Biology subjects

Lycett, G. J.

Publications and source records attributed to Lycett, G. J..

5 recordsLinked to original sources

bioRxiv2023

Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background which does not express the gene and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (5.98), chloropyriphos (6.64) and fenthion (3.18) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies. Author SummaryInsecticides are vital disease control tools against pathogen-transmitting mosquitoes. However, they are becoming less effective as mosquitoes develop resistance. Among the molecular changes that contribute to resistance, increased production of enzymes that break down/sequester the insecticide is common. In Ae. aegypti mosquitoes, which spread many arboviruses, over-expression of the carboxylesterase enzyme, CEae3A, has been associated with resistance to certain insecticides used for vector control, particularly organophosphate compounds. However, multiple resistance enzymes/mechanisms are likely to be present in resistant mosquitoes at the same time. To examine the effect of CCEae3A expression in isolation, we utilised the An. gambiae mosquito with its convenient access to GAL4/UAS technology to regulate gene expression. This enabled production of CCEae3A in a normally insecticide-sensitive mosquito strain, permitting expression without interference from other resistance mechanisms. As anticipated, resistance to organophosphates was observed in larvae expressing CCEae3A. In adults, resistance was also found against compounds from organophosphate, carbamate and pyrethroid insecticide classes, including two compounds for which there had been no previous association. As well directly linking CCEae3A expression to specific insecticide resistance, this transgenic line can be included in a panel expressing alternative enzymes to screen new insecticidal compounds for liability to existing resistance mechanisms.

zoology↗

bioRxiv2021

Actions of camptothecin derivatives on larvae and adults of the arboviral vector Aedes aegypti

Mosquito-borne viruses including dengue, Zika and Chikungunya viruses as well as parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti (New Orleans strain) were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. Of the 400 compounds screened, we identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that significantly reduced Ae. aegypti larval motility compared to DMSO controls. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50s of 25.5 {+/-} 5.0 M and 22.3 {+/-} 5.4 M respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to Ae. aegypti adults at 100 M and 10 M and completely blocked egg laying when fed at 100 M. Camptothecin and its derivatives, inhibitors of topoisomerase I, have known activity against several agricultural pests and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries. Both humans and mosquitoes express the highly conserved topoisomerase I target, however, the design of derivatives with differing pharmacokinetic properties may offer a promising route towards the development of insect-specificity of this chemistry.

pharmacology and toxicology↗

bioRxiv2020

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and smartphone-based detection of larval insecticide resistance

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots. Author summaryWe have developed an automated platform for recording the motility of mosquito larvae and applied it to larvae of a mosquito vector of malaria and a mosquito vector of dengue, Zika, yellow fever and other human diseases. The platform facilitates high-throughput, chemical screening for new compounds to control mosquito larvae and also detects differences in response in larval progeny from pryethroid resistant adult mosquitoes. Pyrethroid-impregnated bednets have helped to halve the deaths from malaria in recent years but pyrethroid resistance is an important threat to this progress. Our approach assays insecticide actions faster than the current WHO standard test and we show that it can be adapted for use with a smartphone, which offers the prospect of a future field assay to monitor developing resistance associated with behavioural response with the added benefit of precise satellite-based location.

pharmacology and toxicology↗

bioRxiv2019

Development of a functional genetic tool for Anopheles gambiae oenocyte characterisation: application to cuticular hydrocarbon synthesis

Oenocytes are an insect cell type having diverse physiological functions ranging from cuticular hydrocarbon (CHC) production to insecticide detoxification that may impact their capacity to transmit pathogens. To develop functional genetic tools to study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to create a transgenic mosquito Gal4 driver line that mediates tissue-specific expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are fluorescently tagged through all life stages and demonstrate clearly the two characteristic oenocyte cell-types arising during development. The driver was then used to characterise the function of two oenocyte expressed An. gambiae cyp4g genes through tissue-specific expression of UAS-RNAi constructs. Silencing of cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in both single gene knockdowns when assayed in young pupa or surviving adults respectively, indicating both genes are required for complete CHC production in An. gambiae and demonstrate synergistic activity in young pupae. Comparative CHC profiles were very similar for the two knockdowns, indicating overlapping substrate specificities of the two enzymes. Differences were observed for example with reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first time that two cyp4gs have both been shown to be required for complete CHC production in an insect. Moreover, the generation of tagged cells and identification of an enhancer region can expediate oenocyte specific transcriptomics. The novel driver line can also be used to explore oenocyte roles in pheromone production, mating behaviour and longevity in the malaria mosquito.

molecular biology↗

bioRxiv2017

Improved Aedes aegypti mosquito reference genome assembly enables biological discovery and vector control

Female Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, has been slowed by the lack of a high-quality genome assembly. Here we combine diverse genome technologies to produce AaegL5, a dramatically improved and annotated assembly, and demonstrate how it accelerates mosquito science and control. We anchored the physical and cytogenetic maps, resolved the size and composition of the elusive sex-determining \"M locus\", significantly increased the known members of the glutathione-S-transferase genes important for insecticide resistance, and doubled the number of chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites. Using high-resolution QTL and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. We predict that AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly arboviral vector.

genomics↗