Indirect transfer of pyriproxyfen to European honeybees via an autodissemination approach

Abstract

The frequency of arboviral disease epidemics is increasing and vector control remains the primary mechanism to limit arboviral transmission. Container inhabiting mosquitoes such as Aedes albopictus and Aedes aegypti are the primary vectors of dengue, chikungunya, and Zika viruses. Current vector control methods for these species are often ineffective, suggesting the need for novel control approaches. A proposed novel approach is autodissemination of insect growth regulators (IGRs). The advantage of autodissemination approaches is small amounts of active ingredients compared to traditional insecticide applications are used to impact mosquito populations. While the direct targeting of cryptic locations via autodissemination seems like a significant advantage over large scale applications of insecticides, this approach could actually affect nontarget organisms by delivering these highly potent long lasting growth inhibitors such as pyriproxyfen (PPF) to the exact locations that other beneficial insects visit, such as a nectar source. Here we tested the hypothesis that PPF treated male Ae. albopictus will contaminate nectar sources, which results in the indirect transfer of PPF to European honey bees (Apis mellifera). We performed bioassays, fluorescent imaging, and mass spectrometry on insect and artificial nectar source materials to examine for intra- and interspecific transfer of PPF. Data suggests there is direct transfer of PPF from Ae. albopictus PPF treated males and indirect transfer of PPF to A. mellifera from artificial nectar sources. In addition, we show a reduction in fecundity in Ae. albopictus and Drosophila melanogaster when exposed to sublethal doses of PPF. The observed transfer of PPF to A. mellifera suggests the need for further investigation of autodissemination approaches in a more field like setting to examine for risks to insect pollinators.

Fig 1

(A) Examination for effects on the fecundity of D. melanogaster females when exposed to PPF. Bars represent the mean ± SEM number of eggs oviposited by PPF treated (N = 4) and untreated females (N = 4). The horizontal line connecting bars represent significant differences using a pairwise Wilcoxon test, P = 0.02. (B) Examination of the reduction of the fecundity of Ae. albopictus females when exposed to PPF. Bars represent the mean ± SEM number of eggs oviposited by PPF treated (N = 5) and untreated (N = 5) females. The horizontal line connecting bars represent significant differences using a pairwise Wilcoxon test, P = 0.008. (C-E) Artificial nectar source components and setup. (F) Examination for non-specific transfer of PPF from PPF treated Ae. albopictus males to artificial nectar sources using the cotton wick from the artificial nectar source in bioassays. Horizontal line connecting bars represent significant differences between treatments using pairwise Wilcoxon tests, P ≤ 0.02, Bonferroni corrected. (G) Image of A. mellifera acquiring sucrose from an artificial nectar source in laboratory cages. (H) The number of A. mellifera feeding on the artificial nectar source in laboratory cages determined by counts conducted every ten minutes.

Fig 2. Insects and materials added to each cage type, and the workflow for examination of the transfer of PPF-fluorescent powder mixture to nectar sources, insects, and oviposition cups using imaging, bioassays, and mass spectrometry analysis.

Fig 3

Image of cotton wick from a cage with PPF-fluorescent powder mixture treated Ae. albopictus males (A) under visible and (B) UV light. Image of cotton wick from a cage with untreated Ae. albopictus males (C) under visible and (D) UV light. Image of the filter paper ring surrounding the cotton wick from a cage with PPF treated Ae. albopictus males (E) under visible and (F) UV light. Image of the filter paper ring surrounding the cotton wick from a cage with untreated Ae. albopictus males (G) under visible and (H) UV light. The scale bars represent 5 mm.

Fig 4

Images of an Ae. albopictus PPF-fluorescent powder mixture treated (A and B) and untreated (C and D) male collected from a laboratory cage under visible and UV light, showing the presence and absence of PPF-fluorescent powder after males had been in cages for five days, respectively. Images of A. mellifera collected from (E and F) cages with PPF treated Ae. albopictus males and (G and H) from cages with untreated Ae. albopictus males demonstrating the transfer of PPF-fluorescent powder mixture to A. mellifera in the presence of treated males. Images of Ae. albopictus females (I and J) collected from cages with treated Ae. albopictus males and (K and L) from cages with untreated Ae. albopictus males demonstrating the transfer of PPF-fluorescent powder mixture to con-specific females in the presence of PPF treated males.

Fig 5

(A) Immature Ae. albopictus mortality in bioassays conducted using insects and materials collected from cage types 1–4. All data is represented by the mean immature mortality ± SEM. Letters above each bar represent significant differences as determined by t-tests, P < 0.05, (B) Survival plots of male Ae. albopictus (N = 4), (C) female Ae. albopictus (N = 4), and (D) A. mellifera (N = 3). All survival plots are mean numbers of surviving insects on each day for each cage type.

Fig 6. Mass spectrometry HPLC PPF quantification from Ae. albopictus, A. mellifera, and materials collected from cages with PPF treated and untreated males (Cage types 3 & 4).

All values represent the mean ± SEM of PPF in parts per million (PPM). The mean ± SEM of concentrations of PPF are also listed ng/mL to the right of each bar.