using-the-gal4-uas-system-for-functional-genetics-in-anopheles-gambiae

Using the GAL4-UAS System for Functional Genetics in <em>Anopheles gambiae</em>

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

Liverpool School of Tropical Medicine

liverpool school of tropical medicine

Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize temephos oxon to the temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.

Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus.

Corrigendum to "Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus" [Insect Biochem. Mol. Biol. 74 (July 2016) 61-67].

Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar.

Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal).

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; ) pyrethroid resistance was detected in 2004 in  (formerly known as the M form of the  complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that  alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in r frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan.

Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities.

Intense use of insecticides has resulted in the selection of extreme levels of resistance in insect populations. Therefore understanding the molecular basis of insecticide resistance mechanisms becomes critical. Penetration resistance refers to modifications in the cuticle that will eventually slow down the penetration of insecticide molecules within insects' body. So far, two mechanisms of penetration resistance have been described, the cuticle thickening and the altering of cuticle composition. Cuticular modifications are attributed to the over-expression of diversified genes or proteins, which belong to structural components (cuticular proteins mainly), enzymes that catalyze enzymatic reactions (CYP4G16 and laccase 2) or ABC transporters that promote cuticular translocation. In the present review we summarize recent studies and discuss future perspectives.

Insect cuticle: a critical determinant of insecticide resistance.

Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide.

Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance.

The surface of insects is coated in cuticular hydrocarbons (CHCs); variations in the composition of this layer affect a range of traits including adaptation to arid environments and defence against pathogens and toxins. In the African malaria vector,  quantitative and qualitative variance in CHC composition have been associated with speciation, ecological habitat and insecticide resistance. Understanding how these modifications arise will inform us of how mosquitoes are responding to climate change and vector control interventions. CHCs are synthesised in sub-epidermal cells called oenocytes that are very difficult to isolate from surrounding tissues. Here we utilise a transgenic line with fluorescent oenocytes to purify these cells for the first time. Comparative transcriptomics revealed the enrichment of biological processes related to long chain fatty acyl-CoA biosynthesis and elongation of mono-, poly-unsaturated and saturated fatty acids and enabled us to delineate, and partially validate, the hydrocarbon biosynthetic pathway in .

Isolation and transcriptomic analysis of  oenocytes enables the delineation of hydrocarbon biosynthesis.

Integrated pest management: Novel tools, remaining challenges, and intriguing non-target effects.

Aedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. chikungunya). Development of insecticide resistance in the vector is a major concern as its control mainly relies on the use of biocides. Data on the species' resistance status are essential for efficient and sustainable control. To date the insecticide resistance status of Ae. albopictus populations from Greece against major insecticides used in vector control remains largely unknown.

Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control.

Article	Year
Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus. Insect biochemistry and molecular biology\| PubMed ID: 27180726	2016
Corrigendum to "Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. al... Insect biochemistry and molecular biology\| PubMed ID: 28578775	2017
Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal). PLoS neglected tropical diseases\| PubMed ID: 28742096	2017
Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29674455	2018
Insect cuticle: a critical determinant of insecticide resistance. Current opinion in insect science\| PubMed ID: 30025637	2018
Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance. PLoS neglected tropical diseases\| PubMed ID: 32357192	2020
Isolation and transcriptomic analysis of oenocytes enables the delineation of hydrocarbon biosynthesis. eLife\| PubMed ID: 32538778	2020
Integrated pest management: Novel tools, remaining challenges, and intriguing non-target effects. Current opinion in insect science\| PubMed ID: 32546375	2020
Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control. Parasites & vectors\| PubMed ID: 32600453	2020

Linda Grigoraki

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