Pyrethroid induced behavioral responses of Anopheles dirus, a vector of malaria in Thailand

Contact and noncontact behavioral actions of wild-caught Anopheles dirus in response to the operational field dose of three synthetic pyrethroids (bifenthrin, α-cypermethrin and λ-cyhalothrin) were evaluated using an exito-repellency test chamber. DEET was used as the repellency standard for comparison with the other three synthetic pyrethroids. Results showed that test specimens rapidly escaped from the test chamber when exposed to direct contact with a surface treated with each of the three synthetic pyrethroids and DEET. Alpha-cypermethrin demonstrated the strongest irritant action (84.9% escape), followed by DEET (77.0%), λ-cyhalothrin (68.6%) and bifenthrin (68.3%). In the noncontact configuration, fewer mosquitoes escaped from the test chambers as compared to contact trials, although a significant escape response was still observed as compared to the controls (P<0.05). We conclude that An. dirus exhibits both irritant and repellent actions in response the three pyrethroids testing in this study. The information obtained will allow us to better understand the behavioral responses of vectors to various chemicals and provide guidance when designing control strategies for targeting specific disease vectors.     

Pyrethroid susceptibility and behavioral avoidance in Anopheles epiroticus,a malaria vector in Thailand

The physiological susceptibility to insecticides and the behavioral responses of four wild‐caught populations of female Anopheles epiroticus to synthetic pyrethroids (deltamethrin, permethrin, and alpha‐cypermethrin) were assessed. Test populations were collected from different localities along the eastern coast, Trat (TR), Songkhla (SK), and Surat Thani (ST) and one population from the western coast, Phang Nga (PN). Results showed that all four populations of An. epiroticus were susceptible to all three synthetic pyrethroids tested. Behavioral responses to test compounds were characterized for all four populations using an excito‐repellency test system. TR displayed the strongest contact excitation (‘irritancy’) escape response (76.8% exposed to deltamethrin, 74.1% permethrin, and 78.4% alpha‐cypermethrin), followed by the PN population (24.4% deltamethrin, 35% permethrin, and 34.4% for alpha‐cypermethrin) by rapidly escaping test chambers after direct contact with surfaces treated with each active ingredient compared with match‐paired untreated controls. Moderate non‐contact repellency responses to all three compounds were observed in the TR population but were comparatively weaker than paired contact tests. Few mosquitoes from the SK and ST populations escaped from test chambers, regardless of insecticide tested or type of trial. We conclude that contact excitation was a major behavioral response in two populations of An. epiroticus, whereas two other populations showed virtually no escape response following exposure to the three pyrethroids. The explanation for these large unexpected differences in avoidance responses between pyrethroid‐susceptible populations of the same species is unclear and warrants further investigation.     

Behavioral responses of Aedes aegypti,Aedes albopictus,Culex quinquefasciatus,and Anopheles minimus against various synthetic and natural repellent compounds

The behavioral responses of colony populations of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus to four essential oils (citronella, hairy basil, catnip, and vetiver), two standard repellents (DEET and picaridin), and two synthetic pyrethroids (deltamethrin and permethrin) were conducted in the laboratory using an excito‐repellency test system. Results revealed that Cx. quinquefasciatus and An. minimus exhibited much stronger behavioral responses to all test compounds (65–98% escape for contact, 21.4–94.4% escape for non‐contact) compared to Ae. aegypti (3.7–72.2% escape (contact), 0–31.7% (non‐contact)) and Ae. albopictus (3.5–94.4% escape (contact), 11.2–63.7% (non‐contact)). In brief, essential oil from vetiver elicited the greatest irritant responses in Cx. quinquefasciatus (96.6%) and An. minimus (96.5%) compared to the other compounds tested. The synthetic pyrethroids caused a stronger contact irritant response (65–97.8% escape) than non‐contact repellents (0–50.8% escape for non‐contact) across all four mosquito species. Picaridin had the least effect on all mosquito species. Findings from the current study continue to support the screening of essential oils from various plant sources for protective properties against field mosquitoes.     

The effects of plant essential oils on escape response and mortality rate of Aedes aegypti and Anopheles minimus

The High Throughput Screening System (HITSS) has been applied in insecticide behavioral response studies with various mosquito species. In general, chemical or natural compounds can produce a range of insect responses: contact irritancy, spatial repellency, knock‐down, and toxicity. This study characterized these actions in essential oils derived from citronella, hairy basil, catnip, and vetiver in comparison to DEET and picaridin against Aedes aegypti and Anopheles minimus mosquito populations. Results indicated the two mosquito species exhibited significantly different (P<0.05) contact irritant escape responses between treatment and control for all tested compound concentrations, except with the minimum dose of picaridin (P>0.05) against Ae. aegypti. Spatial repellency responses were elicited in both mosquito species when exposed to all compounds, but the strength of the repellent response was dependent on compound and concentration. Data show that higher test concentrations had greatest toxic effects on both mosquito populations, but vetiver had no toxic effect on Ae. aegypti and picaridin did not elicit toxicity in either Ae. aegypti or An. minimus at any test concentration. Ultimately, this study demonstrates the ability of the HITSS assay to guide selection of effective plant essential oils for repelling, irritating, and killing mosquitoes.     

Excito-Repellent Responses between <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> Permethrin Susceptible and Resistant Mosquitoes

The behavioral responses exhibited between four permethrin-resistant field-derived populations and a long-established, permethrin susceptible colony population of Culex quinquefasciatus mosquitoes was assessed for behavioral responses to permethrin in an excito-repellency (ER) test system. After a 30-min exposure to two different insecticide concentrations (200 and 500 mg/m²) in paired contact and noncontact test designs, the initial knockdown and escape (exit) responses were recorded followed by mortality after 24-h. All five test populations rapidly escaped from the chambers treated with permethrin regardless of background insecticide susceptibility status or chemical concentration. The greatest contact escape response was seen from the colonized population with 88 % of specimens escaping the chamber within 30-min. Contact irritancy was the predominant response compared to relatively weak noncontact spatial repellency in all test populations at both low and high concentrations. At the lower concentration, the contact escape responses did not appear influenced by background susceptibility status, whereas at the higher concentration the permethrin-resistant field populations exhibited significantly reduced avoidance behavior compared to the laboratory susceptible strain.     

Trophic behavior and biting activity of the two sibling species of the Anopheles minimus complex in western Thailand.

The trophic behavior and host preference of two sibling species, Anopheles minimus s.s. (= An. minimus species A) and species C, were observed during a two-year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Anopheles minimus s.s. and species C were more prevalent during the hot and wet periods of the year. Both species demonstrated exophagic and zoophilic activities. Feeding activity of An. minimus C was unique compared to An. minimus sensu lato from other localities in Thailand. Outdoor blood feeding by An. minimus C occurred throughout the night with one distinct feeding peak immediately after sunset (1800 h), whereas indoor feeding showed two small peaks at 2000 and 2400 h. The small number of An. minimus s.s collected during this study precluded a determination of peak activity patterns. A better understanding of mosquito behavior related to host and patterns of feeding activity will facilitate and improve the efficiency of vector control operations.     

Behavioral responses of Anopheles species (Culicidae: Diptera) with varying surface exposure to pyrethroid‐treated netting in an excito‐repellency test system

Indoor Residual Spray (IRS) with insecticides has been a procedure used for decades to protect humans from biting mosquitoes and potential vectors of pathogens. The objective of this study was to determine the responses of three wild‐caught species of malaria vectors exposed to pyrethroids of three different surface coverage percents using an excito‐repellency test box. Each species was exposed to three insecticide‐treated surfaces at varying exposure levels (full coverage, 50%, and 25% of the maximum allowable by the test system) to a single standard field dose of either lambda‐cyhalothrin or alpha‐cypermethrin. Larger numbers of mosquitoes escaped the treated chambers in the direct contact test compared to the spatial repellent chambers in all three different treated surface exposures. No significant differences in the percent of escaped mosquitoes were detected in the 50% and full coverage surface coverage exposures, whereas the 25% coverage produced significantly lower avoidance responses for both compounds. This study found that varying levels of surface exposure with synthetic pyrethroids can impact the behavioral avoidance responses of Anopheles; however, it may also be possible to reduce the amount of coverage to achieve similar avoidance actions. This information may assist policy makers in designing more cost effective strategies involving residual insecticides to control mosquito vectors.     

Effects of in vivo exposure to DEET on blood feeding behavior and fecundity in Anopheles quadrimaculatus (Diptera: Culicidae)

This study determined the effects of contact with DEET on guinea pig skin on mortality, probing time, blood feeding rate, engorgement time, and fecundity responses in female Anopheles quadrimaculatus Say. Exposure, in this manner, to 10% DEET (in ethanol) for 5 min, resulted in 98% mortality in mosquitoes after 24h. The median probing time (PT(50)) required by females, when exposed to 0.1%, 1.0%, and 10% DEET, was significantly (P<0.0001) longer (12.5, 12.1, and 19.1s, respectively) than the 6.8s required by females to probe ethanol-treated skin (control). Similarly, mean blood feeding rates in populations of females exposed to 1.0% DEET for < or = 5 min (14.4%) was 6x lower (P<0.001) (85.5%) than in females exposed to ethanol-treated skin, whereas the mean engorgement time on skin treated with 1.0% DEET (66.3s) was significantly shorter (P<0.0001) than for females feeding on the control guinea pigs (105.9s). The mean number of mature o?cytes per female (fecundity) in treatment (1.0% DEET) and control mosquitoes was not significantly different. The responses to DEET observed in this study suggest that repeated exposure of female A. quadrimaculatus populations to this repellent, in laboratory bioassays, could result in confounding of toxicant and repellent effects and inaccurate estimates of DEET repellency.     

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.     

Genetic structure and gene flow of Anopheles minimus and Anopheles harrisoni in Kanchanaburi Province, Thailand

Isozyme frequencies were compared in seven field collections of Anopheles minimus complex using starch gel electrophoresis. Mosquito collections were sampled from four districts in Kanchanaburi Province where malaria is endemic. From eight enzyme systems, nine loci and seven polymorphisms were detected, indicating limited genetic differentiation among the seven collections (F(ST) = 0.061). The highest percent polymorphic loci were observed in Bong Ti Noi (BTN) Village (55.6%), whereas the least percent polymorphism was seen in Tha Kradan (TK) Village (22.2%). Comparing villages Pra Jedee (PJ) with Pu Teuy C (PTC) and Huai Khayeng (HK) with Pra Jedee (PJ), gene flow among collections varied from 3.72 to 62.25 reproductive migrants per generation. Among the seven collections, no correlation was seen between genetic and geographical distances (P > 0.05). Anopheles minimus (former species A) and Anopheles harrisoni (former species C) from Pu Teuy fit most closely in the same cluster, possibly indicating relatively recent divergence between taxa. The genetic and epidemiological ramifications of these findings are discussed.     

Population-genetic evidence for two species in Anopheles minimus in Thailand

ABSTRACT. Sympatric occurrence of homozygotes for two electro-morphs controlled by a locus for octanol dehydrogenase, and the absence of heterozygotes, at two localities, indicates two isomorphic species within the taxon Anopheles minimus Theobald in Thailand. This view is supported by significant, relative deficiences of heterozygotes at other electromorphic loci. Gene frequency data are reported for seven electro-morphic loci in An.minimus sensu lato from eleven localities: one of the newly recognized species predominated in all but one locality and the second was confined to two localities. This species pair of An.minimus s.l. was clearly distinguished from An.aconitus Donitz, An.pampanai Biittiker & Beales and An.varuna Iyengar, three species closely related to An.minimus s.l. in the series Myzomyia of Anopheles subgenus Cellia.     

Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic,metabolic and target site characterizations of three populations

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide‐treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock‐down of 50% (KDT₅₀) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT₅₀ than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of β‐esterases and oxidases, but not glutathione‐S‐transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide‐treated bednets increases.     

Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia

Species of the Anopheles fluviatilis James and Anopheles minimus complexes (Diptera: Culicidae) are difficult to distinguish morphologically. Members of the two complexes have been confused and, consequently, their distributions and roles in malaria transmission are uncertain. We identified numerous mosquitoes from China, Thailand, Vietnam, India, Nepal, Pakistan and Iran by single-strand conformation polymorphism (SSCP) and/or sequencing, and analysed the variation in the 28S D3 region of ribosomal DNA for members of the Minimus Subgroup and also the internal transcribed spacer region 2 (ITS2) for members of the An. fluviatilis complex. The D3 region is highly conserved between taxa and therefore could serve as a standard for molecular diagnosis of the subgroup members. D3 sequence, bionomics and malaria transmission data provide further evidence that An. fluviatilis S in India is conspecific with An. minimus C in South-east Asia. An. fluviatilis T has three ITS2 haplotypes (designated T1, T2 and Y) and its distribution in India, Nepal, Pakistan and Iran is confirmed. An. fluviatilis U is well defined on cytogenetic grounds in Uttar Pradesh, India, but is very close to An. fluviatilis T and the two species may hybridize in some regions. Variant ITS2 sequences suggest the possible existence of two additional taxa within the An. fluviatilis complex, one in Iran and another in India, provisionally designated An. fluviatilis forms V and X, respectively. The distributions of members of the An. fluviatilis and An. minimus complexes in south-central Asia are summarized.     

Diurnal test periods influence behavioral responses of Aedes aegypti (Diptera: Culicidae) to repellents

There is a broad understanding of the influence of environmental factors on various aspects of normal mosquito behavior. How these external factors influence responses to repellent compounds is far less clear. The objective of this study was to investigate the effect of different daytime periods combining the normal circadian activity of a laboratory colony of Aedes aegypti (L.) with behavioral responses of mosquitoes exposed to three different compounds possessing repellent properties. Using an excito-repellency test chamber with different test designs (contact irritancy + repellency and noncontact repellency), female mosquitoes were exposed to each chemical or matching blank control during four different 3-h time intervals beginning 0600 to 1800?h. Mosquitoes showed more significant avoidance responses (escape movement away from the chemical) when exposed to either DEET or hairy basil during the afternoon periods. With deltamethrin, there was no significant difference in repellent escape movement during any period of testing. Escape activity with deltamethrin was significantly greater during all diurnal periods in contact tests compared to DEET and hairy basil. From this study, it was shown that time of diurnal testing can significantly influence behavioral responses of Ae. aegypti exposed to chemical-based repellents. Therefore, the assessment of chemicals (toxins, repellents, attractants) and must carefully consider time-of-test as a potential confounding factor during evaluation and comparisons.     

Susceptibility and avoidance behavior by Culex quinquefasciatus Say to three classes of residual insecticides.

The behavioral responses ofthree colonized strains of Culex quinquefasciatus, two from recent field collections in Thailand (Nonthaburi and Mae Sot) and one from a long-established colony from the National Institute of Health (NIH), Ministry of Public Health, Thailand, were compared during and after exposure to deltamethrin (0.02 g/m2), propoxur (0.2 g/m2), and fenitrothion (0.2 g/m2) using an excito-repellency escape chamber system. We observed striking differences in behavioral response and excito-repellency between mosquito strains and test compounds. Greater escape responses were observed in the NIH strain during direct contact with deltamethrin and fenitrothion compared with the two field populations. Deltamethrin was the most irritant, followed by fenitrothion. Escape responses with propoxur were significantly delayed but increased slightly towards the end of the 30-min exposure period, more notably in the Nonthaburi strain (P < 0.05). Non-contact repellent responses were generally much weaker than irritancy, with the greatest escape response seen with NIH and Nonthaburi. Deltamethrin showed the weakest repellent response overall (< 10% escape), while propoxur again demonstrated a delayed effect (NIH and Mae Sot) before escape occurred. We conclude that irritant and repellent behavioral responses by Cx. quinquefasciatus are important components for assessing the impact of residual spraying in mosquito control programs. A better understanding of chemical properties that elicit behavioral responses in mosquitoes should be considered in formulating control strategies designed to control mosquitoes or mitigate disease transmission risk.     

Biting patterns and host preference of Anopheles epiroticus in Chang Island,Trat Province,eastern Thailand

A study of species diversity of Anopheles mosquitoes, biting patterns, and seasonal abundance of important mosquito vectors was conducted in two villages of Chang Island, Trat Province, in eastern Thailand, one located along the coast and the other in the low hills of the central interior of the island. Of 5,399 captured female anophelines, 70.25% belong to the subgenus Cellia and remaining specimens to the subgenus Anopheles. Five important putative malaria vectors were molecularly identified, including Anopheles epiroticus, Anopheles dirus, Anopheles sawadwongporni, Anopheles maculatus, and Anopheles minimus. Anopheles epiroticus was the most commonly collected species in the coastal site, whereas An. dirus was found to be most abundant in the forest‐hill site. From both locations, a greater number of mosquitoes was collected during the dry season compared to the wet. Anopheles epiroticus showed greater exophagic and zoophilic behavior with the highest blood feeding densities occurring between 18:00 and 19:00. In contrast, An. dirus demonstrated an activity peak between midnight and 01:00. We conclude that An. epiroticus and An. dirus, in coastal and inland areas, respectively, appear to be the most epidemiologically important malaria vectors on Chang Island. As no studies of vector competency specific to Chang Island have been conducted, our conclusions that these two species play a primary role in malaria transmission are based on evidence from other localities in Thailand and mainland Southeast Asia. This information serves as a basis for designing improved vector control programs that target specific species, and if integrated with other interventions could result in the elimination of malaria transmission on the island.     

Evidence of Field-Evolved Resistance to Bifenthrin in Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) Populations in Western Nebraska and Kansas

Pyrethroid insecticides have been used to control larvae or adults of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, a key pest of field corn in the United States. In response to reports of reduced efficacy of pyrethroids in WCR management programs in southwestern areas of Nebraska and Kansas the present research was designed to establish a baseline of susceptibility to the pyrethroid insecticide, bifenthrin, using susceptible laboratory populations and to compare this baseline with susceptibility of field populations. Concentration-response bioassays were performed to estimate the baseline susceptibility. From the baseline data, a diagnostic concentration (LC₉₉) was determined and used to test adults of both laboratory and field populations. Larval susceptibility was also tested using both laboratory and field populations. Significant differences were recorded in adult and larval susceptibility among WCR field and laboratory populations. The highest LC₅₀ for WCR adults was observed in populations from Keith 2 and Chase Counties, NE, with LC₅₀s of 2.2 and 1.38 μg/vial, respectively, and Finney County 1, KS, with 1.43 μg/vial, as compared to a laboratory non-diapause population (0.24 μg/vial). For larvae, significant differences between WCR field and laboratory populations were also recorded. Significant differences in mortalities at the diagnostic bifenthrin concentration (LC₉₉) were observed among WCR adult populations with western Corn Belt populations exhibiting lower susceptibility to bifenthrin, especially in southwestern Nebraska and southwestern Kansas. This study provides evidence that resistance to bifenthrin is evolving in field populations that have been exposed for multiple years to pyrethroid insecticides. Implications to sustainable rootworm management are discussed.     

Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin-susceptibility bioassay (i.e. LT50 22 min for UNS, vs. 42min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P<0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross-resistance to other pyrethroids.     

Contact Irritant Responses of Aedes aegypti Using Sublethal Concentration and Focal Application of Pyrethroid Chemicals

Background

Previous studies have demonstrated contact irritant and spatial repellent behaviors in Aedes aegypti following exposure to sublethal concentrations of chemicals. These sublethal actions are currently being evaluated in the development of a push-pull strategy for Ae. aegypti control. This study reports on mosquito escape responses after exposure to candidate chemicals for a contact irritant focused push-pull strategy using varying concentrations and focal application.

Methods

Contact irritancy (escape) behavior, knockdown and 24 hour mortality rates were quantified in populations of female Ae. aegypti under laboratory conditions and validated in the field (Thailand and Peru) using experimental huts. Evaluations were conducted using varying concentrations and treatment surface area coverage (SAC) of three pyrethroid insecticides: alphacypermethrin, lambacyhalothrin and deltamethrin.

Results

Under laboratory conditions, exposure of Ae. aegypti to alphacypermethrin using the standard field application rate (FAR) resulted in escape responses at 25% and 50% SAC that were comparable with escape responses at 100% SAC. Significant escape responses were also observed at <100% SAC using ½FAR of all test compounds. In most trials, KD and 24 hour mortality rates were higher in mosquitoes that did not escape than in those that escaped. In Thailand, field validation studies indicated an early time of exit (by four hours) and 40% increase in escape using ½FAR of alphacypermethrin at 75% SAC compared to a matched chemical-free control. In Peru, however, the maximum increase in Ae. aegypti escape from alphacypermethrin-treated huts was 11%.

Conclusions/Significance

Results presented here suggest a potential role for sublethal and focal application of contact irritant chemicals in an Ae. aegypti push-pull strategy to reduce human–vector contact inside treated homes. However, the impact of an increase in escape response on dengue virus transmission is currently unknown and will depend on rate of biting on human hosts prior to house exiting.     

Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.