Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Concomitant knockdown resistance allele, L982W + F1534C, in Aedes aegypti has the potential to impose fitness costs without selection pressure.

The Aedes aegypti mosquito, is an arbovirus vector that can spread dengue, chikungunya, Zika, and yellow fever. Pyrethroids are widely used to control mosquitoes. The voltage-gated sodium channel (Vgsc) is the target of pyrethroids, and amino acid substitutions in this channel attenuate the effects of pyrethroids. This is known as knockdown resistance (kdr). Recently, we found that Ae. aegypti with concomitant Vgsc mutations L982W + F1534C exhibit extremely high levels of pyrethroid resistance. L982 is located in a highly conserved region of Vgsc in vertebrates and invertebrates. This study aimed to evaluate the viability of Ae. aegypti, with concomitant L982W + F1534C mutations in Vgsc. We crossed a resistant strain (FTWC) with a susceptible strain (SMK) and reared it up to 15 generations. We developed a rapid and convenient genotyping method using a fluorescent probe (Eprobe) to easily and accurately distinguish between three genotypes: wild-type and mutant homozygotes, and heterozygotes. As generations progressed, the proportion of wild-type homozygotes increased, and only 2.9% of mutant homozygotes were present at the 15th generation; the allele frequencies of L982W + F1534C showed a decreasing trend over generations. These observations show that these concomitant mutations have some fitness costs, suggesting that mosquitoes can potentially recover pyrethroid susceptibility over time without pyrethroid selection pressure in the field.

Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States.

House flies (Musca domestica L) are nuisances and vectors of pathogens between and among humans and livestock. Population suppression has been accomplished for decades with pyrethroids and acetylcholinesterase (AChE) inhibitors, but recurrent selection has led to increased frequency of alleles conferring resistance to those two classes of active ingredients (Geden et al., 2021). A common mechanism of resistance to both classes involves an altered target site (mutations in Voltage gated sodium channel (Vgsc) for pyrethroids or in Ace for AChE inhibitors). As part of ongoing efforts to understand the origin, spread and evolution of insecticide resistance alleles in house fly populations, we sampled flies in 11 different US states, sequenced, and then estimated frequencies of the Vgsc and Ace alleles. There was substantial variation in frequencies of the four common knockdown resistance alleles (kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L10414F) and 1B (T929I + L1014F) across the sampled states. The kdr allele was found in all 11 states and was the most common allele in four of them. The super-kdr allele was detected in only six collections, with the highest frequencies found in the north, northeast and central United States. The kdr-his allele was the most common allele in PA, NC, TN and TX. In addition, a novel super-kdr-like mutation in mutually exclusive exon 17a was found. The overall frequencies of the different Ace alleles, which we name based on the amino acid present at the mutation sites (V260L, A316S, G342A/V and F407Y), varied considerably between states. Five Ace alleles were identified: VAGF, VAVY, VAGY, VAAY and VSAY. Generally, the VSAY allele was the most common in the populations sampled. The susceptible allele (VAGF) was found in all populations, ranging in frequency from 3% (KS) to 41% (GA). Comparisons of these resistance allele frequencies with those previously found suggests a dynamic interaction between the different alleles, in terms of levels of resistance they confer and likely fitness costs they impose in the absence of insecticides.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

First detection of voltage-gated sodium channel mutations in Phlebotomus argentipes collected from Bangladesh.

BACKGROUND & OBJECTIVES: Phlebotomus argentipes is the main vector of visceral leishmaniasis in Bangladesh and is controlled using deltamethrin, a synthetic pyrethroid, through indoor residual spraying (IRS). A mutation at L1014 (leucine at codon 1014) of the voltage-gated sodium channel (VGSC), known as a knockdown resistance (kdr) gene, is thought to be an important pyrethroid resistance mechanism. This study detected mutations at codon 1014, and at codons 1011, 1016, and 1020, which are kdr sites in other insects. The kdr relationship with deltamethrin resistance in P. argentipes from an IRS-targeted site in Bangladesh was also evaluated. METHODS: Sand flies were collected from Magurjora village, Mymensingh district, Bangladesh in November 2012. A WHO cone bioassay test using deltamethrin was conducted and specimens were grouped as 'live' or 'dead'. After morphological identification, genomic DNA was used to genotype a partial VGSC gene from P. argentipes. The kdr/ pyrethroid resistance relationship was evaluated using Fisher's exact test. RESULTS: Targeted codons were genotyped from 8 'live' and 63 'dead' P. argentipes. All 'live' specimens had mutant alleles (L1014F and L1014S) at codon 1014. The mutant allele rate was 94% for 'live' specimens and 55% for 'dead' specimens. The mutant allele survival odds were higher for the wild-type L1014L allele, and L1014F odds were lower for L1014S. There were no mutations at codons 1011, 1016, and 1020. INTERPRETATION & CONCLUSION: The L1014 mutations suggested that pyrethroid resistance had appeared in Bangladesh. Further research on kdr mutations in P. argentipes is important for the appropriate IRS.

First detection of a Vssc allele V1016G conferring a high level of insecticide resistance in Aedes albopictus collected from Europe (Italy) and Asia (Vietnam), 2016: a new emerging threat to controlling arboviral diseases.

IntroductionAedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission.AimTo determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions.MethodsWe sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel (Vssc) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed.ResultsMost populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance (kdr) allele V1016G in Vssc for the first time in Ae. albopictus. Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S.ConclusionThe V1016G allele was detected in bothAsian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus, particularly where this mosquito species is the main vector of arboviruses.

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.

Two novel house fly Vssc mutations, D600N and T929I, give rise to new insecticide resistance alleles.

The house fly, Musca domestica, is a serious pest because it transmits a large diversity of human and veterinary diseases. Insecticides, particularly pyrethroids, are commonly used to control house flies. However, the evolution of pyrethroid resistance has reduced the effectiveness of these insecticides. A major mechanism of resistance to pyrethroids is target site insensitivity caused by the mutations in the voltage-sensitive sodium channel (Vssc) gene (e.g. kdr [L1014F] and super-kdr [M918T+L1014F]). Recently, two novel Vssc alleles, super-kdr+D600N and kdr+T929I were detected in a field collected resistant house fly population in Kansas, USA in 2013. To determine the levels of resistance that these new alleles confer to pyrethroids, we isolated strains having the unique Vssc alleles, but being otherwise congenic to the susceptible strain, aabys. We compared levels of resistance conferred to 14 pyrethroids and determined the inheritance of resistance to 8 pyrethroids. Our results revealed that super-kdr+D600N conferred higher levels of resistance to seven pyrethroids relative to super-kdr, and kdr+T929I showed super-kdr-like levels of resistance in house flies. Our results are compared with previous studies and reveal that addition of T929I to the kdr mutation (L1014F) increased resistance to all pyrethroids (except etofenprox), and enhanced resistance by ~1000-fold to acrinathrin and flumethrin. The implications of these results on the evolution of resistance are discussed.

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.

Pyrethroid resistance in Culex pipiens mosquitoes.

Mosquitoes within the Culex pipiens complex are widely distributed and important in the transmission of many human diseases. Insecticides, pyrethroids in particular, remain a mainstay for control of these important vectors. In this paper we review what is known about the levels, mechanisms and fitness costs of pyrethroid resistance in Cx. pipiens. Pyrethroid resistance in Cx. pipiens is a global problem, and resistance ratios of up to 7000-fold have been found in larvae of field collected mosquitoes. However, there is considerable variation between populations, indicating significant geographic heterogeneity of the resistance. The two major mechanisms of resistance to pyrethroids in Culex are mutations in Vssc (target site insensitivity) and overexpression of cytochrome P450(s) (increased detoxification). The most frequently reported Vssc mutation is L1014F (i.e. kdr), which has been found throughout the world. The L1014S mutation has been found in Cx. p. pallens from Japan and China, and in Cx. p. pipiens from China. The L1014C mutation has only been reported for Cx. p. pipens molestus from China and the V1016G mutation has only been reported from Saudi Arabia. Studies on the P450s of Cx. pipiens have identified several that are overexpressed (measured as transcript levels) in pyrethroid resistant strains. CYP9M10 is consistently overexpressed in pyrethroid resistant Cx. pipiens from at least seven countries, suggesting this P450 might be of global importance in resistance. Both CYP9M10-mediated pyrethroid resistance and kdr have fitness costs in the absence of insecticides under certain environmental conditions. Research needs and future directions are discussed.

Recent advances in the study of knockdown resistance mutations in Aedes mosquitoes with a focus on several remarkable mutations.

The Aedes mosquito, which transmits the dengue fever virus and other viruses, has acquired resistance to pyrethroid insecticides in a naturally selective manner. Massive use of insecticides has led to the worldwide expansion of resistant populations. The major factor in pyrethroid resistance is knockdown resistance (kdr) caused by amino acid mutation(s) in the voltage-gated sodium channel, which is the target site of this insecticide group. Some kdr mutations can lead to a dramatic increase in resistance, and multiple mutations can increase the level of pyrethroid resistance by 10 to several-hundred. In this review, we summarize the kdr identified in Aedes mosquitoes with a focus on the recent advances in the study of kdr.

Blowflies are potential vector for avian influenza virus at enzootic area in Japan.

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

Tiger prowling: Distribution modelling for northward-expanding Aedes albopictus (Diptera: Culicidae) in Japan.

The Asian tiger mosquito, Aedes albopictus, is a significant public health concern owing to its expanding habitat and vector competence. Disease outbreaks attributed to this species have been reported in areas under its invasion, and its northward expansion in Japan has caused concern because of the potential for dengue virus infection in newly populated areas. Accurate prediction of Ae. albopictus distribution is crucial to prevent the spread of the disease. However, limited studies have focused on the prediction of Ae. albopictus distribution in Japan. Herein, we used the random forest model, a machine learning approach, to predict the current and potential future habitat ranges of Ae. albopictus in Japan. The model revealed that these mosquitoes prefer urban areas over forests in Japan on the current map. Under predictions for the future, the species will expand its range to the surrounding areas and eventually reach many areas of northeastern Kanto, Tohoku District, and Hokkaido, with a few variations in different scenarios. However, the affected human population is predicted to decrease owing to the declining birth rate. Anthropogenic and climatic factors contribute to range expansion, and urban size and population have profound impacts. This prediction map can guide responses to the introduction of this species in new areas, advance the spatial knowledge of diseases vectored by it, and mitigate the possible disease burden. To our knowledge, this is the first distribution-modelling prediction for Ae. albopictus with a focus on Japan.

Reconsideration of importance of the point mutation L982W in the voltage-sensitive sodium channel of the pyrethroid resistant Aedes aegypti (L.)(Diptera: Culicidae) in Vietnam.

Pyrethroid resistance in Aedes aegypti is widespread in southern Vietnam because the photostable 2nd generation pyrethroids have been used in large amounts over extensive areas for malaria and dengue vector control. In our previous report in 2009, F1534C, one of the point mutations in the voltage-sensitive sodium channel (VSSC) in Ae. aegypti, was widespread at high frequency in south and central area. However, no significant correlation between the frequency of F1534C and pyrethroid susceptibility was detected primarily because the F1534C mutation frequency in the southern highland area was very low, despite that the bioassay indicated high pyrethroid resistance. The point mutation in the VSSC, L982W, which was not the target mutation in our previous study, was recently determined to be an important mutation causing high-pyrethroid resistance in Vietnamese Ae. aegypti. In the present study, a re-investigation of L982W in the mosquito samples collected in 2006-2008 revealed a greater distribution of this mutation (allelic percentage 59.2%) than F1534C (21.7%) and the greater proportion of homozygous L982W as compared to F1534C provided a plausible answer to the question concerning the unknown resistance factor in the southern highland area. L982W frequencies were uniformly higher in the southern part of Vietnam, including the highland area with a significantly high positive correlation with pyrethroid resistance in Ae. aegypti.

Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain "Candidatus Borrelia fainii" Qtaro.

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

Entomological surveillance for flaviviruses at migratory bird stopover sites in Hokkaido, Japan, and a new insect flavivirus detected in Aedes galloisi (Diptera: Culicidae).

To investigate the possible spread of West Nile virus (WNV) into Japan, we carried out entomological surveillance for flaviviruses at migratory bird stopover sites in Hokkaido, Japan, during 2003-2006. A total of 3,826 mosquitoes, identified as 15 species in five genera, were collected and 2,465 of these were grouped into 123 pools that were assayed for cytopathic effects on mosquito and mammalian cell cultures and for flavivirus RNA by reverse transcription-polymerase chain reaction using flavivirus universal primer sets for fragments of the NS3 and NS5 genes. Neither WNV nor other mosquito-vertebrate transmitted flaviviruses were detected in mosquitoes collected at any of the sites in Hokkaido, but five Culex flaviviruses and one novel Aedes galloisi flavivirus were identified from Culex pipiens L. s. l. and Aedes galloisi Yamada, respectively. Genetic and phylogenetic analyses based on the partial NS5 nucleotide sequences classified Aedes galloisi flavivirus with the insect flavivirus, but distant from Cell fusing agent, Kamiti river virus, and Culex flaviviruses, showing <74% sequence identities. Polymerase chain reaction-based bloodmeal analysis of 79 females showed that all of the Aedes and Ochlerotatus mosquitoes fed on mammals (deer and humans), whereas, Cx. pipiens s. l. mosquitoes fed on both of avian (ducks and sparrows, 85.7%) and mammalian hosts (dog, 14.3%). We suggest that to date WNV has not become established in Japan.

Potential efficacy of olyset mosquito netting against Calliphora nigribarbis (Diptera: Calliphoridae) invasion into livestock barns.

Calliphora nigribarbis Vollenhoven is a possible mechanical transmitter of highly pathogenic avian influenza. Based on laboratory tests, we evaluated the efficacy of a long-lasting permethrin-treated mosquito netting, known as the Olyset net, for the prevention of this species entering livestock barns. Flies were trapped in Olyset net cages, and two statistics for knockdown and lethal efficacies were obtained. Median knockdown time in the cage (KT50) was estimated to be 341 s for females, and median lethal time after exposure to the mesh (LT50) was estimated to be 30 s and <15 s for females and males, respectively. These LT50s were faster than those measured for anesthetized stationary flies brought in contact with the Olyset net (> 120 s for both sexes),indicating that a fly's spontaneous contact with the Olyset net accelerates insecticide adhesion. The rate of permethrin adhesion to C. nigribarbis after its spontaneous contact with the Olyset net was estimated to be 3.7 ng/s for females, in reference to the 50% lethal dose (LD50) value (112 ng/female), which was obtained from the topical application bioassay of permethrin. The lethality exhibited after brief spontaneous contact with the Olyset net suggests its potential utility in poultry farms against C. nigribarbis invasion.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

Novel CONCOMITANT mutations L932F and I936V in the Voltage-Gated Sodium Channel and Its Association With Pyrethroid Resistance in Culex quinquefasciatus (Diptera: Culicidae).

Highly residual pyrethroids such as permethrin have been used for controlling mosquitoes that transmit infectious diseases. However, the selective pressure from such insecticides may result in cross-resistance against other pyrethroids used for household insecticides. In this study, we investigated the susceptibility of Culex quinquefasciatus Say collected from Brazil and Myanmar to permethrin in addition to four types of household pyrethroids. Both strains exhibited high resistance against all pyrethroids tested, indicating cross-resistance. Furthermore, we detected the knockdown resistance (kdr) mutations L932F+I936V in the voltage-gated sodium channel gene (VGSC) in the Brazilian strain. Notably, the L932F+I936V haplotype has previously been observed in in silico data, but it should be detected not directly from living insects. In comparison, a common kdr mutation, L1014F, was detected from the Myanmar strain. Although L1014F was also detected from the Brazilian strain, the allele frequency was too low to affect resistance. Both strains harbored the resistance-associated haplotypes of the cytochrome P450 gene, CYP9M10. The Brazilian strain demonstrated comparable resistance against pyrethroids as that of the Myanmar strain even when a cytochrome P450 inhibitor, piperonyl butoxide was added to the bioassay. Our results suggested that the L932F+I936V mutations confer the Brazilian strain of Cx. Quiquefasciatus with resistance at a comparable level to that conferred by the well-recognized kdr mutation L1014F in the Myanmar strain. The identification of unexplored mutations may improve the diagnosis and understanding of resistance of this medically important species.

Common substitution mutation F348Y of acetylcholinesterase gene contributes to organophosphate and carbamate resistance in Cimex lectularius and C. hemipterus.

Bed bug control highly depends on insecticides with a limited number of modes of action, especially since the global prevalence of pyrethroid resistance. De facto insecticide options against bed bugs in Japan are acetylcholinesterase inhibitors (AChEis) that consist of organophosphates and carbamates. However, the status of AChEi resistance and the mechanisms involved have not been ascertained. An amino acid substitution mutation, F348Y (or F331Y in standard numbering), occurring at an acyl-binding site of the paralogous AChE gene (p-Ace), was identified among AChEi-resistant colonies of both common and tropical bed bugs (Cimex lectularius and C. hemipterus, respectively). This mutation was genetically associated with propoxur and fenitrothion resistance in F348Y-segregating colonies of C. hemipterus. Inhibition of heterologously expressed C. lectularius p-Ace with insecticides revealed that the sensitivities of F348Y-carrying AChE decreased by orders of 10- to more than 100-fold for diazoxon, carbaryl, fenitroxon, paraoxon, chlorpyrifos-methyl, malaoxon, azamethiphos, methyl-paraoxon, and propoxur. In contrast, the mutant AChE showed a slightly decreased degree of sensitivity for dichlorvos and almost unchanged sensitivity for metoxadiazone. Further studies are needed to ascertain whether the practical efficacies of dichlorvos and metoxadiazone are ensured against F348Y-carrying bed bugs and whether other resistance mechanisms are involved.