and pesticides, and 5) differences in experimental methods.1,3

In Europe, N. ceranae prevalence and damage appear linked to region and temperature. N. ceranae spores are less tolerant of cold temperatures than N. apis spores. Conversely, N. ceranae is able to replicate at higher temperatures than N. apis. Models thus predict that N. apis will dominate in cold climates and N. ceranae in warm. Indeed, N. ceranae predominates and is linked to colony loss in Mediterranean hives.3 In North America, surveys show that N. ceranae is widespread and can cooccur with N. apis, though N. apis appears to have been displaced in some regions. Both Nosema species have been linked to colony collapse disorder along with two other viruses.1 However, regionally distributed, long-term studies are needed to fully characterize the risks and costs of single-species and mixed infections.

At present, the collective research suggests that Nosema ceranae infection is usually not acutely lethal for hives in North America. Instead, the chronic nature of infections weakens hives, reducing colony workforce and honey production and amplifying the effects of other stressors. The pathology of Nosema spp. explains some of these infection costs. However, many questions regarding Nosema spp. management still remain unanswered.1

Nosema spp. replicate inside the honey bee digestive tract, and infection is energetically costly for bees.

Nosema spp. infections spread through spores. Healthy bees are exposed when they eat contaminated food or groom sick nestmates. Once eaten, specific environmental cues in the digestive tract cause the spores to germinate. Each spore shoots out a long, thin tubule called a polar filament. The polar filament punctures one of the bee's intestinal cells and injects the infectious spore contents into cell. The empty husk of the Nosema spore is left behind while the infectious content replicates inside the bee's cell. Ultimately, new spores are produced. Some of these spores infect neighboring intestinal cells, amplifying the infection. Other spores are released in the bee's digestive system when the host cell breaks. These spores are expelled with the bee's feces and await ingestion by a new host. Molecular analyses suggest that N. ceranae infections can spread to other organs, though further microscopy studies are needed.1

Nosema infection is energetically costly for infected bees. First, intestinal lesions caused by the infection may interfere with a bee's ability to digest and absorb food. Second, intracellular parasites steal energy molecules from host cells to support their own reproduction. Under experimental conditions, infected workers and drones starve faster than their healthy counterparts, highlighting the energetically deprived state of infected bees.1,5

Queens can be infected through artificial insemination, suggesting that Nosema ceranae can be sexually transmitted. Additional evidence suggests that queens can transmit infection to their offspring during egg laying.3,6 N. ceranae can infect larval workers and drones, but less is known about the health outcomes of bees that acquire infection during larval development.3

Symptoms of Nosema spp. infection depend on bee age and caste. Other stressors can exacerbate Nosema infection.

Costs of Nosema spp. infection depend on bee age and caste. Most studies have examined infection in adult bees, especially workers. Adult workers infected with N. apis or N. ceranae display accelerated behavioral maturation, making the normal transition from nursing to foraging behavior at a faster pace than healthy bees. Because foraging is risky, the precocious foraging observed in infected bees is effectively an early death sentence. N. ceranae-infected foragers are also less productive than healthy foragers: they take shorter foraging flights and are less likely to return to the colony. Not only do Infected workers contribute less to overall colony productivity, but also colonies must replace infected workers sooner. Models suggest that if workers and food stores are lost faster than they can be replaced, hives may collapse. Thus, Nosema and other stressors that cause precocious foraging have the potential to drag hives into a resource vortex that concludes in colony death.1,3

Infected drones have shorter life expectancies and show altered mating flight behavior.1,5 Caged drones transmit N. ceranae infection to nestmates more easily than do workers, suggesting that drones are colony “superspreaders." Nosema spp. also appears to shorten queen life expectancy through supercedure. Indeed, N. ceranae affects queen physiology and pheromonal profile, and the resulting signals may contribute to natural queen replacement.3

Nosema spp. has a complex relationship with other stressors, including viruses, other parasites, pesticides and poor nutrition. In many cases (but not all), Nosema infection is intensified by other stressors, and bees have worse survival and productivity outcomes.3

Nosema ceranae infects wild bees worldwide.

N. ceranae has the potential to infect multiple native bee genera in North America, including bumble bees which already carry their own specialized microsporidian parasite, N. bombi. N. ceranae has been detected in bumble bees in England and China. Furthermore, N. ceranae now infects South American bumble bees, stingless bees and a social wasp. N. ceranae is also known to infect 4 honey bee species: A. cerana, A. mellifera, A. dorsata and A. florea. The importance and pathology of Nosema spp. infections in wild bee populations are understudied.7

Integrated pest management for Nosema spp.

Integrated Pest Management (IPM) is a science-based approach that combines multiple tactics to control pests. Before treatments are applied, pests must be accurately identified, and their levels must surpass a threshold where the anticipated damage is likely to exceed the cost of treatment. Crucial studies are still needed to realize a complete Nosema IPM strategy1,8 (Figure 2). Beekeepers lack adequate means to discriminate between N. apis and N. ceranae, and treatment thresholds have not been established. Commercial formulations of fumagillin are the most widely used treatments for Nosema control. However, these pose risks to bee and human health, and may be less effective against N. ceranae. Many new treatments show promise but require further field trials. Some are not yet commercially available.

Nosema identification and quantification: Instructions for collecting bees, preparing a sample and quantifying Nosema infection levels in colonies are available here.9 US beekeepers also have the option of sending bee samples to the Beltsville Bee Research Laboratory for free Nosema spp. detection.10

Beekeepers can detect Nosema spp. spores by using a light microscope. Infections can be quantified with a special slide called a hemocytometer. Since foragers generally have the highest spore counts, sampling foragers is the most sensitive way to detect infection. Nosema spore counts frequently vary among foragers collected from the same colony. Thus, it is important to sample several foragers (at least 30). Bees from the same colony can be pooled to give an average spore count per hive, or bees can be tested individually. (The latter method is time consuming). Notably, colony spore levels fluctuate throughout the year in a Nosema species-dependent manner. In temperate climates, N. apis levels are usually lowest in the summer, show a small peak in the fall, and increase over the winter into the early spring. Spring levels can rise abruptly while poor weather restricts flight. Less is known about N. ceranae dynamics which show geographic variation. In the eastern US, N. ceranae levels may be higher early in the season.11 Of note, N. apis, but not N. ceranae, is associated with dysentery.3 However, diarrhea is not a foolproof indicator of infection since poor quality food can also cause dysentery.

Current diagnostic methods accessible to beekeepers have three important limitations. First, Nosema quantification methods are time-consuming. Second, N. apis and N. ceranae spores look similar, and microscopic inspection cannot reliably distinguish between species. DNA-testing is the only accurate way confirm single or mixed infections. This is clearly a diagnostic limitation for beekeepers. Third, spore concentration thresholds for colony treatment have not been determined.

Scientists have developed a “dipstick" test that can detect very low levels of N. ceranae infection.12,13 Similar to pregnancy tests which detect pregnancy-specific compounds in urine, these tests detect N. ceranae-specific compounds in prepared samples. As of 2020, however, these tests are not commercially available.

Cultural control methods:

  • Manage hive stressors: Best beekeeping practices should be used to minimize hive stress. Nosema spp. infection can be exacerbated by multiple factors, including pathogens, parasites, pesticides and poor nutrition (especially pollen deprivation). Notably, diverse pollen diets support bee immunity.3

  • Consider colony genetic background. Both intra- and intercolony genetic variability contribute to Nosema resistance. Honey bee queens normally mate with multiple males. Multiple mating introduces genetic diversity within a colony and contributes to hive productivity and disease resistance. Using artificial insemination, researchers manipulated whether colonies were fathered by drones from genetically diverse or genetically similar sources.14 Colonies with genetically diverse fathers had a lower Nosema prevalence. Thus, adequate queen mating, either through artificial insemination or open mating, is important for colony resilience. There is also limited evidence that bees derived from Russian lineages (compared with Italian lineages) are less susceptible to N. ceranae.3 Further studies are needed in this area.

  • Replace older queens. Queen age and time of N. ceranae infection have a complex relationship. Replacing older queens (>1 or 2 years) may help reduce colony Nosema loads while improving other colony health and productivity metrics.3

Mechanical control methods: Nosema spores (as well as other pathogens, parasites, pests and chemical residues) contaminate hives. To avoid spreading disease, beekeepers should avoid swapping frames between colonies and exercise caution when combining hives. Disease can also spread when strong colonies rob weak or dead hives. If possible, weak colonies should be quarantined, and dead colonies should be removed from apiaries promptly. Sterilizing hive equipment with heat or fumigation is likely impractical for most beekeepers.

Chemical approaches: While replicating inside a bee, intracellular Nosema states are susceptible to orally administered chemical treatments. Environmental Nosema spores contaminating the hive, however, escape these control measures. Oral treatments frequently reduce colony spore loads, only for bees to become reinfected by environmental spores after the chemicals wear off. The chemicals themselves may also have sublethal effects on bee health.

  • Soft chemicals: A growing number of natural compounds, extracts and microbial supplements are being tested for efficacy against N. ceranae (and potentially N. apis). Treatments showing efficacy were recently reviewed by Burnham (2019) and are summarized in Table 1.8 Robust field studies demonstrating improved colony survival and productivity are still needed for many of these substances.

    Table

    Furthermore, standardizing natural extract or supplement composition poses a challenge for establishing efficacy. For example, propolis extracts show efficacy against N. ceranae, but propolis composition is regionally variable and affected by extraction methods. Burnham (2019) also noted that several commercial products are “advertised as anti-infective [but] do not have any beneficial effects on honey bees infected with N. ceranae. Nosestat® and Vitafeed Gold® were evaluated in a field trial and found to have no impact on colony productivity and Nosema spore levels. ApiHerb® and Nonosz® are also sold to improve bee health and perhaps treat nosemosis, but additional research and more scientific evidence is needed in order to support claims of efficacy."

    Interestingly, several naturally based chemicals that inhibit varroa mites, including oxalic acid, formic acid, thymol and resveratrol, also show activity against Nosema. Some of these chemicals can be applied both orally and through fumigation. Burnham suggests that future studies examine the efficacy of combination oral and fumigation approaches; together, these may target varroa mites, kill replicating spores inside bees and decrease the viability of spores contaminating hive surfaces.

    Burnham's full paper is freely available here.

  • Synthetic compounds: Fumagillin has been widely used by US beekeepers for the last 60 years to control Nosema infection, and many beekeepers prophylactically treat their hives twice a year. US commercial products include Fumagilin-B® and Fumadil-B® Medivet recently discontinued Fumagilin-B® production. However, Fumadil-B® is commercially available, and many beekeepers may still have Fumagilin-B® in their personal stores.

    Commercial preparations sell fumagillin is in salt form (fumagllin dicyclohexylamine [DCH]) for easy dissolution in sugar syrup. Fumagilin effectively reduces both N. apis and N. ceranae spore loads in bees in the short term. However, DCH has demonstrated toxicity against bees and mammals, and fumagillin has the potential to impede an enzyme found in both invertebrates and vertebrates.15 Thus, chemical build-up in colonies and contamination of hive products are concern for both bee and human health. (Beekeepers are restricted to spring and fall fumagillin applications to minimize chemical contamination of honey collected for human consumption). Finally, caged studies suggest that N. ceranae levels may strongly rebound once fumagillin treatment wears off.16 Field studies are needed to confirm this finding.

Treatments on the horizon: Molecular therapies (RNA interference or RNAi) may be used in the future to control Nosema spp. in colonies.8 No molecular therapies are commercially available at this time.

References

  1. Holt, H. L. & Grozinger, C. M. Approaches and Challenges to Managing Nosema (Microspora: Nosematidae) Parasites in Honey Bee (Hymenoptera: Apidae) Colonies. J. Econ. Entomol. 109, (2016).

  2. White, G. F. Nosema-Disease. (1919).

  3. Martín-Hernández, R. et al. Nosema ceranae in Apis mellifera: a 12 years postdetection perspective. Environ. Microbiol. 20, 1302–1329 (2018).

  4. Chemurot, M., De Smet, L., Brunain, M., De Rycke, R. & de Graaf, D. C. Nosema neumanni n. sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in Uganda. Eur. J. Protistol. 61, 13–19 (2017).

  5. Holt, H. L., Villar, G., Cheng, W., Song, J. & Grozinger, C. M. Molecular, physiological and behavioral responses of honey bee (Apis mellifera) drones to infection with microsporidian parasites. J. Invertebr. Pathol. 155, (2018).

  6. Traver, B. E. & Fell, R. D. Low natural levels of Nosema ceranae in Apis mellifera queens. J. Invertebr. Pathol. 110, 408–410 (2012).

  7. Grupe II, A. C. & Quandt, C. A. A growing pandemic: A review of Nosema parasites in globally distributed domesticated and native bees. PLOS Pathog. 16, e1008580 (2020).

  8. Burnham, A. J. Scientific Advances in Controlling Nosema ceranae (Microsporidia) Infections in Honey Bees (Apis mellifera) . Frontiers in Veterinary Science 6, 79 (2019).

  9. Bee-Health. Testing for Nosema spores. eXtension (2019).

  10. USDA Agricultural Research Service. Bee disease diagnosis service. Bee Research Laboratory: Beltsville, MD (2017).

  11. Fries, I. et al. Standard methods for Nosema research. J. Apic. Res. 52, 1–28 (2013).

  12. Aronstein, K. A., Webster, T. C. & Saldivar, E. A serological method for detection of Nosema ceranae. J. Appl. Microbiol. 114, 621–625 (2013).

  13. Bee-Health. Detect Nosema parasite in time to save bee colonies. eXtension (2019).

  14. Desai, S. D. & Currie, R. W. Genetic diversity within honey bee colonies affects pathogen load and relative virus levels in honey bees, Apis mellifera L. Behav. Ecol. Sociobiol. 69, 1527–1541 (2015).

  15. van den Heever, J. P. et al. The effect of dicyclohexylamine and fumagillin on Nosema ceranae-infected honey bee (Apis mellifera) mortality in cage trial assays. Apidologie 47, 663–670 (2016).

  16. Huang, W.-F., Solter, L. F., Yau, P. M. & Imai, B. S. Nosema ceranae Escapes Fumagillin Control in Honey Bees. PLOS Pathog. 9, e1003185 (2013).