Neonicotinoid-induced pathogen susceptibility is mitigated by Lactobacillus plantarum immune stimulation in a Drosophila melanogaster model

Abstract

Pesticides are used extensively in food production to maximize crop yields. However, neonicotinoid insecticides exert unintentional toxicity to honey bees (Apis mellifera) that may partially be associated with massive population declines referred to as colony collapse disorder. We hypothesized that imidacloprid (common neonicotinoid; IMI) exposure would make Drosophila melanogaster (an insect model for the honey bee) more susceptible to bacterial pathogens, heat stress, and intestinal dysbiosis. Our results suggested that the immune deficiency (Imd) pathway is necessary for D. melanogaster survival in response to IMI toxicity. IMI exposure induced alterations in the host-microbiota as noted by increased indigenous Acetobacter and Lactobacillus spp. Furthermore, sub-lethal exposure to IMI resulted in decreased D. melanogaster survival when simultaneously exposed to bacterial infection and heat stress (37 °C). This coincided with exacerbated increases in TotA and Dpt (Imd downstream pro-survival and antimicrobial genes, respectively) expression compared to controls. Supplementation of IMI-exposed D. melanogaster with Lactobacillus plantarum ATCC 14917 mitigated survival deficits following Serratia marcescens (bacterial pathogen) septic infection. These findings support the insidious toxicity of neonicotinoid pesticides and potential for probiotic lactobacilli to reduce IMI-induced susceptibility to infection.

Figure 1

IMI exposure in D. melanogaster results in dose dependent toxicity. Survival curves for newly eclosed wildtype Canton-S flies fed food containing vehicle or food containing varying concentrations of IMI. Data are displayed from at least 5 independent experiments (20–25 flies each group per experiment). All statistical symbols are representative of comparison made using the log-rank (Mantel-Cox) test. ****p < 0.0001. ns = not significant.

Figure 2

IMI exposure results in an increased abundance of indigenous Acetobacter and Lactobacillus spp. in D. melanogaster. Third-instar larvae that were reared on food containing vehicle or 10 μM IMI were surface-sterilized, homogenized, plated on selective medium, and incubated at 37 °C. Colony forming units (CFU) were subsequently enumerated after 48 h of incubation. Mean ± standard deviation (unpaired, two-tailed t-tests) of 3 independent experiments (n = 15 for each group). ****p < 0.0001.

Figure 3

The Imd pathway is necessary to mitigate IMI-induced toxicity in D. melanogaster. (A–C) Survival curves for newly eclosed D. melanogaster Imd pathway mutant flies (Rel ^−/−, Hop2 ^−/−, Upd1 ^−/−, Upd2/3 ^−/−) exposed to 100 μM IMI compared to their wildtype background controls (Canton-S, FM7A, and w¹¹¹⁸, respectively). Data are displayed from at least 3 independent experiments (20–25 flies for each group per experiment). Statistical analyses shown are from log-rank (Mantel-Cox) tests. (D–F) First-instar Imd pathway mutant larvae and their respect background controls were exposed to 1 μM and 10 μM IMI and percentage of larvae that subsequently eclosed. Data are means ± standard deviations (Mann-Whitney tests [D,E]; Kruskal-Wallis tests [F]) of results from 5 biological replicates (10 larvae per biological replicate; n = 5 for each group). *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001. ns = not significant.

Figure 4

IMI exposed D. melanogaster respond similarly to heat stress as Imd pathway mutants. (A) Survival curves for newly eclosed Imd pathway mutants (Rel ^−/−) and WT Canton-S exposed to heat stress (37°) with or without concurrent exposure to IMI. Data are displayed from at least 3 independent experiments (15–25 flies reared in separate food vials on different occasions for each experiment). Statistical analyses are representation of comparisons made to heat-stressed WT Canton-S controls using the log-rank (Mantel-Cox) test. (B) TotA gene expression of newly eclosed D. melanogaster that were heat stressed (37°) with or without concurrent exposure to IMI compared to non-heat stressed (25°) controls. All samples were taken 6 h after experimental start time. Gene expression was quantified using RT-qPCR and is relative to vehicle flies not exposed to heat stress. Means ± standard deviations (two-way ANOVA) from 3 biological replicates (each consisting of 10 flies) with triplicate technical repeats are shown. ****p < 0.0001.

Figure 5

IMI-exposed D. melanogaster are more susceptible to septic infection with Serratia marcescens. (A) Survival curves for newly eclosed Imd pathway mutant (Rel ^−/−) and WT Canton-S flies subjected to septic infection (S. marcescens NCIMB 11782) with or without concurrent exposure to IMI. All statistical symbols are representative of comparison made to infected WT Canton-S controls using the log-rank (Mantel-Cox) test. (B) Pathogen load of S. marcescens NCIMB 11782 during septic infection of WT flies was determined by plating surface-sterilized whole fly homogenates on LB agar medium. Colony forming units (CFU) per fly obtained at each time point represents the mean ± standard deviation (unpaired, two-tailed t-tests) of 9 biological replicates (n = 36 for each group). (C) Pathogen load of S. marcescens Db11 during oral infection of WT larvae was determined by plating surface-sterilized whole larvae homogenates on LB with 100 μg/ml streptomycin medium. CFU per larvae obtained at each time point represents the mean ± standard deviation (unpaired, two-tailed t-tests) of 9 biological replicates for each time point (n = 36 total for each group). (D,E) TotA and Dpt gene expression of newly eclosed WT flies that were subjected to septic injury with or without S. marcescens NCIMB 11782 infection and with or without concurrent exposure to IMI. All samples were taken 6 h after experimental start time. Gene expression was quantified by RT-qPCR and is relative to vehicle flies that were subjected to septic injury with a sterile needle. Means ± standard deviations (two-way ANOVA) from 3 biological replicates (each consisting of 10 flies) with triplicate technical repeats are shown. *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Lp39 can tolerate, but not bind or metabolize IMI. (A) Percent imidacloprid (IMI) was determined in stationary phase live bacterial cultures relative to pesticide-only controls following 24 h co-incubations in 50 mM HEPES. Data are depicted as means ± standard deviations (one-way ANOVA) of 2 independent experiments (2 biological replicates each). (B) Growth curves of Lp39 in MRS and MRS supplemented with vehicle or imidacloprid (IMI). Data are depicted as means ± standard deviations of 3 biological replicates with triplicate technical replicates.

Figure 7

Lp39 supplementation mitigates IMI-induced D. melanogaster survival deficits following infection. (A) Survival curves for newly eclosed WT Canton-S flies that were subjected to septic infection with Serratia marcescens NCIMB 11782 while concurrently exposed to IMI and/or supplemented with Lp39 separately or in combination, compared to infected WT-Canton control and vehicle (septic injury alone). All statistical symbols are representative of comparison made to infected WT Canton-S controls. Statistical significance was determined using the log-rank (Mantel-Cox) test. (B) Dpt expression of newly eclosed Dpt-RFP reporter flies was determined following 15 h oral supplementation of Lp39 or vehicle. WT Canton-S flies were used as a negative control. Gene expression was quantified by fluorescence intensity using a microplate reader. Mean ± standard deviations (two-way ANOVA) from 7 biological replicates (10 flies each replicate) per group are shown. (C) Newly enclosed Dpt-RFP and negative control WT Canton-S flies were orally supplemented with Lp39 or vehicle for 15 h. Midguts were dissected, nuclear counterstained with Hoechst 33342, trihydrochloride, tetrahydrate and viewed by confocal microscopy. Scale bar = 20 µm. **p < 0.01, ***p < 0.001, ****p < 0.0001.