Experimental and molecular genetic analysis of the impact of pyrethroid and non-pyrethroid insecticide impregnated bednets for mosquito control in an area of pyrethroid resistance

Experimental huts in C?te d'Ivoire were used to evaluate the pyrethroid alpha-cypermethrin, the non-ester pyrethroid etofenprox, the organophosphate pirimiphos-methyl and the carbamate carbosulfan on bednets against pyrethroid-resistant Anopheles gambiae Giles. To test for selection for the resistance gene by the treated nets, A. gambiae collected live or dead from the huts were kept and analysed for the presence of the kdr gene using a new polymerase chain reaction followed by sequence-specific oligonucleotide probing (PCR-SSOP) for kdr-genotyping. Deliberately holed bednets freshly treated with pirimiphos-methyl or carbosulfan caused over 90% kill of A. gambiae s.s. and Culex spp. However, the mortality with alpha-cypermethrin or etofenprox treated nets was similar to that with untreated nets. Bloodfeeding of A. gambiae s.s. on the sleepers under the nets was only significantly reduced by alpha-cypermethrin and carbosulfan. Tests of the residual activity of the bednets after seven months showed that pirimiphos-methyl had lost its efficacy while carbosulfan still performed well. Once again the pyrethroid treated nets gave similar results to the untreated nets. Selection for the kdr-allele by alpha-cypermethrin and etofenprox, but not by carbosulfan, was indicated by PCR-SSOP genotyping of mosquitoes. Thus carbamates such as carbosulfan, or organophosphates of longer persistence than pirimiphos-methyl and of low mammalian toxicity, would seem to be a promising alternative to be used on bednets, particularly in areas of pyrethroid resistance.     

Insecticide susceptibility status in two medically important mosquito vectors,Anopheles gambiae,and Culex quinquefasciatus to three insecticides commonly used in Niger State,Nigeria

High resistance ability on insecticides among major mosquito vectors of diseases in Nigeria is of growing concern for severe control strategies. The objective of this study was to assess the susceptibility status of females Anopheles gambiae and Culex quinquefasciatus complexes mosquitoes to permethrin (21.5 μg/bottle-pyrethroids), propoxur(12.5 μg/bottle-carbamate) and malathion (50 μg/bottle organophosphate), in Niger State, North-Central, Nigeria. Anopheline and Culecine larvae were collected from the larval habitats of the studied sites (Bosso, Katcha, Lapai, and Shiroro) larvae and pupae were identified guided by standard keys and reared to adults in troughs. Insecticide susceptibility bioassays were performed according to the CDC bottle bioassay standard operating procedures on 3 days old, sugar-fed female Anopheles and Culex mosquitoes. Post-exposure mortality after 24hr and knockdown values for KDT₅₀ were calculated. Knock-down at 1-hour insecticide exposure ranged (84–96 %) permethrin, (94–100 %) propoxur and (100 %) malathion for An. gambiae and (86–97 %) permethrin, (92–100 %) propoxur and (96–100 %) malathion for Cx. quinquefasciatus. Mortality, after 24hr post-exposure was 100 % in malathion, indicating the high effect of the insecticide. Tested samples were found potentially resistant to permethrin recorded against mosquitoes collected from all study sites, in two locations of the study sites to propoxur and one location site to malathion. All the tested mosquitoes were found to be potentially resistant to permethrin, however, mosquitoes tested in Katcha and Shiroro resist potentially to propoxur. Except, Culex quinqufasciatus from Lapai that partially resist malathion, all the tested mosquitoes were found to be susceptible to malathion, across the study sites.     

Microplate assay of elevated esterase activity in individual pyrethroid-resistant mosquitoes

Involvement of esterase-mediated hydrolysis as a mechanism of pyrethroidresistance in three species of mosquitoes,viz., Aedes aegypti, Culex quinquefasciatus andAnopheles Stephensi was investigated by microplate assay of Β-esterases in individual larva and adult female and male mosquitoes. Assuming an absorbance value of 0.4 and above at 555 nm as the threshold level of elevated esterase activity which confers resistance, frequency distributions of such individual test mosquitoes were constructed in resistant and susceptible populations. The results indicate the involvement of ester hydrolysis of Pyrethroids as a predominant mechanism of pyrethroid-resistance in the larvae ofCulex quinquefasciatus but not inAedes aegypti. However, a marginal role of esterases is indicated in the larvae ofAnopheles stephensi.     

The kdr pyrethroid resistance gene in Anopheles gambiae: tests of non-pyrethroid insecticides and a new detection method for the gene

The organophosphate pirimiphos-methyl and the carbamate carbosulfan were evaluated in comparison to the pyrethroid alphacypermethrin and the 'near-pyrethroid' etofenprox against pyrethroid resistant Anopheles gambiae and Culex spp. in an experimental hut station located in central C?te d'Ivoire. Bednets were impregnated with the above mentioned compounds and randomly allocated to the huts. On 40 consecutive mornings, after sleepers had occupied the huts overnight, mosquitoes were collected from the huts, identified and scored as live or dead (including delayed mortality). An. gambiae s.l. that had been collected were tested for the presence of the kdr allele in heterozygous or homozygous form. Both non-pyrethroid treatments caused very high mortality, whereas mortality with alpha-cypermethrin and etofenprox generally did not differ from the levels observed with untreated control nets in this experiment. The nets had holes cut in them and there was considerable bloodfeeding on the sleepers, which was only significantly reduced for An. gambiae by carbosulfan and alpha-cypermethrin. PCR genotyping suggested that there was selection for the kdr resistance allele by the pyrethroid treated nets. Organophosphates and carbamates may therefore present an alternative to be used on bednets especially in areas of pyrethroid resistance, but the safety of these insecticides will have to be carefully considered.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

Resistance of field-collected populations of Culex pipiens pallens (Diptera: Culicidae) to insecticides in the Republic of Korea

Toxicities of 10 insecticides were examined against late third instars of Culex pipiens pallens, the northern house mosquito, using a direct-contact mortality bioassay. Several strains of mosquitoes were tested (insecticide-susceptible KS-CP strain and five geospatially distant field-collected strains (DG-CP, US-CP, BS-CP, GS-CP, and SG-CP)) and identified by polymerase chain reaction. Marked regional variations of insecticide susceptibility were observed. Extremely high to low levels of resistance were measured: bifenthrin, resistance ratio (RR) = 1-521; β-cyfluthrin, RR = 16-397; α-cypermethrin, RR = 9-343; deltamethrin, RR = 1-40; etofenprox, RR = 2-42; permethrin, RR = 3-46; chlorpyrifos, RR = 2-675; fenitrothion, RR = 0.5-364; and fenthion, RR = 2-360. All strains were susceptible to one or more of the insecticides examined. These results indicate that careful selection and rotational use of these insecticides may result in continued satisfactory control against field populations of northern house mosquitoes.     

Effects of permethrin at different temperatures on pyrethroid-resistant and susceptible strains of Anopheles

The influence of temperature (16, 22, 28, 37 degrees C) on effects of permethrin was investigated for susceptible and pyrethroid-resistant strains of the mosquitoes Anopheles gambiae and An. stephensi (Diptera: Culicidae). Young unfed female adult mosquitoes were exposed to 0.25% permethrin test papers or to polyester netting treated with permethrin 500mg a.i./m2. The time to 50% knockdown (KT50) declined as temperature increased, i.e. there was a positive temperature coefficient of this effect of the pyrethroid. Resistance ratios (comparing KT50 values) between resistant and susceptible An. stephensi ranged between 2.5 and 4.4 at the different temperatures. Comparative tests of pyrethroid tolerance of different strains would be valid over the 22-28 degrees C range but, when using a discriminating dose to detect resistance, more precise temperature control is desirable. Mortality 24h after exposure to 0.25% permethrin of both susceptible and resistant strains of An. stephensi showed a negative correlation with temperature between 16 and 22 degrees C and a positive correlation at higher temperatures. In An. gambiae, however, the correlation was positive over the whole range. Irritancy of permethrin-treated netting to Anopheles females (measured as time lapse until first flight take-off, and the number of take-offs during 7.5 min exposure) was positively correlated with temperature in all four strains and was much greater for the susceptible than the resistant strains.     

The Cry48Aa-Cry49Aa binary toxin from Bacillus sphaericus exhibits highly restricted target specificity

The Cry48Aa/Cry49Aa binary toxin of Bacillus sphaericus was recently discovered by its ability to kill Culex quinquefasciatus mosquito larvae through a novel interaction between its two components. We have investigated the target specificity of this toxin and show it to be non-toxic to coleopteran, lepidopteran and other dipteran insects, including closely related Aedes and Anopheles mosquitoes. This represents an unusually restricted target range for crystal toxins from either B. sphaericus or Bacillus thuringiensis. Gut extracts from Culex and Aedes larvae show differential processing of the Cry48Aa protein, with the location of cleavage sites in Culex reflecting those previously shown for the activation of Cry4 toxins in mosquitoes. Pre-activation of Cry48Aa/Cry49Aa with Culex extracts, however, fails to induce toxicity to Aedes larvae. Co-administration of Cry49Aa with Cry4Aa gives higher than predicted toxicity, perhaps suggesting weak synergism against Culex larvae between Cry49Aa and other three-domain Cry toxins.     

Infectivity of two strains of Plasmodium vivax to Anopheles albitarsis mosquitoes from Colombia

Anopheles albitarsis obtained from Villavicencio, Colombia, were colonized in the laboratory using force-mating techniques. Laboratory reared mosquitoes were allowed to feed on Aotus monkeys infected with the Salvador II or the Rio Meta strains of Plasmodium vivax from El Salvador and Colombia, respectively. In comparison with other species, the An. albitarsis were less susceptible than Anopheles freeborni, Anopheles culicifacies and strains of Anopheles albimanus from El Salvador, Panama and Colombia and more susceptible than a strain of An. albimanus from Haiti.     

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.     

Efficacy of culture filtrates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against larvae of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

Efficacy of culture filtrates of five strains of Metarhizium anisopliae isolated from insects were evaluated against Anopheles stephensi and Culex quinquefasciatus. The culture filtrates released from the strains of M. anisopliae in the YpSs and chitin broths were filtered and used for the bioassays after a growth of 7 days. Among the culture filtrates of five strains, M. anisopliae 892 was found to be more effective against both the mosquitoes. The LC(50) values of culture filtrates of M. anisopliae 892 in chitin broth was lower than the LC(50) of culture filtrates in YpSs broth against first and fourth instars of both the mosquitoes. The LC(50) values of culture filtrates were significantly different between first and fourth instars of A. stephensi (t test; P = 0.0001) and C. quinquefasciatus (t test; P = 0.02). The larvae of A. stephensi were more susceptible than C. quinquefasciatus except in two cases. This is the first report of efficacy of culture filtrates produced by M. anisopliae in chitin broth against mosquitoes and have potential as a biological control agent of mosquitoes.     

A generalized approach to detection of organophosphate resistance in mosquitoes

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.     

蚊虫抗药性分子机理

蚊虫由于特殊的行为、生理及与人类生活密切相关,从而成为人类许多疾病的媒介,如疟疾、登革热、日本脑炎、丝虫病等,大多由库蚊Culex、伊蚊Aedes或按蚊Anopheles传播。世界上每年大量的化学杀虫剂用于农业和公共卫生,直接或间接给蚊虫带来选择压力而引发抗性。抗性机制基本可以分为两大类:代谢抗性(如非特异性羧酸酯酶,P450单加氧酶,谷胱甘肽S-转移酶的扩增)和靶标抗性(如乙酰胆碱酯酶、GABA受体和电压门控钠离子通道的突变)。文章对这些机理的研究进展进行综述。     

The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance

The primary routes of insecticide resistance in all insects are alterations in the insecticide target sites or changes in the rate at which the insecticide is detoxified. Three enzyme systems, glutathione S-transferases, esterases and monooxygenases, are involved in the detoxification of the four major insecticide classes. These enzymes act by rapidly metabolizing the insecticide to non-toxic products, or by rapidly binding and very slowly turning over the insecticide (sequestration). In Culex mosquitoes, the most common organophosphate insecticide resistance mechanism is caused by co-amplification of two esterases. The amplified esterases are differentially regulated, with three times more Est beta 2(1) being produced than Est alpha 2(1). Cis-acting regulatory sequences associated with these esterases are under investigation. All the amplified esterases in different Culex species act through sequestration. The rates at which they bind with insecticides are more rapid than those for their non-amplified counterparts in the insecticide-susceptible insects. In contrast, esterase-based organophosphate resistance in Anopheles is invariably based on changes in substrate specificities and increased turnover rates of a small subset of insecticides. The up-regulation of both glutathione S-transferases and monooxygenases in resistant mosquitoes is due to the effects of a single major gene in each case. The products of these major genes up-regulate a broad range of enzymes. The diversity of glutathione S-transferases produced by Anopheles mosquitoes is increased by the splicing of different 5' ends of genes, with a single 3' end, within one class of this enzyme family. The trans-acting regulatory factors responsible for the up-regulation of both the monooxygenase and glutathione S-transferases still need to be identified, but the recent development of molecular tools for positional cloning in Anopheles gambiae now makes this possible.     

DDT resistance in Anopheles gambiae declines with mosquito age

Adult Anopheles gambiae Giles mosquitoes from Zanzibar were tested on a standard discriminating dose of DDT which reliably kills susceptible mosquitoes. Adults from wild-caught larvae reared in the laboratory, and from the F1 progeny of wild-caught adults, showed less than 5% test mortality when newly-emerged, but mortality rose with age to over 90% when they were 12-14 days old. Wild-caught mixed-age adults showed an intermediate mortality rate of 25%, close to the rate predicted from laboratory results for a fully resistant population with an age-structure typical of this species in natural conditions. It is inferred that older, genetically resistant insects may be scored as susceptible, so that routine susceptibility tests with wild-caught adults underestimate the frequency of resistance. In Zanzibar, such tests probably helped to persuade spraying authorities to continue spraying DDT in spite of resistance. Resistance which is restricted to younger insects may nonetheless reduce the effectiveness of spraying.     

Insecticide susceptibility and resistance of Culex tritaeniorhynchus (Diptera: Culicidae) larvae collected from Gwangju,Republic of Korea

The susceptibility of Culex tritaeniorhynchus collected from Gwangju, Jeollabuk Province, Republic of Korea (ROK) to insecticides was evaluated under laboratory conditions using ten insecticides (7 pyrethroids and 3 organophosphates) that are currently applied by local public health centers in the ROK. Based on the values of median lethal concentration (LC₅₀), Cx. tritaeniorhynchus larvae were most susceptible to chlorpyrifos (0.006 ppm), fenitrothion (0.022 ppm), fenthion (0.035 ppm) and bifenthrin (0.038 ppm), and were least susceptible to esbiol (1.722 ppm). In comparative resistance tests, the resistance ratios (RRs) of seven insecticides were compared among each other using two strains of Cx. tritaeniorhynchus that were collected from the same locality during 1992 and 2010. Culex tritaeniorhynchus demonstrated significantly increased RRs to pyrethroids over time, while demonstrating decreased RRs among the organophosphates. Among the pyrethroids, permethrin had the highest RR values of 182.1‐ and 833.3‐fold differences, followed by etofenprox with RRs of 138.4‐ and 224.1‐fold differences in values of LC₅₀ and concentration that produced 90% mortality (LC₉₀), respectively. Culex tritaeniorhynchus strains demonstrated the least amount of change in susceptibility to the organophosphates, chlorpyrifos, fenitrothion and fenthion with 0.020‐, 0.019‐ and 0.001‐fold differences in resistance ratios (RRLC₅₀), respectively.     

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV) has been isolated from mosquitoes such as Aedes, Mansonia uniformis, and Culex perfuscus; However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported. In June and July2018, 22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China. Among them, six strains of ZIKV were isolated from mosquitoes: four from An. sinensis and two from Cx. tritaeniorhynchus. Molecular genetic analysis showed that the ZIKV isolated from An. sinensis and Cx. tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV. In addition, the ZIKV strains isolated from An. sinensis and Cx. tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015. This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province, China; This is also the first time that ZIKV has been isolated from An. sinensis and Cx. tritaeniorhynchus. Given that An. sinensis and Cx. tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia, the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2005

Adult mosquito surveillance was conducted during 2005 at 22 US military installations and two training sites located in six provinces in the Republic of Korea. Adult mosquitoes were collected in New Jersey light traps from 1 May to 15 October to determine threshold levels to initiate pesticide applications based on trap indices. A total of 27 363 adult mosquitoes (22 368 [81.7%] females and 4995 [18.3%] males) comprising 15 species (including five members of the Anopheles sinensis group) belonging to seven genera were collected. The most common species collected were members of the Anopheles sinensis Wiedemann group (50.2%), followed by Culex tritaeniorhynchus Giles (30.6%), Aedes vexans nipponii (Theobald) (11.8%), and Culex pipiens Coquillett (6.3%) females. Trap indices varied widely for species over their range, due in part to geographical distribution and degree of association with urban or rural communities.