한국에 서식하는 모기종 중 흔히 볼 수 있는 금빛숲모기(Aedes vexans nipponii)는 미국에서 West Nile Virus의 주요 매개종으로 알려져 있다. 또한 지카바이러스를 포함한 30가지가 넘는 바이러스의 매개체로 작용할 가능성이 있다고 보고되고 있다. 이들을 방제하기 위한 방법으로 화학적 방제는 매우 효과적일 수 있으나 지속적인 살충제 사용은 저항성 문제를 불러일으킬 수 있다. 현재 금빛숲모기의 경우 이에 대한 관련 연구가 거의 이루어지지 않은 상황이다. 본 연구는 근연종인 흰줄숲모기와 이집트숲모기의 연구를 참고하여 합천에 위치한 축사에서 채집된 금빛숲모기 샘플로부터 기존에 알려진 Acetylcholinesterase-1(Ace-1)의 target site mutation인 G119, T506과 knockdown resistance(kdr) target site mutation인 V410, L982, S989, I1011, L1014, V1016, T1520, I1532, F1534, D1763을 분석하였다. 분석결과, 저항성을 가진 개체는 발견되지 않았으며 mutation site에 관해 정확히 알려진 바가 없으므로 bioassay를 통한 추가조사가 필요할 것으로 판단된다.

A residual contact vial plus water (RCVpW) bioassay method was established to monitor insectiside resistance in field populations of the melon thrips, Thrips palmi. Resistance level against six major insecticides were evalutated in five regions to test applicability of RCVpW as an on-site resistance monitoring tool. Reduced mortality in response to six test insecticides were exhibited compared to the RDA susceptable strain showing 100 % mortality, indicating different degree of resistance. An apparently reduced mortality to emamectin benzoate and chlofenapyr was observed in some field populations, suggesting uneven distribution of resistance to these insecticides in field populations. In addition, spinosad resistance was high and widely distributed in the test regions. Synergistic bioassay revealed that cytochrome P450-mediated metabolic factor is involved in spinosad resistance in the Korean population.

One of major mechanisms of insecticide resistance is the reduced susceptibility caused by mutations on the main target sites, such as sodium channel and acetylcholinesterase. Bioassay is a useful method to diagnose insecticide resistance of mosquitoes; however, it is hard to establish regional/annual resistance database based on bioassay. Recently, various markers of mutation and copy number variation have been identified through insecticide mechanism studies. Thus, molecular detection of resistance based on the resistance marker is now feasible, which can be readily implemented as a novel resistance monitoring tool to complement or replace the conventional bioassay method. In Korea, the density of vector mosquitoes native to the subtropical areas has increased due to climate change. Therefore, it is required to establish an efficient resistance monitoring system based on molecular markers to facilitate the construction of a nation-wide resistance map for more effective control of mosquitos. In addition, alternative insecticides should be introduced to the areas where mosquitoes develop high levels of resistance to maximize control efficacy against resistant populations

Residual contact vial (RCV) method was used to monitor insecticide resistance in field populations of the melon thrips, Thrips palmi. Median lethal doses (LD50) at 8 h post-treatment of six insecticides (chlorfenapyr, cyantraniliprole, cypermethrin, dinotefuran, emamectin benzoate and spinosad), which are commonly used for T. palmi control, were determined at 8 h post-treatment using a susceptible RDA strain. The diagnostic doses for on-site resistance monitoring of the six insecticides, which were determined as two-fold higher doses of LD90 for the RDA strain, were in the range of 0.299 to 164,25 μg-1cm2. To test the applicability of RCV for T. palmi, insecticide resistance levels in three field populations (Gyeonggi; GG_AS, Chungbuk; CB_CJ, Jeonbuk; JB_KJ) were evaluated. Field populations showed reduced mortality (0-50% mortality) to spinosad, cypermethrin, cyantraniliprole and emamectin benzoate, that they have different degree of resistances to these insecticides. In particular, all test field populations exhibited 0% mortality to spinosad, suggesting wide spread of spinosad resistance in the field. Moreover, no detectable mortality to emamectin benzoate was observed in JB_KJ strain, suggesting uneven distribution of emamectin benzoate resistant population of T. palmi. To provide more precise information on resistance profiles and distribution in T. palmi populations, it would be necessary to conduct a large scale resistance mapping for broad geographical regions.

The cotton bollworm, Helicoverpa armigera is one of the most polyphagous and cosmopolitan pest species in world wide including Korea. It also showed high level of resistance against various types of insecticide. To set the integrated resistant management program, first, we tried to identify the insecticide resistant mechanism via transcriptome analysis of coding and non-coding RNAs using 4 strains (Australian susceptible and resistant strains, Korean and Brazilian resistant strains, additional a sub-lethal dose insecticide treatment in Korean resistant strain). From the illumina based RNAseq data sets with genome information, some resistant involved detoxification genes and long non-coding RNAs were discovered. Second, following molecular markers were developed from resistant strain specific amino acid substitution from those candidate genes. Third, functionally identified genes’ markers were applied in field populations using some molecular diagnostic tools such as LAMP (loop mediated isothermal amplification). Now additional functional analysis is going on for these un-characterized candidate genes and long non-coding RNAs.

Growing resistance to insecticides, especially pyrethroids, poses an increased risk for insect control world-wide. Thus, alternative compounds are urgently needed. Accordingly, my laboratory has been investigating synthetic materials, as well as natural products, which are an attractive source of new chemistries. We have examined the insecticidal and synergistic activity of new compounds using Drosophila melanogaster and the mosquitoes Aedes aegypti and Anopheles gambiae as model insects. In addition, we have also been investigating the modes of action of experimental molecules in various physiological preparations. Natural product research included thymol, which was toxic to Ae. aegypti adults at doses (3-50 μg/mg) similar to published toxicities for the cabbage looper larvae, Trichoplusia ni. On homomultimeric Anopheles gambiae GABA receptor-chloride channel complex expressed in Xenopus laevis oocytes, thymol acts as a positive allosteric modulator, increasing the potency and maximal effectiveness of applied GABA, consistent with the sluggish paralysis it elicits in exposed insects. Other studies investigated matrine; a bioactive component extracted from Sophora flavescens that is used as the main ingredient in Chinese bio-pesticidal products. Matrine caused flaccid paralysis in headless fourth instar larvae of Ae. aegypti (50% paralysis in 5 hours at 8 ppm) and was toxic to adult females by contact (topical LD50 = 258 ng/mg). Adult toxicity was increased about 2-fold by pretreatment with the mono-oxygenase inhibitor, piperonyl butoxide. Interestingly, this compound was much less effective on D. melanogaster in either glass contact or feeding bioassays. Adult mosquito knockdown was rapid, with little or no expression of hyperactivity or hyperexcitability. Matrine (ca. 1 mM) had effects similar to thymol on expressed An. gambiae GABA receptors, and reduced EPSP amplitude at the Musca domestica neuromuscular junction, without any evidence of neuroexcitation or membrane depolarization. These physiological actions are sufficient to explain the whole animal intoxication by matrine, but require relatively high concentrations to manifest themselves. Other studies tested a series of potassium channel blockers, including 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34), 11-dansylaminoundecanoic acid (DAUDA), and 5-hydroxydecanoic acid (5-HDC) as insecticides and synergists of pyrethroids. We also evaluated the action of these compounds in patch clamp recordings of engineered HEK cells expressing Anopheles gambiae Kv2.1 channels. Patch clamp studies revealed that fatty acid compounds without functional groups in the alkyl chain (e.g., decanoate, DAUDA) yielded a more potent blocking action on Kv2.1 currents than substituted fatty acids (e.g., 5-HDC). Also, in comparison to 2-methoxy-N-((1-phenylcyclopentyl)methyl)benzamide (2S-65465), a known Kv2 channel blocker (IC50 = 100 nM), decanoate and DAUDA were 6-fold and 12-fold less active, respectively, as blockers of potassium current. TRAM-34 was the least potent inhibitor tested in patch clamp studies (IC50 = 30 uM). When tested on D. melanogaster CNS, the compounds typically gave an initial increase in firing rate, followed by a decrease, both effects at micromolar concentrations. Various toxicity assays showed the same potency ranking as that obtained through patch-clamp recordings, indicating a possible connection between channel block and whole organism effects. In order to possibly improve the insecticidal activity of the fatty acids, we synthesized derivatized analogs (e.g., methyl esters) presumed to better penetrate the cuticular barrier, but activity did not improve. Finally, in contrast to a published patent, we found that fatty acids did not show much synergism with pyrethroids. The implications of this work for resistance management will be discussed.

Toxicities of Carbofuran showed different patterns by three application methods, Drench(as systemic effect), Leaf dipping(as contact and systemic effects) and Topical(as contact effect) applications. LC50 dropping pattern was L-shape in Drench, low slope line in Topical and intermediate of them in Leaf dipping. Drench application was the most Carbofuran treatment method to Brown planthoppers. Dropping patterns of LC50 were almost same between resistant and susceptible strains. The checking time of Carbofuran toxicity was suggested to be 2nd day after application by SD analysis of mortality. Resistance ratios were peak on 3rd day after application in Drench, 4th day(Susceptible) and 5th day(Resistant) in Leaf dipping and showed steady increase line pattern in Topical application.

For our survey of insecticidal resistance of Palm thrips (Thrips palmi Karny), we established the discriminating time (DT) and concentration (DC) of nine insecticides, and we conducted a bioassay about seven local populations via leaf-dipping methods. The discriminating times of the recommended concentration (RC) were 24 h at emamectin benzoate EC and spinetoram SC, 48 h at chlorfenapyr EC, 72 h at spinosad SC, cyantraniliprole EC, acetamiprid WP, dinotefuran WG, imidacloprid WP and thiacloprid SC after treatment. The DC estimated the concentration which showed the difference within the mortalities of these local populations. The DCs were emamectin benzoate EC 0.013 mg L-1 (RC, 10.8 mg L-1), spinetoram SC 0.125 mg L-1 (RC, 25.0 mg L-1), chlorfenapyr EC 0.25 mg L-1 (RC, 50.0 mg L-1), spinosad SC 0.083 mg L-1 (RC, 50.0 mg L-1) and cyantraniliprole EC 5.0 mg L-1 (RC, 50.0 mg L-1), and DCs of neonicotinoids were their RCs, that is, acetamiprid WP (RC, 40.0 mg L-1), dinotefuran WG (RC, 20.0 mg L-1), imidacloprid WP (RC, 50.0 mg L-1) and thiacloprid SC (RC, 50.0 mg L-1). From our investigation into the resistance of the local populations with DT and DC application, the neonicotinoid insecticides have shown a high resistant level for all the local populations, and the other insecticides have demonstrated low or non-resistance. In the use of neonicotinoid insecticides to control Palm thrips, one must take caution. As a result, the establishment of DT and DC in the single dose bioassay method was helpful for surveying the insecticide response dynamics and the development of an insecticide resistance management strategy.

Anopheles sinensis is an important vector for Plasmodium vivax and thus has been targeted with organophosphates in Republic of Korea. Using the direct contact mortality bioassay, two field populations of An. sinensis from Paju-si and Yeoncheon-gun were characterized for their resistance to organophosphats with RR ratio values (Resistant ratio to imidacloprid) of 446.9 to 756.3 folds and 502.1 to 861.7 folds, respectively. Genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the acethylcholine esterase to detect Gly119Ser mutations. The homozygous susceptible Ser/Ser genotype in Paju-si and Yeoncheon-gun was 20.0% and 20.9% and the resistance genotypes were 80.0% and 79.1%, respectively. The homozygous Gly/Ser resistance genotype was the most prevalent as 60.7% in Paju-si and 41.9% in Yeoncheon-gun. Hence, this study suggests that malaria vector control programs should be prepared for the management of organophosphate insecticide resistance.

Anopheles sinensis is an important vector for Plasmodium vivax and thus has been targeted with pyrethroids in Republic of Korea. Using the direct contact mortality bioassay, two field populations of An. sinensis from Paju-si and Yeoncheon-gun were characterized for their resistance to pyrethroids with RR ratio values (Resistant ratio to imidacloprid) of 96.8 to 167.2 folds and 34.2 to 98.4 folds, respectively. Genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the sodium channel genes to detect L1014S mutations. The homozygous susceptible Leu/Leu genotype in Paju-si and Yeoncheon-gun was 3.3% and 37.1% and the resistance genotypes were 96.7% and 62.9%, respectively. The homozygous Phe/Phe resistance genotype was the most prevalent as 45.0% in Paju-si and 31.5% in Yeoncheon-gun. Hence, this study suggests that malaria vector control programs should be prepared for the management of pyrethroid insecticide resistance.

Increasing numbers of insecticide resistant insect is one of the main problem in current agriculture. To manage the insecticide resistance, IRAC suggest some tips for farmers : First, sequence or alteration of insecticide based on MOA. Second, choosing the effective insecticide. In this point raised following question : How to distinguish the effective or non-effective insecticide. Until now, bioassay is the right answer. However that is time-consuming, labor-intensive and costly process. So we have been steadily advancing molecular based insecticide resistance diagnosis for supporting and/or substitution of classical bioassay. It can summarized following steps : First, identification of insecticide resistance mechanism. Second, marker development. Third, development molecular diagnosis such as PASA, LAMP etc. Forth, application on field collected insects. Here we discuss some case of these study particular several major pests in highland agriculture.

Worldwide, increasing numbers insecticide resistant insect is one of the main problem in this agricultural era. To manage the insecticide resistance, IRAC suggest some tips for farmers : 1. Sequence or alteration of insecticide based on MOA 2. Choosing the effective insecticide. In this point raised following question : How to distinguish the effective or non-effective insecticide. Until now, bioassay is the right answer. However that is a very time-consuming, labor-intensive, and costly process, so we were hoping that molecular based diagnosis could be alternate. It can summarized following steps : 1. Identification of insecticide resistance mechanism 2. Marker development 3. Molecular diagnosis 4. Selection of non-effective insecticide. Here we focused on the development of insecticide resistance marker. Point mutation in target site of insecticide is a good example. Up or down-regulation of specific gene and/or existence of chimeric gene is another example from Helicoverpa armigera.

The small brown planthopper (SBPH), Laodelphax striatellus Fallen, is an important pest that causes severe yield losses by transmitting plant viruses to rice. For the efficient control of SBPH in Korea, the respective resistance levels in the immigrant and indigenous populations need to be discerned. The resistance levels to 10 insecticides (three carbamates, an organophosphate, four neonicotinoids, and a phenylpyrazole) were evaluated in 21 field populations collected from either SBPH-immigrating or indigenous regions during two different seasons (early spring vs. late summer). Imidacloprid resistance was most widely observed in many regional populations, followed by thiamethoxam resistance. Interestingly, the resistance level to imidacloprid was significantly higher in both immigrant and late-summer-collected populations than in indigenous and early spring-collected populations, respectively [3.3- (p = 0.018) and 2.6-fold (p = 0.026)]. Moreover, the late summer immigrant population exhibited higher imidacloprid resistance (2.4-fold) than the early spring-collected population from the same region, suggesting that the migratory SBPH that immigrated into Korea already exhibited imidacloprid resistance traits and were further selected after inhabitation. All field populations showed little resistance to fipronil (0.1- to 0.7-fold), suggesting that it is the most effective among the tested insecticides to control field populations of SBPH. The coefficient of variation of the resistance ratio (RR) among different regional populations and the correlation coefficient of RR among different insecticides have been suggested as supplementary parameters when determining appropriate insecticides as respective indicators for the dispersion status of resistance among SBPH populations and the possibility of cross resistance among tested insecticides.

Transcriptome analysis was conducted for the identification of genes associated with insecticide resistance in Frankliniella occidentalis. Resistant strain (FO_RDAHC) exhibited 39.2- ~ 533-fold resistance to acrinathrin, spinosad, emmamectin benzoate and thiamethoxam compared with a susceptible FO_RDA strain. Average 7.6 million reads (± 5,068,895 reads) were obtained from the pyrosequencing and were assembled into the draft CDS database. Gene annotation was conducted by BLAST (UniProt), Pfam, FUNCAT and COG analysis. In the deferentially expressed gene (DEG) analysis, 838 genes were up-regulated and 815 genes were down-regulated over 2-fold ratio in FO_RDAHC strain. Highly up-regulated genes included genes encoding several cuticle-related proteins, cytochrome P450s, esterases and transporter genes. An autotransporter protein gene exhibited the highest up-regulation (596 fold) whereas a GMC oxido-reductase revealed the highest down-regulation (12 fold). Further study would be necessary to validate the actual transcript levels of DEGs and to investigate their functional roles in insecticide resistance.

An easy and rapid resistance detection protocol for the Western flower thrips Frankliniella occidentalis was established based on the residual contact vial bioassay (RCV), in which insecticide resistance levels can be estimated at 8 h-post treatment of diagnostic doses. The RDA strain was used as a reference susceptible strain, which has been reared under laboratory conditions over 10 years without exposure to any insecticides. Seven insecticides were tested for the determination of diagnostic dose. Among them, five insecticides (chlorfenapyr, acrinathrin, spinosad, emmamectin benzoate and thiamethoxam, ranged as 0.03 ~ 0.42 μg-1cm2) were applicable to the RCV. However, two insecticides (omethoate and imidacloprid) were not able to be used for the RCV because the treated inner surface of glass vials by these insecticides were too viscous, causing non-specific mortality. The RCV detection kit was employed for the estimation of resistance levels for the five insecticides in five local populations. Almost field-collected populations revealed high levels of resistance to the four insecticides (acrinathrin, spinosad, emmamectin benzoate and thiamethoxam) by showing less than 50% mortality. The baseline resistance detection by RCV method will facilitate the selection of proper insecticides for farmers to manage insecticide resistant-populations of F. occidentalis.

Nilaparvata lugens Stål is one of the important migratory pests of rice paddy fields in Korea. Resistance levels to nine insecticides were monitored in 12 local strains and correlation analysis was conducted to determine cross-resistance relationships among the tested insecticides. The local strains revealed 1.3- to 28.0-, 1.6- to 6.0-, 2.8- to 237.0-, 0.6- to 0.9-, and 0.7- to 1.3-fold resistance to carbamates, organophosphates, neonicotinoids, fipronil and etofenprox, respectively. Organophosphates revealed moderate correlations with benzofuranyl methylcarbamate (r = 0.566 - 0.614, p > 0.01). Three neonicotinoids were not correlated each other, but imidacloprid and clothianidin were moderately correlated with several benzofuranyl methylcarbamate and organophosphate insecticides (r = 0.590 - 0.705, p > 0.05), indicating that unknown common factors (such as detoxification enzymes) might contribute to resistance to both insecticides. Fipronil and etofenprox exhibited low levels of resistance and cross-resistance with other insecticides, suggesting their potential as an effective insecticide for field application. Resistance level monitoring and correlation analysis would be valuable for the selection of appropriate insecticides to control insecticide-resistant N. lugenes, a typical migratory pest in Korea.

2009~2011년 동안 국내 주요 배추 재배지 5개 지역(평창, 홍천, 봉화, 무주, 제주)에서 살충제에 대한 복숭아혹진딧물의 저항성 발달 정도를 조사하고, 야외 개체군에 적용 가능한 살충제 저항성 마커를 개발하기 위해 본 연구를 수행하였다. 조사된 5개 지역 개체군 모두 여러 살충제 종류에 대하여 다양한 저항성을 보였다. 다양한 저항성을 보인 5개 지역 야외 개체군으로부터 여라 살충제에 대하여 복합적으로 저항성을 보이는 복합저항성 계통(MR)을 선발하였고, 이 MR과 모든 지역 채집 개체군에 대해 등전점전기영동과 정량염기서열분석(quantitative sequencing, QS)을 통하여 에스터레이즈 과발현과 살충제 작용점 내 돌연변이를 확인하였다. MR을 포함한 모든 야외 개체군에서 에스터레이즈의 과발현과 아세틸콜린에스터레이즈 1 유전자(ace1)의 StoF 돌연변이를 확인할 수 있었다. 넉다운 저항성 돌연변이로 잘 알려진 파라 타입 나트륨 채널 유전자(para)의 LtoF 돌연변이는 모든 지역 채집 개체군은 물론 비펜스린에 대해 3,461배 저항성을 보이는 MR에서도 발견되지 않았다. 그 외에 MtoL 돌연변이를 발견하였는데, 생물검정 결과 저항성 수준과 돌연변이 발생 빈도가 일치하였다. 따라서 생물검정 대신, 이러한 분자 마커를 활용 한다면 더 효율적으로 살충제 저항성 평가가 가능할 것이다. 이러한 분자 마커들(ace1의 StoF, para의 MtoL)은 정량염기서열분석, PCR amplification of specific alleles (PASA) 등의 진단 방법에 쉽게 응용이 가능 하고, 이러한 방법은 야외 복숭아혹진딧물 저항성 관리에 적용이 가능 할 것이다.

Vector mosquitoes are related with various diseases on human, such as malaria (Anopheles sinensis (L.), An. sinensis), Japanese encephalitis (Culex tritaeniorhynchus (L.), Cx. tritaeniorhynchus), yellow fever (Aedes albopictus (L.)), dengue hemorrhagic fever (Ae. albopictus) and filariasis (Ae. albopictus), as well as nuisance insect pests (Culex pipiens pallens Foskal, Cx. p. pallens). Continued and repeated use of conventional insecticides such as organophosphorus and carbamates, pyrethroids has often resulted in the widespread development of resistance and has undesirable effects on non-target organisms and environment. Particularly, widespread insecticide resistance has been a major obstacle in the cost-effective integrated vector management program. In theRepublic of Korea (ROK), studies on mass vector mosquito control using insecticides were mainly carried out until 1970’s such as a fuselage-mounted ULV spray system to C-46 aircraft, effectiveness of ground aerosols ULV premiumsumihtion, a helicopter application of ULV Dibrom and residual toxicity of organophosphates in animal shelters for Japanese Encephalitis (JE) and Plasmodium vivax malaria vector mosquito control. Regional insecticide resistance of vector mosquitoes has been annually evaluated since ,early 1980’s, the first detailed study on susceptibility of seven mosquito species to 13 organophosphorus insecticides revealed the DDT and lindane resistance of Cx. tritaeniorhyunchus. Marked annual and regional variations of insecticide resistance have been observed. In comparative resistance, the resistance ratios of various insecticides were compared among each other using two strains of vector mosquitoes that were collected from same locality. An. sinensis from Koyang-si in 1992 and 1981 demonstrated >100 folds and >3000 folds of higher resistance to DDT and to bio-resmethrin. An. sinensis from Paju-si in 2008 demonstrated 30 folds of higher susceptibility to fenthion than those collected in seven years ago. Cx. tritaeniorhynchus from Gwanju in 2010 and in 1992 showed >100 folds of higher resistance to pyrethroids. In comparative regional resistance monitoring, Cx. p. pallens from six localities in the ROK in 2008 and six geospatially-distant field Cx. tritaeniorhynchus strains in 2011 showed marked regional resistance variations. Field populations of Cx. tritaeniorhynchus from agricultural areas demonstrated extremely higher insecticide resistance to pyrethroids than those from non-agricultural areas. Cx. tritaeniorhynchus populations from rice paddies had been under heavy selection pressure due to pyrethroids, which were used for a long time to control agricultural pests in the localities. These results indicate that careful selection and rotational use of these insecticides may results in continued satisfactory control against field populations of vector mosquitoes

Insecticide resistance and activation of the metabolic detoxification enzymes of female Culex pipiens pallens by the blood meal were assessed using a micro-application bioassay and micro-plate enzyme activity assays. Four group of Cx. pipiens pallens were used, a susceptible non-engorging group at seven days after emerging, SNE7 Cp; a resistant non-engorging group at seven days after emerging, RNE7 Cp; a resistant engorged group at one day after blood feeding and a resistant engorged group at seven days after blood feeding, REG7 Cp. Insecticide resistance of Cx. pipiens pallans was increased by the blood feeding. Based on LC50 values, SNE7 Cp demonstrated >50 fold of higher susceptibility to all tested insecticides when compared with RNE7 Cp. RNE7 Cp showed higher susceptibility to all tested insecticides than REG1 Cp and REG7 Cp with a relative susceptibility LC50 (SRLC50) of 25.8 to 50.0 and 25.0 to 48.8. In micro-plate enzyme assays, the metabolic detoxification enzyme activity of Cx. pipiens pallans adult females was increased by the blood feeding. Activation of non-specific esterases (EST), glutathione-S-transferase (GST) and mixed function oxidase (MFO) in RNE7 were higher than in SNE7 and the all tested enzymes in REG1 and REG7 demonstrated significantly higher enzyme activation than RNE7, except for activation of GST in REG1. Activation of MFO in REG1 and REG7 were 209.4- and 74.6- fold higher than in REG7, respectively. Non-specific esterases (EST) and glutathione-S-transferase exhibited < 10 fold of higher Rr values. These results may be significant in terms of the criteria that are used to evaluate resistance, because blood fed female mosquitoes may show enhanced expression of the resistance phenotype, possibly allowing for earlier detection of insecticide resistance.