PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

<p>Abstract</p> <p>Background</p> <p><it>Anopheles culicifacies s.l</it>., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (<it>kdr</it>) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of <it>kdr </it>mutation (L1014F) in a field population of <it>An. culicifacies s.l</it>. and three new PCR-based methods for <it>kdr </it>genotyping.</p> <p>Methods</p> <p>The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant <it>An. culicifacies s.l</it>. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for <it>kdr </it>genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.</p> <p>Results</p> <p>The genotyping of this <it>An. culicifacies s.l</it>. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the <it>kdr </it>allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.</p> <p>Conclusion</p> <p>The Leu-Phe mutation, which generates the <it>kdr </it>phenotype in many insects, was detected in a pyrethroid and DDT resistant <it>An. culicifacies s.l</it>. population. Three PCR-based methods were developed for <it>kdr </it>genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at <it>kdr </it>locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.</p

Global Transcriptome Profiling of the Pine Shoot Beetle, Tomicus yunnanensis (Coleoptera: Scolytinae)

Background: The pine shoot beetle Tomicus yunnanensis (Coleoptera: Scolytinae) is an economically important pest of Pinus yunnanensis in southwestern China. Developed resistance to insecticides due to chemical pesticides being used for a long time is a factor involved in its serious damage, which poses a challenge for management. In addition, highly efficient adaptation to divergent environmental ecologies results in this pest posing great potential threat to pine forests. However, the molecular mechanisms remain unknown as only limited nucleotide sequence data for this species is available. Methodology/Principal Findings: In this study, we applied next generation sequencing (Illumina sequencing) to sequence the adult transcriptome of T. yunnanensis. A total of 51,822,230 reads were obtained. They were assembled into 140,702 scaffolds, and 60,031 unigenes. The unigenes were further functionally annotated with gene descriptions, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genome (KEGG). In total, 80,932 unigenes were classified into GO, 13,599 unigenes were assigned to COG, and 33,875 unigenes were found in KO categories. A biochemical pathway database containing 219 predicted pathways was also created based on the annotations. In depth analysis of the data revealed a large number of genes related to insecticides resistance and heat shock protein genes associated with environmental stress. Conclusions/Significance: The results facilitate the investigations of molecular resistance mechanisms to insecticides an

Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na+ channel of Anopheles culicifacies from Orissa, India

<p>Abstract</p> <p>Background</p> <p>Knockdown resistance in insects resulting from mutation(s) in the voltage gated Na⁺channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common <it>kdr </it>mutation in insects, was reported in <it>Anopheles culicifacies</it>-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an <it>An. culicifacies </it>population from Malkangiri district of Orissa, India.</p> <p>Methods</p> <p><it>Anopheles culicifacies sensu lato (s.l.) </it>samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The <it>An. culicifacies </it>population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.</p> <p>Results</p> <p>DNA sequencing of <it>An. culicifacies </it>individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.</p> <p>Conclusions</p> <p>This is the first report of the presence of L1014S (homologous to the <it>kdr-e </it>in <it>An. gambiae</it>) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of <it>An. culicifacies </it>in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.</p

SUPPORT Tools for evidence-informed health Policymaking (STP) 17: Dealing with insufficient research evidence

This article is part of a series written for people responsible for making decisions about health policies and programmes and for those who support these decision makers

Absence of knockdown resistance suggests metabolic resistance in the main malaria vectors of the Mekong region

<p>Abstract</p> <p>Background</p> <p>As insecticide resistance may jeopardize the successful malaria control programmes in the Mekong region, a large investigation was previously conducted in the Mekong countries to assess the susceptibility of the main malaria vectors against DDT and pyrethroid insecticides. It showed that the main vector, <it>Anopheles epiroticus</it>, was highly pyrethroid-resistant in the Mekong delta, whereas <it>Anopheles minimus sensu lato </it>was pyrethroid-resistant in northern Vietnam. <it>Anopheles dirus sensu stricto </it>showed possible resistance to type II pyrethroids in central Vietnam. <it>Anopheles subpictus </it>was DDT- and pyrethroid-resistant in the Mekong Delta. The present study intends to explore the resistance mechanisms involved.</p> <p>Methods</p> <p>By use of molecular assays and biochemical assays the presence of the two major insecticide resistance mechanisms, knockdown and metabolic resistance, were assessed in the main malaria vectors of the Mekong region.</p> <p>Results</p> <p>Two FRET/MCA assays and one PCR-RFLP were developed to screen a large number of <it>Anopheles </it>populations from the Mekong region for the presence of knockdown resistance (<it>kdr</it>), but no <it>kdr </it>mutation was observed in any of the study species. Biochemical assays suggest an esterase mediated pyrethroid detoxification in <it>An. epiroticus </it>and <it>An. subpictus </it>of the Mekong delta. The DDT resistance in <it>An. subpictus </it>might be conferred to a high GST activity. The pyrethroid resistance in <it>An. minimus s.l</it>. is possibly associated with increased detoxification by esterases and P450 monooxygenases.</p> <p>Conclusion</p> <p>As different metabolic enzyme systems might be responsible for the pyrethroid and DDT resistance in the main vectors, each species may have a different response to alternative insecticides, which might complicate the malaria vector control in the Mekong region.</p

Pyrethroid resistance in Anopheles gambiae leads to increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana

<p>Abstract</p> <p>Background</p> <p>Entomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of <it>Anopheles gambiae sensu stricto</it>, and to test a potential field application method with respect to the viability and virulence of two fungal species</p> <p>Methods</p> <p>Pieces of white polyester netting were dipped in <it>Metarhizium anisopliae </it>ICIPE-30 or <it>Beauveria bassiana </it>IMI391510 mineral oil suspensions. These were kept at 27 ± 1°C, 80 ± 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of <it>An. gambiae s.s</it>., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net.</p> <p>Results</p> <p>The resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, <it>B. bassiana </it>was significantly more virulent than <it>M. anisopliae </it>for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension.</p> <p>Conclusions</p> <p>The insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.</p