Stop the crop: Insights into the insecticidal mode of action of cinnamodial against mosquitoes.

Cinnamodial (CDIAL) is a drimane sesquiterpene dialdehyde found in the bark of Malagasy medicinal plants (Cinnamosma species; family Canellaceae). We previously demonstrated that CDIAL was insecticidal, antifeedant, and repellent against Aedes aegypti mosquitoes. The goal of the present study was to generate insights into the insecticidal mode of action for CDIAL, which is presently unknown. We evaluated the effects of CDIAL on the contractility of the ventral diverticulum (crop) isolated from adult female Ae. aegypti. The crop is a food storage organ surrounded by visceral muscle that spontaneously contracts in vitro. We found that CDIAL completely inhibited spontaneous contractions of the crop as well as those stimulated by the agonist 5-hydroxytryptamine. Several derivatives of CDIAL with known insecticidal activity also inhibited crop contractions. Morphometric analyses of crops suggested that CDIAL induced a tetanic paralysis that was dependent on extracellular Ca2+ and inhibited by Gd3+, a non-specific blocker of plasma membrane Ca2+ channels. Screening of numerous pharmacological agents revealed that a Ca2+ ionophore (A23187) was the only compound other than CDIAL to completely inhibit crop contractions via a tetanic paralysis. Taken together, our results suggest that CDIAL induces a tetanic paralysis of the crop by elevating intracellular Ca2+ through the activation of plasma membrane Ca2+ channels, which may explain the insecticidal effects of CDIAL against mosquitoes. Our pharmacological screening experiments also revealed the presence of two regulatory pathways in mosquito crop contractility not previously described: an inhibitory glutamatergic pathway and a stimulatory octopaminergic pathway. The latter pathway was also completely inhibited by CDIAL.

Exploring sources of inaccuracy and irreproducibility in the CDC bottle bioassay through direct insecticide quantification.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) bottle bioassay is a commonly used susceptibility test for measuring insect response to insecticide exposure. However, inconsistencies and high variability in insect response when conducting CDC bottle bioassays have been reported in previous publications. We hypothesized that the CDC bottle bioassay results may be compromised when expected and actual insecticide concentrations in the bottles are not equivalent and that inadequate bottle cleaning and/or loss during insecticide introduction and bottle storage steps could be responsible. We explored this hypothesis by quantifying insecticides using gas chromatography tandem mass spectrometry (GC-MS/MS) in bottles that had been cleaned, prepared, and stored according to the CDC guidelines. METHODS: We investigated the bottle cleaning, preparation, and storage methods outlined in the CDC bottle bioassay procedure to identify sources of irreproducibility. We also investigated the effectiveness of cleaning bottles by autoclaving because this method is commonly used in insecticide assessment laboratories. The two insecticides used in this study were chlorpyrifos and lambda-cyhalothrin (λ-cyhalothrin). Insecticides were removed from glass bioassay bottles by rinsing with ethyl-acetate and n-hexane and then quantified using GC-MS/MS. RESULTS: The CDC bottle bioassay cleaning methods did not sufficiently remove both insecticides from the glass bottles. The cleaning methods removed chlorpyrifos, which has higher water solubility, more effectively than λ-cyhalothrin. Chlorpyrifos experienced significant loss during the bottle-coating process whereas λ-cyhalothrin did not. As for bottle storage, no significant decreases in insecticide concentrations were observed for 6 h following the initial drying period for either insecticide. CONCLUSIONS: The CDC bottle bioassay protocol is susceptible to producing inaccurate results since its recommended bottle cleaning method is not sufficient and semi-volatile insecticides can volatilize from the bottle during the coating process. This can lead to the CDC bottle bioassay producing erroneous LC50 values. High levels of random variation were also observed in our experiments, as others have previously reported. We have outlined several steps that CDC bottle bioassay users could consider that would lead to improved accuracy and reproducibility when acquiring toxicity data.