Transcriptomic and proteomic analysis of pyrethroid resistance in the CKR strain of Aedes aegypti.

Aedes aegypti is an important vector of human viral diseases. This mosquito is distributed globally and thrives in urban environments, making it a serious risk to human health. Pyrethroid insecticides have been the mainstay for control of adult A. aegypti for decades, but resistance has evolved, making control problematic in some areas. One major mechanism of pyrethroid resistance is detoxification by cytochrome P450 monooxygenases (CYPs), commonly associated with the overexpression of one or more CYPs. Unfortunately, the molecular basis underlying this mechanism remains unknown. We used a combination of RNA-seq and proteomic analysis to evaluate the molecular basis of pyrethroid resistance in the highly resistant CKR strain of A. aegypti. The CKR strain has the resistance mechanisms from the well-studied Singapore (SP) strain introgressed into the susceptible Rockefeller (ROCK) strain genome. The RNA-seq and proteomics data were complimentary; each offering insights that the other technique did not provide. However, transcriptomic results did not quantitatively mirror results of the proteomics. There were 10 CYPs which had increased expression of both transcripts and proteins. These CYPs appeared to be largely trans-regulated, except for some CYPs for which we could not rule out gene duplication. We identified 65 genes and lncRNAs as potentially being responsible for elevating the expression of CYPs in CKR. Resistance was associated with multiple loci on chromosome 1 and at least one locus on chromosome 3. We also identified five CYPs that were overexpressed only as proteins, suggesting that stabilization of CYP proteins could be a mechanism of resistance. Future studies to increase the resolution of the resistance loci, and to examine the candidate genes and lncRNAs identified here will greatly enhance our understanding of CYP-mediated resistance in A. aegypti.

Sodium channel activation underlies transfluthrin repellency in Aedes aegypti.

BACKGROUND: Volatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency. CONCLUSIONS/SIGNIFICANCE: These results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Fitness costs of individual and combined pyrethroid resistance mechanisms, kdr and CYP-mediated detoxification, in Aedes aegypti.

BACKGROUND: Aedes aegypti is an important vector of many human diseases and a serious threat to human health due to its wide geographic distribution and preference for human hosts. A. aegypti also has evolved widespread resistance to pyrethroids due to the extensive use of this insecticide class over the past decades. Mutations that cause insecticide resistance result in fitness costs in the absence of insecticides. The fitness costs of pyrethroid resistance mutations in A. aegypti are still poorly understood despite their implications for arbovirus transmission. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated fitness based both on allele-competition and by measuring specific fitness components (i.e. life table and mating competition) to determine the costs of the different resistance mechanisms individually and in combination. We used four congenic A. aegypti strains: Rockefeller (ROCK) is susceptible to insecticides; KDR:ROCK (KR) contains only voltage-sensitive sodium channel (Vssc) mutations S989P+V1016G (kdr); CYP:ROCK (CR) contains only CYP-mediated resistance; and CYP+KDR:ROCK (CKR) contains both CYP-mediated resistance and kdr. The kdr allele frequency decreased over nine generations in the allele-competition study regardless of the presence of CYP-mediated resistance. Specific fitness costs were variable by strain and component measured. CR and CKR had a lower net reproductive rate (R0) than ROCK or KR, and KR was not different than ROCK. There was no correlation between the level of permethrin resistance conferred by the different mechanisms and their fitness cost ratio. We also found that CKR males had a reduced mating success relative to ROCK males when attempting to mate with ROCK females. CONCLUSIONS/SIGNIFICANCE: Both kdr and CYP-mediated resistance have a fitness cost affecting different physiological aspects of the mosquito. CYP-mediated resistance negatively affected adult longevity and mating competition, whereas the specific fitness costs of kdr remains elusive. Understanding fitness costs helps us determine whether and how quickly resistance will be lost after pesticide application has ceased.

Fitness studies of insecticide resistant strains: lessons learned and future directions.

The evolution of insecticide resistance is generally thought to be associated with a fitness cost in the absence of insecticide exposure. However, it is not clear how these fitness costs manifest or how universal this phenomenon is. To investigate this, we conducted a literature review of publications that studied fitness costs of insecticide resistance, selected papers that met our criteria for scientific rigor, and analyzed each class of insecticides separately as well as in aggregate. The more than 170 publications on fitness costs of insecticide resistance show that in 60% of the experiments there is a cost to having resistance, particularly for measurements of reversion of resistance and reproduction. There were differences between classes of insecticides, with fitness costs seen less commonly for organochlorines. There was considerable variation in the experiments performed. We suggest that future papers will have maximum value to the community if they quantitatively determine resistance levels, identify the resistance mechanisms present (and the associated mutations), have replicated experiments, use related strains (optimally congenic with the resistance mutation introgressed into different genetic backgrounds) and measure fitness by multiple metrics. Studies on the fitness costs of insecticide resistance will continue to enlighten our understanding of the evolutionary process and provide valuable information for resistance management. © 2021 Society of Chemical Industry.

CYP-mediated resistance and cross-resistance to pyrethroids and organophosphates in Aedes aegypti in the presence and absence of kdr.

Aedes aegypti thrives in urban environments and transmits several debilitating human viral diseases. Thus, our ability to control this mosquito species in endemic areas is of utmost importance. The use of insecticides, mostly pyrethroids and organophosphates (OPs), has long been the primary means of controlling A. aegypti, but widespread insecticide resistance has emerged. The two main mechanisms of pyrethroid resistance in A. aegypti are CYP-mediated detoxification and mutations in the target site, voltage-sensitive sodium channel (Vssc), referred to as knockdown resistance (kdr). Knowledge about the contributions and interactions of these mechanisms to resistance is important for the understanding of the molecular and evolutionary basis of insecticide resistance, and to determine the effectiveness of insecticides. In this study, we address two aims: 1) determine the patterns of CYP-mediated cross-resistance to pyrethroid and OP insecticides, both in the presence and absence of kdr (S989P + V1016G), and 2) determine whether the interaction between the two mechanisms yields a greater than, less than, or additive effect on resistance. We tested seven pyrethroids and four OPs against three congenic strains of A. aegypti: ROCK (susceptible), CYP:ROCK (CR) (resistant due to CYP-mediated detoxification without kdr), and CYP + KDR:ROCK (CKR) (resistant due to both CYPs and kdr), and compared these to the congenic KDR:ROCK strain that was previously reported. We found that resistance ratios (RRs) were variable between pyrethroids and strains, ranging from 6.2- to 42-fold for CR, and 70- to 261-fold for CKR. In general, we found that CYP-mediated resistance alone contributes less to resistance than kdr. The effect of the combined mechanisms on resistance was significantly greater than additive for all pyrethroids except (1R)-trans-fenfluthrin. CYP-mediated pyrethroid resistance conferred cross-resistance to both methyl paraoxon and fenitrothion, and negative cross-resistance to methyl parathion and naled. Based on our results, we recommend that etofenprox and cyfluthrin be avoided for A. aegypti control in areas where these two resistance mechanisms are prevalent.

CYP-mediated permethrin resistance in Aedes aegypti and evidence for trans-regulation.

Aedes aegypti poses a serious risk to human health due to its wide global distribution, high vector competence for several arboviruses, frequent human biting, and ability to thrive in urban environments. Pyrethroid insecticides remain the primary means of controlling adult A. aegypti populations during disease outbreaks. As a result of decades of use, pyrethroid resistance is a global problem. Cytochrome P450 monooxygenase (CYP)-mediated detoxification is one of the primary mechanisms of pyrethroid resistance. However, the specific CYP(s) responsible for resistance have not been unequivocally determined. We introgressed the resistance alleles from the resistant A. aegypti strain, Singapore (SP), into the genetic background of the susceptible ROCK strain. The resulting strain (CKR) was congenic to ROCK. Our primary goal was to determine which CYPs in SP are linked to resistance. To do this, we first determined which CYPs overexpressed in SP are also overexpressed in CKR, with the assumption that only the CYPs linked to resistance will be overexpressed in CKR relative to ROCK. Next, we determined whether any of the overexpressed CYPs were genetically linked to resistance (cis-regulated) or not (trans-regulated). We found that CYP6BB2, CYP6Z8, CYP9M5 and CYP9M6 were overexpressed in SP as well as in CKR. Based on the genomic sequences and polymorphisms of five single copy CYPs (CYP4C50, 6BB2, 6F2, 6F3 and 6Z8) in each strain, none of these genes were linked to resistance, except for CYP6BB2, which was partially linked to the resistance locus. Hence, overexpression of these four CYPs is due to a trans-regulatory factor(s). Knowledge on the specific CYPs and their regulators involved in resistance is critical for resistance management strategies because it aids in the development of new control chemicals, provides information on potential environmental modulators of resistance, and allows for the detection of resistance markers before resistance becomes fixed in the population.

Voltage-sensitive sodium channel mutations S989P + V1016G in Aedes aegypti confer variable resistance to pyrethroids, DDT and oxadiazines.

BACKGROUND: Aedes aegypti is a vector of several important human pathogens. Control efforts rely primarily on pyrethroid insecticides for adult mosquito control, especially during disease outbreaks. A. aegypti has developed resistance nearly everywhere it occurs and insecticides are used. An important mechanism of resistance is due to mutations in the voltage-sensitive sodium channel (Vssc) gene. Two mutations, in particular, S989P + V1016G, commonly occur together in parts of Asia. RESULTS: We have created a strain (KDR:ROCK) that contains the Vssc mutations S989P + V1016G as the only mechanism of pyrethroid resistance within the genetic background of Rockefeller (ROCK), a susceptible lab strain. We created KDR:ROCK by crossing the pyrethroid-resistant strain Singapore with ROCK followed by four backcrosses with ROCK and Vssc S989P + V1016G genotype selections. We determined the levels of resistance conferred to 17 structurally diverse pyrethroids, the organochloride DDT, and oxadiazines (VSSC blockers) indoxacarb (proinsecticide) and DCJW (the active metabolite of indoxacarb). Levels of resistance to the pyrethroids were variable, ranging from 21- to 107-fold, but no clear pattern between resistance and chemical structure was observed. Resistance is inherited as an incompletely recessive trait. KDR:ROCK had a > 2000-fold resistance to DDT, 37.5-fold cross-resistance to indoxacarb and 13.4-fold cross-resistance to DCJW. CONCLUSION: Etofenprox (and DDT) should be avoided in areas where Vssc mutations S989P + V1016G exist at high frequencies. We found that pyrethroid structure cannot be used to predict the level of resistance conferred by kdr. These results provide useful information for resistance management and for better understanding pyrethroid interactions with VSSC. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases.

Aedes aegypti and A. albopictus mosquitoes are vectors of important human disease viruses, including dengue, yellow fever, chikungunya and Zika. Pyrethroid insecticides are widely used to control adult Aedes mosquitoes, especially during disease outbreaks. Herein, we review the status of pyrethroid resistance in A. aegypti and A. albopictus, mechanisms of resistance, fitness costs associated with resistance alleles and provide suggestions for future research. The widespread use of pyrethroids has given rise to many populations with varying levels of resistance worldwide, albeit with substantial geographical variation. In adult A. aegypti and A. albopictus, resistance levels are generally lower in Asia, Africa and the USA, and higher in Latin America, although there are exceptions. Susceptible populations still exist in several areas of the world, particularly in Asia and South America. Resistance to pyrethroids in larvae is also geographically widespread. The two major mechanisms of pyrethroid resistance are increased detoxification due to P450-monooxygenases, and mutations in the voltage sensitive sodium channel (Vssc) gene. Several P450s have been putatively associated with insecticide resistance, but the specific P450s involved are not fully elucidated. Pyrethroid resistance can be due to single mutations or combinations of mutations in Vssc. The presence of multiple Vssc mutations can lead to extremely high levels of resistance. Suggestions for future research needs are presented.

Detection of eastern equine encephalitis virus antibodies in moose (Alces americana), Maine, 2010.

Moose sera were collected from harvested animals during the 2010 hunting season in Maine. Of the 145 serum samples screened by plaque reduction neutralization test (PRNT), 16 (11%) had antibodies to eastern equine encephalitis virus (EEEV). Positive samples were collected from Aroostook County (n=13), Somerset County (n=2), and Piscataquis County (n=1) in northern and central Maine. Preliminary mosquito surveillance revealed the presence of enzootic and bridge vectors mosquitoes, including Culiseta (Climacura) melanura (Coquillett), Aedes (Aedimorphus) vexans (Meigen), and Coquillettidia (Coquillettidia) perturbans (Walker). Select mosquito species were tested by RT-PCR for the presence of EEEV. None were positive. This is the first report of EEEV in moose from Maine.

Effect of a botanical acaricide on Ixodes scapularis (Acari: Ixodidae) and nontarget arthropods.

We tested the effectiveness of the rosemary oil-based insecticide, Eco-Exempt IC2, to control all stages of Ixodes scapularis (Say) in southern Maine. We selected plots in oak-pine forest where I. scapularis is endemic and recorded the abundance of ticks and nontarget arthropods before and after applications of IC2, bifenthrin (a synthetic pyrethroid), and water (reference treatment). Licensed applicators applied high-pressure spray treatments during the summer nymphal and fall adult seasonal peaks. Both acaricides sprayed during the summer nymphal season reduced nymphal I. scapularis/hour to zero. IC2 was as effective as bifenthrin in controlling nymphs through the rest of the nymphal season and also controlled adult ticks 9 mo postspray compared with 16 mo for bifenthrin, and both acaricides reduced larvae through 14 mo postspray. Both acaricides sprayed during the fall adult season reduced adult I. scapularis/hour to zero; IC2 controlled adult ticks 6 mo postspray compared with 1 yr for bifenthrin. Both fall-applied acaricides controlled nymphs 9 mo postspray and reduced larvae up to 10 mo postspray. Impacts on some nontarget arthropods was assessed. Colleoptera, Hymenoptera, and Collembola declined 1 wk postspray in acaricide-treated plots, and in IC2 plots all numbers rebounded by 20 d postspray. For bees and other flower-visiting insects there were no detectable reductions in nests produced, number emerged from nests, or number of foraging visits to flowering plants in IC2 or bifenthrin plots. IC2 was phytotoxic to the leafy portions of select understory plants that appeared to recover by the next growing season.

An epizootic of eastern equine encephalitis virus, Maine, USA in 2009: outbreak description and entomological studies.

From July to September, 2009, an outbreak of eastern equine encephalitis virus (EEEv) occurred in five counties in Maine. The virus was isolated from 15 horses, 1 llama, and pheasants in three separate captive flocks. One wild turkey, screened before translocation, also showed exposure to the virus in January 2010. Two pools of Culiseta melanura (Coquillett) tested positive for EEEv during routine seasonal surveillance in York County in September, but none of the mosquitoes collected during rapid response surveys tested positive. There were more Cs. melanura in July, August, and September 2009 than in preceding (2006-08) and subsequent (2010-11) years. August and September Cs. melanura abundances were correlated with July rainfall, and abundance of all species combined was correlated with total rainfall for the meteorological summer. This outbreak represents a substantial expansion of the range of EEEv activity in northern New England.