Permethrin resistance variation and susceptible reference line isolation in a field population of the mosquito,Culex quinquefasciatus （Diptera： Culicidae）

This study examines the genetic variations and mechanisms involved in the development of permethrin resistance in individual mosquitoes from a field population of Culex quinquefasciatus, HAmCq^G0, and characterizes susceptible reference lines of mosquitoes with a similar genetic background to the field HAmCq^G0 strain. Six upregulated cytochrome P450 genes, CYP9M10, CYP9J34, CYP6P14, CYP9J40, CYP6AA7, and CYP4C52v1, previously identified as being upregulated in the larvae of resistant HAmCq68 mosquitoes were examined in the larvae of 3 strains （susceptible S-Lab, parental HAmCq^G0 and permethrin-selected highly resistant HAmCq68） and 8 HAmCq^G0 single- egg raft colonies, covering a range of levels of susceptibility/resistance to perrnethrin and exhibiting different variations in the expression of A and/or T alleles at the L-to-F kdr locus of the sodium channel. The 2 lines with the lowest tolerance to permethrin and bearing solely the susceptible A allele at the L-to-F kdr locus of the sodium channels, from colonies Cx_SERC5 and Cx_SERC8, showed lower or similar levels of all 6 of the P450 genes tested compared with the S-Lab strain, suggesting that these 2 lines could be used as the reference mosquitoes in future studies characterizing insecticide resistance in HAmCq mosquitoes. This study also provides a detailed investigation of the mechanisms involved in insecticide resistance in individuals within a population： individuals with elevated levels of resistance to permethrin all displayed one or more potential resistance mechanisms-either elevated levels of P450 gene expression, or L-to-F mutations in the sodium channel, or both.     

Novel Kdr mutations (K964R and A943V) in pyrethroid‐resistant populations of Triatoma mazzottii and Triatoma longipennis from Mexico and detoxifying enzymes

Although having five different ways of transmission the vector-borne is the principal way of transmission of Chagas disease, which involves insects of the subfamily Triatominae (Hemiptera: Reduviidae). Nineteen of the 31 species that occur in Mexico are associated with humans, and all are capable of transmitting the disease. Pyrethroids are the insecticides recommended for the control of these vectors in Mexico. We determined the susceptibility to the pyrethroids dcltamethrin and permethrin of peridomestic populations of Triatoma mazzottii Usinger and two populations of Triatoma longipetmis Usinger in comparison with a reference strain for each species. Bioassays were performed for the determination of the LD50 for both field populations and reference strains. A maximum of 27 fold resistance to deltamethrin was observed in T. mazzottii, meanwhile, for permethrin, T. longipennis from Jalisco show the highest value of 3.19 fold. There was significantly increased activity of esterases in field populations in comparison with their corresponding reference strain. The results of the search of kdr mutations related to the resistance to deltamethrin and permethrin in the evaluated species show the presence of mutations in the field populations, as is the case with individuals of T. mazzottii, for which the mutation was found A943V, and for the two populations of T. longipennis included in this study, we report the presence of the kdr mutation K964R. Evaluation of the various mechanisms involved in resistance to pyrethroids in triatomines from Mexico could guide us to the real justification for insecticide resistance monitoring.     

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.     

Foraging site recursion by forest elephants Elephas maximus borneensis

Recursion by herbivores is the repeated use of the same site or plants. Recursion by wild animals is rarely investigated but may be ubiquitous. Optimal foraging theory predicts site recursion as a function of the quality of the site, extent of its last use, and time since its last use because these influence site resource status and recovery. We used GPS collars, behaviour and site sampling to investigate recursion to foraging sites for two elephant Elephas maximus borneensis herds in the Lower Kinabatangan Wildlife Sanctuary, Borneo, over a 12 month period. Recursion occurred to 48 out of 87 foraging sites and was most common within 48 hours or between 151-250 days, indicating two different types ofrecursion. Recursion was more likely to occur if the site had previously been occupied for longer. Moreover, the time spent at a site at recursion was the same as the time spent at the site on the first occasion. The number of days that had passed between the first visit and recursion was also positively correlated with how much time was spent at the site at recursion. Habitat type also influenced the intensity of site-use, with more time spent at recursion within riverine/open grass areas along forest margins compared to other habitat types. Recursion is a common behaviour used by the elephants and its pattern suggests it may be a foraging strategy for revisiting areas of greater value. The qualities of recursion sites might usefully be incorporated into landscape management strategies for elephant conservation in the area [Current Zoology 60 （4）： 551-559, 2014].     

Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal,putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites.Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1 st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells.These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.     

Seasonal changes of resistance to fenvalerate and cypermethrin in the larval parasitoid Cotesia plutellae （Hymenoptera： Braconidae）

The seasonal changes of insecticide resistance and stability in hymenopteran Cotesia plutellae, collected from Jianxin, Fuzhou-City, and Shangjie, Minhou-County, Fujian, China, were assessed by using a dry residual film method. The resistance to two insecticides in the field populations of C. plutellae was not stable under insecticide-free conditions in the insectarium. Compared with susceptible F11 progeny of C. plutellae in the insectarium, the resistance ratios （RR） in F0 parents were 18.4 for fenvalerate and 11.4 for cypermethrin based on LC50 at 9 hours, and 32.8 for fenvalerate and 28.5 for cypermethrin based on LC50 at 24 hours when the parasitoids were left in contact with the insecticides for 1 hour and mortalities were recorded at 9 and 24 hours, respectively. However, the RR in a field population of C. plutellae were 9.2 for fenvalerate and 12.7 for cypermethrin, if the parasitoids were left in contact with the insecticides for 24 hours. The resistances to the two pyrethroids in other field populations collected from Jianxin and Shangjie from November 2000 and July 2004 were also determined. Significant seasonal variations of resistance to the two insecticides in the field populations of C. plutellae were found. The RR were 3.0-18.4 for fenvalerate and 4.8-20.6 for cypermethrin in Jianxin populations from November 2000 to April 2002 based on LC50 at 9 h, and 2.3-13.6 for fenvalerate and 3.6-16.0 for cypermethrin in Shangjie populations from May 2002 to July 2004 based on LC50 at 24 hours. The resistance levels were high in spring and autumn and decreased sharply in summer. In addition, significant recovery from the knocked-down caused by the insecticides was found in the F0 and field populations of C. plutellae which were resistant to fenvalerate and cypermethrin if the parasitoids were left in contact with the pyrethroids for 1 hour. However, no recovery was found in susceptible F11 progeny.     

Effect of mixed infection on TB dynamics

Poor living conditions,overcrowding and strain diversity are some of the factors that influence mixed infection in Tuberculosis (TB) at the population level.We formulate a mathematical model for mixed infection in TB using nonlinear ordinary differential equa-tions where such factors were represented as probabilities of acquiring mixed infection.A qualitative analysis of the model shows that it exhibits multiple endemic equilibria and backward bifurcation for certain parameter values.The reactivation rate and trans-mission rate of individuals with mixed infection were of importance as well as the prob-abilities for latent individuals to acquire mixed infection.We calculate the prevalence of mixed infection from the model and the effect of mixed infection on TB incidence,TB prevalence and Mycobacterium tuberculosis (MTB) infection rate.Numerical simu-lations show that mixed infection may explain high TB incidences in areas which have a high strain diversity,poor living conditions and are overcrowded even without HIV.     

Comparison of bednets treated with alphacypermethrin, permethrin or lambdacyhalothrin against Anopheles gambiae in the Gambia

In the Gambian village of Saruja, where malaria is transmitted mainly by mosquitoes of the Anopheles gambiae complex, a trial was undertaken of the acceptability and efficacy of bednets treated with one of three pyrethroid insecticides – alphacypermethrin 40 mg/m², permethrin 500 mg/m² and lambdacyhalothrin 10 mg/m^2. Fewer mosquitoes were found alive under nets treated with insecticide than under control nets. Significantly more dead mosquitoes were found under nets treated with alphacypermethrin than under nets treated with permethrin or lambdacyhalothrin. Side-effects were reported by a proportion of the users of nets treated with each of the insecticides, but none were severe and their prevalence was similar between treatment groups. Unwashed nets treated with alphacypermethrin were more effective at killing anopheline mosquitoes in bioassays than nets treated with permethrin or lambdacyhalothrin. Killing activity was reduced when nets were washed, irrespective of which insecticide was used. Bednets treated with alphacypermethrin are well accepted, effectively killed anopheline mosquitoes and should therefore be evaluated for personal protection against malaria transmission.     

Village trial of bednets impregnated with wash-resistant permethrin compared with other pyrethroid formulations

Abstract. A village-scale field trial of pyrethroid-impregnated mosquito nets was undertaken in The Gambia, West Africa, in the Mandinka village of Saruja (13^o13'N, 14^o55'W) during July-November 1989. Nearly all the villagers possessed and used their own bednets. Anopheles gambiae is the main vector of human malaria in the area.
An experimental wash-resistant formulation of permethrin was compared with standard emulsifiable concentrate (EC) formulations of permethrin and lambda-cyhalothrin, versus placebo-treated bednets. Target concentrations of pyrethroids on bednets were permethrin 500mg/m² and lambda-cyhalothrin 25 mg/m². The experimental design involved random allocation of a treatment to one net per family. Whereas 68% of people questioned said they washed their nets fortnightly, observations during the 16-week trial period showed that only 4/130 (3%) of nets involved in the trial had been washed as frequently as once per month.
Early morning searches for mosquitoes under bednets (1 day/week for 16 weeks) found significantly more mosquitoes (60% An. gambiae ) in placebo-treated nets than in pyrethroid-treated nets. The numbers found with each of the three pyrethroid treatments did not differ significantly from each other. Insecticidal efficacy of the treatments was tested by bioassays using wild-caught unfed mosquitoes exposed to netting for 3min. Linear regression analysis of bioassay mortality against number of times that a net had been washed by villagers showed that nets impregnated with the wash-resistant permethrin retained their insecticidal properties better than nets impregnated with lambda-cyhalothrin or with the standard permethrin formulation.     

Dosage differential effects of permethrin impregnated into bednets on pyrethroid resistant and susceptible genotypes of the mosquito Anopheles stephensi

Abstract Effects of bednets impregnated with permethrin 200 mg and 500mg/m² on pyrethroid resistant and susceptible strains of Anopheles stephensi and their F hybrid progeny were studied, using free-flying female mosquitoes of these three genotypes, in a room with a human subject under a polyester net, having one of his arms in contact with the treated netting. Unexpectedly an apparently higher feeding rate, but lower knockdown and mortality rates, of mosquitoes were obtained for each of the three genotypes with the higher concentration of 500mg/m² compared with the lower dose of 200mg/m². At the lower dose there was 100% mortality 24 h after exposure of all three genotypes, suggesting that there would not be selection for resistance at this dose. However, at the higher dose there was significantly higher mortality of the susceptible strain than of the F hybrids, suggesting incomplete recessiveness of this resistance and that there would therefore be effective selection for resistance by this dose.
When female mosquitoes were confined in bioassay cones on treated netting, the resistant strain of An.stephensi showed significantly less irritability (scored as the time until first flight take-off) in response to each dose, as compared with the susceptible strain and F, hybrids. The higher dose provoked more irritation of each genotype; this could explain the greater knockdown and mortality rates of mosquitoes exposed to the lower dose which was less irritating and hence more effectively insecticidal. Thus a dose of 200mg/m² is preferable to 500mg/m² for malaria vector control.     

Monitoring pyrethroid resistance in field collected Blattella germanica Linn. (Dictyoptera: Blattellidae) in Indonesia

The German cockroach, Blattella germanica , is a major and the most common pest in public areas in Indonesia. Although intensive control measures have been carried out to control the populations of this pest, results have been far from successful, which is believed to be because of its resistance to insecticides. A standard World Health Organization (WHO) glass jar test was carried out to determine the resistance level of this insect to pyrethroid insecticides, the most commonly used insecticides for cockroach control in Indonesia. A susceptible S1 strain collected from Tembagapura Papua was compared with four strains collected from Bandung, West Java: strain S2, from a local restaurant; strain S3, from the Bandung train station; and strains S4 and S5, from two different hotels. All strains showed low resistance to the pyrethroid, except the S5 strain, which had a Resistance Ratio (RR)₅₀ of 95 for permethrin. The addition of piperonyl butoxide (PBO) suggests that the detoxifying enzyme mixed function oxidases (MFO) played an important role in the development of resistance to permethrin in the S5 strain, suggested by the high Synergist Ratio (SR) of 70.4. However, the low level of resistance to cypermethrin was not affected by PBO, suggesting that other mechanisms of pyrethroid resistance are involved. Our study is the first report of German cockroach resistance to permethrin in Indonesia, and the findings can be used in formulating potential strategies for cockroach resistance management.     

Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence for larval selection

Abstract.  Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l . (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT₅₀ of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT₅₀ did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis . They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages.     

Comparative repellency of commercial formulations of deet, permethrin and citronellal against the mosquito Aedes aegypti, using a collagen membrane technique compared with human arm tests

A collagen membrane technique, based on the membrane blood-feeding system of Cosgrove et al . (1994) , was used to compare repellents against Aedes aegypti mosquitoes. Repellency was defined in terms of inhibition of probing (ED₅₀ and ED₉₀) after 5 min exposure. A direct comparison was made with repellency from probing after 5 min on five male volunteers.
Four repellent products were compared with technical DEET as the standard. The liquid formulations tested were: Autan^® (20% deet; Bayer); Repel Plus^® (20% deet plus 0.05% permethrin; Boots); permethrin (Zeneca) and citronellal (Sigma) .
Membrane and arm tests gave similar results. Deet formulations required less active compound than citronellal for the same degree of repellency. Pure deet and Autan gave similar results, dose for dose. Permethrin was highly repellent at very low doses, but Repel Plus did not enhance the immediate repellency of deet. A technique using the same membrane system was developed to evaluate persistence of Autan, which declined to 75% after 1 h against Ae. aegypti , and to about 50% after 2–4 h.     

Insecticide susceptible/resistance status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand during 2003-2005

Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide. Almost all Ae. aegypti collected were incipient resistant or resistant to deltamethrin and permethrin, except those from some areas of Songkhla (southern) and Phan district of Chiang Rai (northern) province. Susceptibility to fenitrothion was found in mosquitoes from Bangkok (central), Chonburi (eastern), Chiang Rai, Kanchanaburi (western), and Songkhla, whereas they were resistant in almost all areas of Nakhon Sawan (north central) and Nakhon Ratchasima (northeastern) provinces. Most of Ae. aegypti were susceptible to propoxur except those from Mae Wong, Nakhon Sawan province. Various levels of insecticide resistance and susceptibility in adjacent areas revealed a focal susceptible/resistance profile in the country. It could be noted that almost all of Ae. albopictus were susceptible to the insecticides tested at the same diagnostic doses. In conclusion, resistance to pyrethroids (permethrin and deltamethrin) has developed in Ae. aegypti in most of the collected areas, suggesting that an alternative choice of insecticide or other control measures should be applied.     

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance,recovery, and death

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.     

First report of L1014F kdr mutation in Culex quinquefasciatus in Mexico

The L1014F mutation in the voltage‐sodium channel gene has been associated with resistance to DDT and pyrethroids in various arthropod species including mosquitoes. We determined the frequency of the L1014F kdr mutation in 16 field populations of Culex quinquefasciatus from Northeastern Mexico collected between 2008 and 2013. The L1014F was present in all populations analyzed with the lowest frequency (3.33%) corresponding to the population from Monclova collected in 2012, and the highest frequency (63.63%) from the Monterrey population collected in 2012. The presence of a kdr mutation in populations of Cx. quinquefasciatus from northeastern Mexico provides evidence of pyrethroid resistance. This requires a special attention, considering that pyrethroid‐based insecticides are commonly used in vector‐control campaigns, especially against Aedes aegypti (L.).