Project Number: 2080-21000-017-12-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2017
End Date: Apr 30, 2020
Bumble bees are among the most charismatic and important of these wild pollinators. Despite strong evidence that certain closely related Bombus species are virtually absent from areas where they were recently abundant, the causes of their decline remain elusive. Here we propose to investigate two factors that are currently at the forefront of concern regarding bumble bee health, pathogen infection by Nosema and neonicotenoid insecticide exposure. The primary goals of our research are to: • determine whether declining bumble bee species are more susceptible to Nosema pathogens than are stable (non-declining) species • compare the effects of exposure to the neonicotenoid insecticide imidacloprid in declining and stable species • determine if there is a stronger impact of Nosema in declining bumble bees exposed to imidacloprid vs. non-declining species exposed to imidacloprid • determine if there is a causal link between imidacloprid exposure and alteration of the immune system in declining vs. non-declining bumble bees We propose to test two as yet untested hypotheses: First, that declining Bombus species are more susceptible to Nosema infection (mediated by a weaker immune defense response) than stable bumble bee species. Our second hypothesis is that imidacloprid pesticide at sublethal doses reduces immunocompetence in bumble bees, thus enhancing proliferation of Nosema infection. We predict that bees treated with imidacloprid will be more susceptible to Nosema regardless of their known decline status—that is, imidacloprid exposure lowers the threshold for pathogen infection in any bumble bee species. The long term goal of our research is to increase our limited knowledge of the direct causes of bumble bee decline in North America with the ultimate goal of protecting bumble bee health. In addition we expect to improve understanding of the effects of a widely used agricultural insecticide on one of our most important wild pollinators, with important implications for broadening science-based guidelines for testing sublethal effects of insecticides on wild pollinators.
To test our hypotheses concerning differential susceptibility of Bombus spp. to Nosema infection & the effects (lethal & sublethal) of neonicotinoid compounds on diverse Bombus species with & without exposure to N. bombi infection, we will administer a series of tests on different N. American Bombus species. Our main experimental objectives are to test for the different bumble bee species’ phenotypic & gene expression responses to pathogen & insecticide exposure & to determine whether the pathogen & insecticide act synergistically to influence bumble bee fitness. Throughout the proposed work on the effects of the multiple stressors of neonicotinoid & pathogen N. bombi, a cross treatment design will yield four treatments: 1. Control; 2. Experimentally inoculated with N. bombi spores; 3. Exposed to a one-time field realistic dose of imidacloprid; 4. Exposed to imidacloprid & subsequently inoculated with N. bombi spores. Treatments will be provided in sucrose solutions, with control bees fed with sucrose-only solutions. After treatments, the intestinal tract, gut DNA (adults), & total RNA (larvae) of the bees will be extracted for three experiments: (i)Compare the bees’ susceptibility to N. bombi infection, measured by quantifying infection intensity. (ii)Examine the bees’ histological (gut epithelial cells & the peritrophic matrix) interaction with N. bombi during infection, & the effects of N. bombi (ii-a) & imidacloprid on bee fitness (ii-b). (iii)Conduct RNA-seq for genome-wide transcription analysis. Experiment i & ii will determine whether the bee species & insecticide uptake influence bumble bee susceptibility & histological response to N. bombi infection. The effect of these stressors on bumble bee fitness will also be examined. Experiment iii will identify genes [i.e. immune genes & other host resistance genes, & genes manipulated by the parasite whose expressions differ among different bee species &/or are affected by either N. bombi exposure or ingestion of imidacloprid (or both). Four bumble bee species will be used throughout the project (Bombus terricola, B. occidentalis, B. bimaculatus & B. bifarius). Additional sequencing & testing of samples from the other two species will be conducted depending on the additional resources available. For each bee species (in each experiment), individual colonies will be divided into different queenless sub-colonies (workers & a cluster of brood, without the queen), each receiving distinct treatments; this will help to reduce the variability resulting from genetic heterogeneity among colonies. For example, if an established laboratory colony consists of 40 workers & 40 larvae, 4 sets of ten workers & 10 larvae will be separated in different containers & receive different treatments. Although the absence of the queens could gradually provoke competition among workers, the effects of their behavioral changes should be negligible during the short experiment time (3-day treatment, sampled after ~2 wks) prior to sample collection (RNA, DNA, & gut tissue samples; details of the treatments & sampling are described in the following sections).For each species,each experiment will be repeated on 3 colonies