Molecular Detection of Benzimidazole Resistance in Haemonchus contortus Larvae of Goats in Chhattisgarh, India

Background: Benzimidazole resistance is one of the key problem in small ruminant production. A rapid, truthful and responsive system is required for detection benzimidazole resistance so that proper regulatory measure can be applied. Allele specific PCR is one of the tools to understand the mechanism and origin of benzimidazole resistance. Methods: A total 198 larvae of Haemonchus contortus were isolated from goats of Chhattisgarh region, central India were genotyped by allele specific polymerase chain reaction (AS-PCR). Faecal samples of goats were collected from three Government farms and adjoining field goats and were subjected for faecal culture, separately. DNA of third stage larva was used for nested PCR for amplification of β- tubulin gene. Restriction Fragment Length Polymorphism (RFLP) was applied on nested PCR product for species identification with RsaI enzyme. AS-PCR was applied on the nested-PCR product to know the genotypic and allelic frequency. Result: The nested PCR amplified product showed approximately 820 bp in all cases and PCR-RFLP revealed 462 bp, 211 bp and 147 bp fragments, which confirmed the species as H. Contortus. Frequency of resistant allele ('r') was 49.7% and 50.3% for susceptible allele ('S '). Frequency of homozygous resistant (rr), heterozygous susceptible (rS) and homozygous susceptible (SS) genotype were 33.83 per cent, 31.81 per cent and 34.34 per cent, respectively. The frequency of homozygous resistant (rr) genotype was low (19.61%) in field compare to farm (48.96%) indicating refugia in field region.

Insecticide resistance selection and reversal in two strains of Aedes aegypti

Background: Laboratory reared mosquito colonies are essential tools to understand insecticide action. However, they differ considerably from wild populations and from each other depending on their origin and rearing conditions, which makes studying the effects of specific resistance mechanisms difficult. This paper describes our methods for establishing multiple resistant strains of Aedes aegypti from two colonies as a new resource for further research on metabolic and target site resistance. Methods: Two resistant colonies of Ae. aegypti, from Cayman and Recife, were selected through 10 generations of exposure to insecticides including permethrin, malathion and temephos, to yield eight strains with different profiles of resistance due to either target site or metabolic resistance. Resistance ratios for each insecticide were calculated for the selected and unselected strains. The frequency of kdr alleles (F1534C and V1016I) in the Cayman strains was determined using TaqMan assays. A comparative gene expression analysis among Recife strains was conducted using qPCR in larvae (CCae3A, CYP6N12, CYP6F3, CYP9M9) and adults (CCae3A, CYP6N12, CYP6BB2, CYP9J28a). Results: In the selected strain of Cayman, mortality against permethrin reduced almost to 0% and kdr became fixated by 5 generations. A similar phenotype was seen in the unselected homozygous resistant colony, whilst mortality in the susceptible homozygous colony rose to 82.9%. The Recife strains showed different responses between exposure to adulticide and larvicide, with detoxification genes in the temephos selected strain staying similar to the baseline, but a reduction in detoxification genes displayed in the other strains. Conclusions: These selected strains, with a range of insecticide resistance phenotypes and genotypes, will support further research on the effects of target-site and/or metabolic resistance mechanisms on various life-history traits, behaviours and vector competence of this important arbovirus vector.

Monitoring Insecticide Resistance and Target Site Mutations of L1014 Kdr And G119 Ace Alleles in Five Mosquito Populations in Korea

Mosquitoes are globally distributed and important vectors for the transmission of many human diseases. Mosquito control is a difficult task and the cost of preventing mosquito-borne diseases is much lower than that for curing the associated diseases. Thus, chemical control remains the most effective tool for mosquito. Due to the long-term intensive use of insecticides to control mosquito vectors, resistance to most chemical insecticides has been reported. This study aimed to investigate the relationship between insecticide resistance and target site mutation of L1014 kdr and G119 ace alleles in 5 species/species group of mosquitoes (Aedes vexans, Ae. albopictus, Anopheles spp., Culex pipiens complex, and Cx. tritaeniorhynchus) obtained from 6 collection sites. For Anopheles spp., the proportion of mosquitoes with mutated alleles in L1014 was 88.4%, homozygous resistant genotypes were observed in 46.7%, and heterozygous resistant genotypes were observed in 41.8%. For the Cx. pipiens complex and Cx. tritaeniorhynchus species, homozygous resistant genotypes were found in 25.9% and 9.8%, respectively. However, target site mutation of L1014 in the Ae. vexans nipponii and Ae. albopictus species was not observed. Anopheles spp., Cx. pipiens complex, and Cx. tritaeniorhynchus mosquitoes were resistant to deltamethrin and chlorpyriphos, whereas Ae. vexans nipponii and Ae. albopictus were clearly susceptible. We also found a correlation between the resistance phenotype and the presence of the L1014 kdr and G119 ace mutations only in the Anopheles spp. population. In this study, we suggest that insecticide resistance poses a growing threat and resistance management must be integrated into all mosquito control programs.

Insecticide resistance selection and reversal in two strains of Aedes aegypti

Background: Laboratory reared mosquito colonies are essential tools to understand insecticide action. However, they differ considerably from wild populations and from each other depending on their origin and rearing conditions, which makes studying the effects of specific resistance mechanisms difficult. This paper describes our methods for establishing multiple resistant strains of Aedes aegypti from two colonies as a new resource for further research on metabolic and target site resistance. Methods: Two resistant colonies of Ae. aegypti, from Cayman and Recife, were selected through 10 generations of exposure to insecticides including permethrin, malathion and temephos, to yield eight strains with different profiles of resistance due to either target site or metabolic resistance. Resistance ratios for each insecticide were calculated for the selected and unselected strains. The frequency of kdr alleles in the Cayman strains was determined using TaqMan assays. A comparative gene expression analysis among Recife strains was conducted using qPCR in larvae (CCae3A, CYP6N12, CYP6F3, CYP9M9) and adults (CCae3A, CYP6N12, CYP6BB2, CYP9J28a). Results: In the selected strain of Cayman, mortality against permethrin reduced almost to 0% and kdr became fixated by 5 generations. A similar phenotype was seen in the unselected homozygous resistant colony, whilst mortality in the susceptible homozygous colony rose to 82.9%. The Recife strains showed different responses between exposure to adulticide and larvicide, with detoxification genes in the temephos selected strain staying similar to the baseline, but a reduction in detoxification genes displayed in the other strains. Conclusions: These selected strains, with a range of insecticide resistance phenotypes and genotypes, will support further research on the effects of target-site and/or metabolic resistance mechanisms on various life-history traits, behaviours and vector competence of this important arbovirus vector.

Impact of Insecticide Resistance on P. falciparum Vectors’ Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya

Introduction. Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors’ biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. Methods. Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. Results. The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p≤0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

Detection of Aedes aegypti Resistance towards Cypermethrin using Polymerase Chain Reaction (PCR) Techniques in Ambarawa Semarang Regency

Ambarawa Regency is one of the endemic sub-districts since 2017 which always accounts for the highest DHF cases in Semarang Regency. Molecular detection of Aedes aegypti resistance towards sipermetrin using Polymerase Chain Reaction (PCR) techniques. The study was conducted to see mutations in the Aedes aegypti VGSC gene. This research was a pure experimental study. The Aedes aegypti samples examined were 10 in every village taken from 3 endemic DHF villages with high fogging intensity in Ambarawa. The research used random sampling techniques taken with ovitrap which was first installed during the month of August. Resistance detection test using the PCR method were conducted at the Banjarnegara Research and Development Center in September. Data were analyzed descriptively to illustrate the results of the study. The results showed that in Tambakboyo Village, 2 samples were susceptible (V / V), 7 samples were detected as homozygous (G / G) resistant, 1 sample was detected as heterozygous (V / G) resistant; in Kupang District 5 samples were detected as being homozygous resistant (G / G) and 5 samples were detected as heterozygous resistant (V / G); and in Panjang Village 1 susceptible sample (V / V), 8 samples were detected as homozygous resistant (G / G), 1 sample was detected as heterozygous resistant (V / G). Based on the results of mutation studies had been found in the VGSC gene in codon V1016G. The proper implementation of management, selection and rotation of insecticides is expected to reduce the risk of resistance in the Aedes aegypti mosquito population.

Anopheline Mosquito Species Composition, Kdr Mutation Frequency, and Parasite Infectivity Status in Northern Tanzania

The scaling-up of malaria control interventions in northern Tanzania has resulted in a decline in malaria prevalence and vector species composition. Despite this achievement, residual malaria transmission remains a concern in the area. The main aim of this study was to investigate malaria vector species composition, parasite infectivity rates, and the presence of insecticide knockdown resistance (kdr) mutations in three sites that have experienced a significant decline in malaria in northern Tanzania. Adult mosquitoes were sampled using light traps in houses and hand-aspirators in cowsheds, whereas the standard dipping method was used for sampling mosquito larvae. Adult mosquitoes identified as Anopheles gambiae s.l. and An. funestus s.l. and larval stages III and IV of An. gambiae s.l. were stored in absolute ethanol for further laboratory molecular identification. The identified species in the An. gambiae complex were An. gambiae s.s., An. merus, An. quadriannulatus, and An. arabiensis, whereas the An. funestus group comprised An. funestus s.s., An. rivulorum, and An. leesoni. For An. gambiae s.s. analyzed from Zeneth, 47.6% were kdr-East homozygous susceptible, 35.7% kdr-East heterozygous resistant, 9.6% kdr-East homozygous resistant, and 7.1% undefined, whereas specimens from Kwakibuyu were 45.5% kdr-East homozygous susceptible, 32.7% kdr-East heterozygous resistant, 16.3% kdr-East homozygous resistant, and 5.5% undefined. There were no kdr-West alleles identified from any specimen. The overall malaria parasite infectivity rate was 0.75%. No infections were found in Moshi. The findings indicate that populations of the major malaria vector mosquitoes are still present in the study area, with An. funestus taking a lead in malaria transmission.

Pesticides and the evolution of the genetic structure of Anopheles coluzzii populations in some localities in Benin (West Africa)

Background Changes in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of the Anopheles coluzzii populations in four localities of Benin. Methods Insecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on the Anopheles mosquitoes collected with the allelic and genotypic frequencies of kdr gene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy–Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study. Results Carbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava. Anopheles coluzzii was strongly represented across all study localities. The L1014F allele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado (p value < 0.05). Surprisingly in Sô-Ava, a relatively high frequency of the L1014F allele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS< 0). No genetic differentiation (FST< 0) was observed in the localities of Zagnanado and Kétou. Conclusion The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides.

Exploring the impact of glutathione S-transferase (GST)-based metabolic resistance to insecticide on vector competence of Anopheles funestus for Plasmodium falciparum

Background: Malaria control heavily relies on insecticide-based interventions against mosquito vectors. However, the increasing spread of insecticide resistance is a major threat. The extent to which such resistance, notably metabolic resistance, influences the development of the Plasmodium parasite and its impact on overall malaria transmission remains poorly characterized. Here, we investigated whether glutathione S-transferase-based resistance could influence Plasmodium falciparum development in Anopheles funestus. Methods: Anopheles funestus females were infected with P. falciparum gametocytes and midguts were dissected at day 7 post infection for detection/quantification of oocysts. Infection parameters were compared between individuals with different L119F-GSTe2 genotypes, and the polymorphism of the GSTe2 gene was analyzed in infected and uninfected mosquito groups. Results: Overall, 403 An. funestus mosquitoes were dissected and genotyped. The frequency of the L119F-GSTe2 resistance allele was significantly higher in non-infected (55.88%) compared to infected (40.99%) mosquitoes (Fisher's exact test, P<0.0001). Prevalence of infection was significantly higher in heterozygous and homozygous susceptible genotypes (P<0.001). However, homozygous resistant and heterozygous mosquitoes exhibited significantly higher infection intensity (P<0.01). No association was observed between the GSTe2 polymorphism and the infection status of mosquitoes. Conclusion: Altogether, these results suggest that GSTe2-based metabolic resistance may affect the vectorial competence of resistant An. funestus mosquitoes to P. falciparum infection, by possibly increasing its permissiveness to Plasmodium infection.