Kanutcharee Thanispong

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

Challenges and prospects for dengue and malaria control in Thailand, Southeast Asia

Despite significant advances in the search for potential dengue vaccines and new therapeutic schemes for malaria, the control of these diseases remains difficult. In Thailand, malaria incidence is falling whereas that of dengue is rising, with an increase in the proportion of reported severe cases. In the absence of antiviral therapeutic options for acute dengue, appropriate case management reduces mortality. However, the interruption of transmission still relies on vector control measures that are currently insufficient to curtail the cycle of epidemics. Drug resistance in malaria parasites is increasing, compromising malaria control and elimination. Deficiencies in our knowledge of vector biology and vectorial capacity also hinder public health efforts for vector control. Challenges to dengue and malaria control are discussed, and research priorities identified.

Irritancy and Repellency Behavioral Responses of Three Strains of Aedes aegypti Exposed to DDT and α-Cypermethrin

ABSTRACT This study quantified both contact irritancy and noncontact repellency behavioral responses of three strains of Aedes aegypti (L.) (one long-term colony and two F1–F2 generation field-caught strains) to field application rates of DDT (2g/m2) and &agr;-Cypermethrin (ACyp) (0.025 g/m2) by using an excito-repellency test chamber. The colony The colony strain (USDA) was completely susceptible to DDT and ACyp. One field strain was collected from Chiang Mai (CM) Province, northern Thailand, and was characterized as tolerant (reduced susceptibility) to DDT and completely susceptible to ACyp. The second field strain, collected from Kanchanaburi (KAN) Province, western Thailand, was highly resistant to DDT but fully susceptible to ACyp. All three strains exhibited marked irritancy to contact with ACyp, with more pronounced escape responses occurring in the two field strains. With DDT, the KAN strain demonstrated the lowest escape response during both contact and noncontact trials, whereas a greater response was seen in trials conducted with CM and USDA strains. With exposure to ACyp, repellency was less profound than irritancy but still resulted in a significant escape response compared with paired controls without insecticide (P < 0.05). DDT elicited both irritancy and repellency responses but comparably greater spatial repellency than ACyp. Findings indicate ACyp functions primarily as a strong contact irritant, whereas DDT functions as a relatively strong noncontact repellent in the strains tested. The higher the degree of physiological resistance to DDT, the greater the apparent suppression of both behavioral avoidance responses. Most importantly, observations using susceptible, tolerant, and resistant Ae. aegypti strains show that behavioral responses that can interrupt human-vector contact still occur regardless of degree of physiological susceptibility to compounds tested.

A High Throughput Screening System for Determining the Three Actions of Insecticides Against Aedes aegypti (Diptera: Culicidae) Populations in Thailand

ABSTRACT Chemicals can protect humans from the bites of hemophagous arthropods through three different primary actions: irritancy (excitation), repellency, or toxicity, actions that can be evaluated using a laboratory-based assay system, In this study, the deterrent and toxic actions of three synthetic pyrethroids and DDT were characterized on six field strains of Aedes aegypti from Thailand under laboratory-controlled conditions using the high throughput screening system. All six strains showed significant contact irritant responses to the three synthetic pyrethroids, but significantly weaker irritant responses to DDT, Marked repellency responses were seen in all six Ae. aegypti test strains exposed to DDT, whereas the synthetic pyrethroids resulted in greater toxicity than DDT under similar test conditions. Although significantly different in actions, irritancy and repellency may reflect and be influenced by the background insecticide susceptibility status of a particular mosquito population. Results from this study can be used to guide decision making regarding more effective Ae. aegypti adult control in Thailand.

Discriminating Lethal Concentrations and Efficacy of Six Pyrethroids for Control of Aedes aegypti in Thailand

Abstract Establishing baseline insecticide discriminating doses is crucial in accurately determining susceptibility status and changing temporal patterns of physiological response in mosquito populations. Pyrethroids are the predominant chemicals used for controlling adult Aedes aegypti and Ae. albopictus, both vectors of dengue viruses, in Thailand. Presently, only 2 pyrethroids, permethrin and λ-cyhalothrin, have published diagnostic dose rates for monitoring Ae. aegypti. This study established the diagnostic lethal concentrations for 6 different pyrethroids available in Thailand for dengue vector control. United States Department of Agriculture insecticide-susceptible strain of Ae. aegypti was used to establish the baseline concentrations for subsequent susceptibility testing of field populations. Our findings showed lower discriminating concentrations for λ-cyhalothrin and permethrin than those recommended by the World Health Organization (WHO), at 2.5- and 1.7-fold lower dosing, respectively. The susceptibility status of 3 different geographical populations of field-collected Ae. aegypti were tested using the standard WHO procedures. All 3 field strains demonstrated varying levels of physiological resistance to each compound. We conclude that establishing the baseline diagnostic concentration of an insecticide is of paramount importance in accurately determining the susceptibility status in field-collected mosquitoes. If possible, discriminating doses should be established for all insecticides and test assays run concurrently with a known susceptible strain for more accurate monitoring of resistance in mosquito populations in Thailand.

Comparative Behavioral Responses of Pyrethroid—Susceptible and—Resistant Aedes aegypti (Diptera: Culicidae) Populations to Citronella and Eucalyptus Oils

ABSTRACT The objective of this study was to compare the behavioral responses (contact irritancy and noncontact spatial repellency) between susceptible and resistant populations of Aedes aegypti (L.) (= Stegomyia aegypti) to essential oils, citronella, and eucalyptus, Eucalyptus globulus, extracts, using an excito-repellency test system. N, N-diethyl-meta-toluamide (DEET) was used as the standard reference repellent. Mosquitoes included two long-standing insecticide susceptible colonies (U.S. Department of Agriculture and Bora Bora) and two pyrethroid-resistant populations recently obtained from Phetchabun and Kanchanaburi provinces in Thailand. Both DEET and citronella produced a much stronger excitation (“irritancy”) and more rapid flight escape response in both pyrethroid-resistant populations compared with the laboratory populations. Noncontact repellency was also greater in the two resistant populations. Eucalyptus oil was found to be the least effective compound tested. Differences in responses between long-established pyrethroid-susceptible colonies and newly established and naturally resistant colonies were clearly demonstrated. These findings also demonstrate the need for further comparisons using natural pyrethroid-susceptible populations for elucidation of factors that might contribute to different patterns of escape behavior.

Establishment of Diagnostic Doses of Five Pyrethroids for Monitoring Physiological Resistance in Aedes Albopictus in Thailand

Monitoring insecticide resistance of Aedes albopictus is required for implementing effective dengue and chikungunya vector control in Thailand. The World Health Organization standard susceptibility test for adult mosquitoes was used to determine the baseline susceptibility of a pyrethroid-susceptible laboratory strain of Ae. albopictus to 5 different pyrethroids (deltamethrin, permethrin, bifenthrin, cypermethrin, and α-cypermethrin). Subsequently, the respective established diagnostic doses (0.026% deltamethrin, 1.024% permethrin, 0.570% bifenthrin, 0.237% cypermethrin, and 0.035% α-cypermethrin) were used to test field-collected Ae. albopictus from Rayong, Koh Chang, and Pong Nom Ron. As expected, the laboratory strain was completely susceptible to all pyrethroid insecticides at the established concentrations. Rayong mosquitoes were found to be highly susceptible to bifenthrin, cypermethrin, and α-cypermethrin. Koh Chang mosquitoes were susceptible to only deltamethrin and permethrin. Pong Nom Ron mosquitoes were resistant to all pyrethroids tested. Routine assessment of these baseline results should guide future resistance monitoring to pyrethroid insecticides in Ae. albopictus in Thailand.

Enhanced mortality in deltamethrin-resistant Aedes aegypti in Thailand using a piperonyl butoxide synergist

Aedes aegypti is the primary vector of dengue viruses in Thailand. Control of this mosquito continues to rely heavily on use of insecticides in various forms and applications. The synergistic effect of piperonyl butoxide (PBO), combined with deltamethrin against eight populations of Ae. aegypti collected from different regions in Thailand is presented. The standard WHO adult contact bioassays found all populations with low to moderate levels of resistance to deltamethrin alone (using a 0.05% discriminating concentration), with final mortalities ranging from 15.6 to 70%, while a laboratory strain was fully susceptible (100% mortality). Pre-exposure of female mosquitoes to 4% PBO for 1 h, followed immediately by exposure to deltamethrin for 1 h, significantly increased mortality in seven populations (64.8-98.1%) with the exception of mosquitoes derived from Lampang Province. The knockdown time (KDT) synergist ratios between deltamethrin only and PBO + deltamethrin ranged from 1.7 to 2.8 for KDT50 and 1.9 to 4.0 for KDT95. Between deltamethrin alone and mosquitoes exposed to PBO + deltamethrin, all resistant populations produced significant differences (P < 0.05) in final 24-h mortality, except marginally for Lampang (P = 0.053). The synergistic effects of PBO with deltamethrin-resistant Ae. aegypti suggest a combination of this synergist with deltamethrin or other pyrethroid compounds can significantly enhance the effectiveness of these insecticides against pyrethroid-resistant Ae. aegypti found commonly in Thailand.

Plasmodium Infections in Anopheles Mosquitoes in Ubon Ratchathani Province, Northeastern Thailand During a Malaria Outbreak

ABSTRACT An unprecedented malaria outbreak occurred in Ubon Ratchathani Province, northeastern Thailand, in 2014. The province showed the highest number of malaria cases of all Thai provinces. Five entomological surveys were conducted at 8 sentinel sites from September 2013 to September 2015 to address the role of different Anopheles species in malaria transmission. Mosquito collections were conducted using human landing catches and cow bait. A total of 10,369 Anopheles mosquitoes were collected and 2,240 were morphologically identified as potential malaria vectors, including An. dirus (n = 78), An. minimus (n = 18), An. sawadwongporni (n = 4), An. barbirostris s.l. (n = 819), An. philippinensis (n = 612), An. nivipes (n = 676), An. annularis (n = 42), An. aconitus (n = 7), and An. rampae (n = 142). Real-time polymerase chain reaction was used to screen for the presence of Plasmodium spp. in salivary glands. The proportion of primary vectors of surveyed villages was very low (<1%), and no Plasmodium-infected specimens were detected among in the 2,240 Anopheles mosquitoes tested. The absence of positive Plasmodium samples during malaria outbreaks suggests that malaria transmission most likely occurred outside the villages, particularly in the deep-forested hilly areas that provided suitable habitats for competent malaria vectors. These results emphasize the need to develop vector control related to village community activities to reduce malaria transmission along Thailand border areas.

Optimal Discriminating Concentrations of Six Synthetic Pyrethroids for Monitoring Insecticide Susceptibility in Anopheles minimus (Diptera: Culicidae), a Primary Malaria Vector in Thailand

Abstract Malaria is a serious mosquito-borne disease transmitted to humans by Anopheles mosquitoes. Seven species of Anopheles are important malaria vectors in Thailand and all remain susceptible to pyrethroid insecticides, despite decades of use in public health mosquito control programs. However, for a more refined and accurate approach to temporal monitoring of susceptibility patterns to commonly used pyrethroids to control adult Anopheles mosquitoes, it is preferred to have specific concentrations for more discriminating testing. A laboratory strain of Anopheles minimus Theobald was used to establish baseline concentrations for susceptibility to six different synthetic pyrethroids—deltamethrin, permethrin, bifenthrin, cypermethrin, α-cypermethrin, and λ-cyhalothrin using the World Health Organization (WHO) adult bioassay system. Final discriminating concentrations of each active ingredient were determined based on doubling LC99 for each chemical to arrive at the following percent concentrations: deltamethrin (0.009%), permethrin (0.551%), bifenthrin (0.12%), cypermethrin (0.104%), α-cypermethrin (0.034%), and λ-cyhalothrin (0.019%). For permethrin, deltamethrin, α-cypermethrin, and λ-cyhalothrin, all revised percentages were lower than currently recommended discriminating concentrations by WHO. Using the newly derived concentrations, insecticide susceptibility tests were followed in a field population of An. minimus s.l. from Kanchanaburi Province. Deltamethrin, bifenthrin, and λ-cyhalothrin produced 100% kill, whereas permethrin, cypermethrin, and α-cypermethrin gave between 92.9 and 97.7% mortality, indicating lower sensitivity (tolerance). Routine monitoring of insecticide susceptibility in An. minimus wild populations throughout Thailand should continue to ensure that insecticides in use are effective to control this vector species.