Drug metabolizing enzymes such as cytochrome P450s have often been implicated in influencing levels of pesticide toxicology and resistance. Consequently, a variety of different P450 genes and variants have been linked to pesticide metabolism. Substantially less is known in regards to which tissues these P450s contribute to pesticide metabolism. Here, we isolate the effect of different tissues in pesticide toxicology by driving the model P450 Cyp6g1 in specific tissues of Drosophila melanogaster. Fluorescent and luminescent assays were used to compare the strength of GAL4 lines specific to the midgut (Mex‐GAL4), Malpighian tubules (UO‐GAL4) and the fat body (LSP2‐GAL4) with the widely used HR‐GAL4 line which drives GAL4 expression in all three tissues simultaneously. These data suggested that GAL4 drivers specific for the midgut and fat body were of approximately equal strength to the HR‐GAL4 line, while the Malpighian tubule specific line was significantly weaker. Multiple toxicology assays using the pesticides bendiocarb, imidacloprid and malathion were then performed to assess which tissues provide the most chemoprotection. In the long‐term feeding assay, transgenic expression of Cyp6g1 specifically in the midgut accounted for the majority of the resistance caused by Cyp6g1 overexpression with the HR‐GAL4 driver. Real‐time toxicology assays on third instar larvae were also performed and showed variable contributions of tissues to acute toxicology response depending on which pesticide was used. These data suggest a strong influence of bioassay parameters such as life stage and dosing method on outcome but suggest a prominent role for the midgut in larval toxicology.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster . An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.

Parhyale hawaiensis is a marine crustacean which has emerged as a powerful model organism to study molecular and cellular mechanisms linked to embryonic development and regenerative processes. Recently, several fluorescence-based optical microscopy techniques have been employed for the study of Parhyale to obtain anatomical descriptions, analyze gene expression patterns and reconstruct cell lineages. Aiming at the expansion of the imaging repertoire for this emerging model organism, we introduce a low-cost hybrid diagnostic system which integrates confocal fluorescence and frequency domain photoacoustic (FDPA) microscopy modalities, concurrently capturing both the radiative and radiationless relaxations of molecules following their excitation by an intensity-modulated laser source. We initially characterize the hybrid microscope in terms of PA signal generation efficiency and lateral spatial resolution, and subsequently evaluate its capabilities for the in vivo imaging of unlabelled and fluorescently labelled Parhyale embryos found at different stages of development. The recorded hybrid images have revealed a remarkable contrast complementarity between the integrated imaging modes, providing valuable information regarding cells’ structure, nuclei location, cellular membranes and yolk distribution. Our findings may pave the way for the broader adoption of inexpensive hybrid optical and PA microscopy methods in developmental biology, significantly upgrading the capabilities of the currently used technologies.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge in mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect D. melanogaster. An RNAi screen was performed, which knocked down individual ABCs in targeted epithelial tissue, examining the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This simultaneously implicated a novel ABC drug transporter CG4562 but also highlighted a predominant role for the P-glycoprotein orthologue Mdr65. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, heterologous expression of the Mdr65 orthologue from the major malaria vector Anopheles gambiae was used to establish an in vivo characterization system for the characterization of P-glycoproteins from non-model insects in D. melanogaster. This study provides the basis for establishing Drosophila as a model for toxicology research regarding drug transporters.