A Closer Look

PARASITE, PATHOGEN, PESTICIDE, POLLINATOR INTERACTIONS

Honey bees are suffering from elevated colony losses in the northern hemisphere possibly because of a variety of emergent microbial pathogens, with which pesticides may interact to exacerbate their impacts.

There is an increasing body of evidence indicating that there are strong associations (synergistic or antagonistic) between honey bees and pathogens, parasites, pesticides and other pollinators that are impacting colony health (Johnson et al. 2009). Alaux et al. (2010) produced physiological evidence that the insecticide imidacloprid and the fungal pathogen Nosema can interact synergistically to affect bee health negatively, including physiological changes initiated by pesticide exposure that decreased bee tolerance toward Nosema infection. Similarly, Pettis et al. (2012) showed an increase in Nosema spore loads in colonies treated with imidacloprid.

Honey bees are suffering from elevated colony losses in the northern hemisphere possibly because of a variety of emergent microbial pathogens, with which pesticides may interact to exacerbate their impacts. To reveal such potential interactions, Doublet et al. (2015) administered at sublethal and field realistic doses one neonicotinoid pesticide (thiacloprid) and two common microbial pathogens, the invasive microsporidian Nosema ceranae and black queen cell virus (BQCV), individually to larval and adult honey bees in the laboratory.  Through fully crossed experiments in which treatments were administered singly or in combination, they found an additive interaction between BQCV and thiacloprid on host larval survival likely because the pesticide significantly elevated viral loads.  In adult bees, two synergistic interactions increased individual mortality: between N. ceranae and BQCV, and between N. ceranae and thiacloprid. The combination of two pathogens had a more profound effect on elevating adult mortality than N. ceranae plus thiacloprid.  Common microbial pathogens appear to be major threats to honey bees, while sublethal doses of pesticide may enhance their deleterious effects on honey bee larvae and adults.

Gregorc et al. (2012) conducted experiments to determine the physiological responses of bees to chemical and biological threats by measuring gene expression after exposure to the ubiquitous (ever present) parasitic Varroa mite and a suite of pesticide threats.  The tested pesticides included two fungicides (myclobutani, chlorothalonil), two herbicides (simazine, glyphosate) and five insecticides/miticides (fluvalinate, imidacloprid, coumaphos, chlorpyrifos, amitraz) and represent a range of modes-of-action and pesticide families. Three of these compounds (amitraz, fluvalinate and coumaphos) are used often by beekeepers to control Varroa mites and/or small hive beetles in colonies. The other chemicals are used commonly in agricultural settings and, with the exception of glyphosate, have been found as residues in honey bee colonies (Mullin et al. 2010). 

Honey bee larvae reared in vitro (in an artificial environment) was exposed to one of these nine pesticides and/or was challenged with the parasitic Varroa mite. Total RNA was extracted from individual larvae and first strand cDNAs were generated.  Gene expression changes in larvae were measured using quantitative PCR (Polymerase Chain Reaction-molecular technique used to amplify/copy a small segment of DNA or RNA) targeting transcripts (record of genetic instructions from DNA to RNA) for pathogens and genes involved in physiological processes, bee health, immunity and/or xenobiotic (foreign chemical substance found within an organism) detoxification. Transcript levels for Peptidoglycan Recognition Protein, a pathogen gene, increased in larvae exposed to Varroa mites and were not changed in pesticide treated larvae. Varroa-parasitized brood had higher transcripts of Deformed Wing Virus than did control larvae. Varroa mite parasitism arguably coupled with virus infection, resulted in significantly higher transcript abundances for the antimicrobial peptides abaecin, hymenoptaecin, and defensin 1 (chemicals involved in the immune system). Transcript levels for Prophenoloxidase-activating enzyme, an immune end product, were elevated in larvae treated with myclobutanil and chlorothalonil (both are fungicides). Transcript levels for Hexameric storage protein (Hsp 70) were significantly upregulated in imidacloprid, fluvalinate, coumaphos, myclobutanil and amitraz treated larvae. Definitive impacts of pesticides and Varroa mite parasitism on honey bee larval gene expression were demonstrated (Gregorc et al. 2012).

The association between the ectoparasitic Varroa mite and viruses is detrimental to colony health. In particular, viral replication in the mite and the transmission of viruses to the hosts through their saliva when feeding on mature and immature bees constitute crucial vectoring mechanisms (de Miranda and Genersch 2010; Mockel et al. 2011).  At least, 18 honey bee viruses have been isolated, characterized and described (Allen and Ball 1996; Genersch 2010) and 11 of them are transmitted by Varroa mites (Kevan et al. 2006). Multiple viral infections are frequently detected in bee colonies (Chen et al. 2004; Ellis and Munn 2005; Tentcheva et al. 2004).

Bahreini and Currie (2015) quantified the costs and benefits of co-parasitism with Varroa mites and Nosema ceranae Fries /Nosema apis Zander on honey bees with different defense levels. Newly-emerged worker bees from either high-mite-mortality-rate (high-MMR) bees or low-mite-mortality-rate (low-MMR) bees were confined in forty bioassay cages which were either inoculated with Nosema spores [Nosema (+) group] or were left un-inoculated [Nosema (-) group]. Caged-bees were then inoculated with Varroa mites [Varroa (+) group] or were left untreated [Varroa (-) group].  This established four treatment combinations within each Nosema treatment group: (1) low-MMR Varroa (-), (2) high-MMR Varroa (-), (3) low-MMR Varroa (+), and (4) high-MMR Varroa (+). Overall mite mortality in high-MMR bees (0.12 mites per day) was significantly greater than in the low-MMR bees (0.06 mites per day). In the Nosema (-) groups bee mortality was greater in high-MMR bees than low-MMR bees but only when bees had a higher mite burden. Overall, high-MMR bees in the Nosema (-) group showed greater reductions in mean abundance of mites over time compared with low-MMR bees, when inoculated with additional mites.  However, high-MMR bees could not reduce mite load as well as in the Nosema (-) group when fed with Nosema spores. Mean abundance of Nosema spores in live bees and dead bees of both strains of bees was significantly greater in the Nosema (+) group.  Molecular analyses confirmed the presence of both Nosema species in inoculated bees but N. ceranae was more abundant than N. apis and unlike N. apis increased over the course of the experiment. Collectively, this study showed differential mite mortality rates among different genotypes of bees, however, Nosema infection restrained Varroa removal success in high-MMR bees.

Singh et al. (2010) found for the first time the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies.  

These viruses were detected in 11 other non-Apis hymenopteran (order containing ants, bees, wasps) species ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of the ecosystem.  Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD (Colony Collapse Disorder) harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non-Apis hymenopteran pollinators also had IAPV, while those near apiaries without IAPV did not. In containment greenhouse experiments, IAPV moved from infected honey bees to bumble bees and from infected bumble bees to honey bees within a week, demonstrating that viruses can be transmitted from one species to another.

The issue of host specificity of honey bee viruses has been raised since it is a key in understanding disease epidemiology and the development of effective disease management practices.  Studies have shown that Deformed wing virus (DWV) and Black Queen Cell Virus (BQCV), two viruses originally indentified in the western honey bee, Apis mellifera, can cause infection in several species of bumble bees, including Bombus terrestris, Bombus pascuorum and Bombus huntii (Genersch et al. 2006; Li et al. 2011; Peng et al. 2011).  Other viruses commonly found in honey bees including Acute bee paralysis virus (ABPV) and Kashmir bee virus (KBV) were also found to infect different species of bumble bees (Bailey and Gibbs 1964; Anderson 1991). A recent study regarding inter-taxa virus transmission in the pollinator community reported the detection of DWV, BQCV, Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and Sacbrood virus (SBV) in multiple non-apis hymenopteran species and in pollen pellets from forager bees (Singh et al. 2010).

Levitt et al. (2013) examined managed Apis mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees- black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, they observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species.  Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, their study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.

Nosema ceranae and deformed wing virus (DWV) are two of the most prevalent pathogens currently attacking honey bees and often simultaneously infect the same hosts. Zheng et al. (2015) investigated the effect of N. ceranae and deformed wing virus interactions on infected honey bees under laboratory conditions and at different nutrition statuses. Their results showed that Nosema could accelerate DWV replication in infected bees in a dose-dependent manner at the early stages of DWV infection. When bees were restricted from pollen nutrition, inoculation with 1 x 104 and 1 x 105 spores/bee could cause a significant increase in DWV titer, while inoculation with 1 x 103 spores/bee did not show any significant effect on the DWV titer. When bees were provided with pollen, only inoculation with 1 x 105 spores/bee showed significant effect on DWV titer.  However, their results showed that the two pathogens did not act synergistically when the titer of DWV reached a plateau. This study suggests that the synergistic effect of N. ceranae and DWV is dosage-and nutrition-dependent and that the synergistic interactions between the two pathogens could have implications on honey bee colony losses.


References

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Zheng, H.Q., H.R. Gong, S.K. Huang, A. Sohr, F.L. Hu and Y.P. Chen 2015. Evidence of the synergistic interaction of honey bee pathogens Nosema ceranae and deformed wing virus. Vet. Microbiol. 177 (1-2): 1-6, doi: 10.1016/j.vetmic.2015.02.003


Clarence Collison

Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.