Species abundance and density of malaria vectors in Western Thailand and implications for disease transmission.

Understanding the dynamics of malaria vectors and their interactions with environmental factors is crucial for effective malaria control. This study investigated the abundance, species composition, seasonal variations, and malaria infection status of female mosquitoes in malaria transmission and non-transmission areas in Western Thailand. Additionally, the susceptibility of malaria vectors to pyrethroid insecticides was assessed. Entomological field surveys were conducted during the hot, wet, and cold seasons in both malaria transmission areas (TA) and non-transmission areas (NTA). The abundance and species composition of malaria vectors were compared between TA and NTA. The availability of larval habitats and the impact of seasonality on vector abundance were analyzed. Infection with Plasmodium spp. in primary malaria vectors was determined using molecular techniques. Furthermore, the susceptibility of malaria vectors to pyrethroids was evaluated using the World Health Organization (WHO) susceptibility test. A total of 9799 female mosquitoes belonging to 54 species and 11 genera were collected using various trapping methods. The number of malaria vectors was significantly higher in TA compared to NTA (P < 0.001). Anopheles minimus and An. aconitus were the predominant species in TA, comprising over 50% and 30% of the total mosquitoes collected, respectively. Seasonality had a significant effect on the availability of larval habitats in both areas (P < 0.05) but did not impact the abundance of adult vectors (P > 0.05). The primary malaria vectors tested were not infected with Plasmodium spp. The WHO susceptibility test revealed high susceptibility of malaria vectors to pyrethroids, with mortality rates of 99-100% at discriminating concentrations. The higher abundance of malaria vectors in the transmission areas underscores the need for targeted control measures in these regions. The susceptibility of malaria vectors to pyrethroids suggests the continued effectiveness of this class of insecticides for vector control interventions. Other factors influencing malaria transmission risk in the study areas are discussed. These findings contribute to our understanding of malaria vectors and can inform evidence-based strategies for malaria control and elimination efforts in Western Thailand.

Inference for entomological semi-field experiments: Fitting a mathematical model assessing personal and community protection of vector-control interventions.

The effectiveness of vector-control tools is often assessed by experiments as a reduction in mosquito landings using human landing catches (HLCs). However, HLCs alone only quantify a single characteristic and therefore do not provide information on the overall impacts of the intervention product. Using data from a recent semi-field study which used time-stratified HLCs, aspiration of non-landing mosquitoes, and blood feeding, we suggest a Bayesian inference approach for fitting such data to a stochastic model. This model considers both personal protection, through a reduction in biting, and community protection, from mosquito mortality and disarming (prolonged inhibition of blood feeding). Parameter estimates are then used to predict the reduction of vectorial capacity induced by etofenpox-treated clothing, picaridin topical repellents, transfluthrin spatial repellents and metofluthrin spatial repellents, as well as combined interventions for Plasmodium falciparum malaria in Anopleles minimus. Overall, all interventions had both personal and community effects, preventing biting and killing or disarming mosquitoes. This led to large estimated reductions in the vectorial capacity, with substantial impact even at low coverage. As the interventions aged, fewer mosquitoes were killed; however the impact of some interventions changed from killing to disarming mosquitoes. Overall, this inference method allows for additional modes of action, rather than just reduction in biting, to be parameterised and highlights the tools assessed as promising malaria interventions.

The impact of insect growth regulators on adult emergence inhibition and the fitness of Aedes aegypti field populations in Thailand.

BACKGROUND: The frequent use of insecticides in vector control causes the development of insecticide resistance. Insect growth regulators (IGRs), which effect insect development, are used as a promising alternative to control resistant insect vectors. This study aimed to develop novel effective tools for Aedes aegypti control by evaluating the efficacy of different IGRs on larval development, blood feeding capacity, fecundity, and fertility in females and sperm productivity in males across geographical regions of Thailand. METHODS: The efficacy of 16 technical grade IGRs were evaluated against laboratory strain Ae. aegypti larvae in order to determine their emergence inhibition (EI) at 50% and 95% under laboratory conditions. Six IGRs were selected for fecundity, fertility, and sperm productivity studies using feed-through treatments at EI95 concentration levels against adult Ae. aegypti field strains. RESULTS: The results from larval bioassay tests indicate that juvenile hormone mimics (EI50 = 0.010-0.229 ppb; EI95 = 0.066-1.118 ppb) and chitin synthesis inhibitors affecting CHS1 (EI50 = 0.240-2.412 ppb; EI95 = 0.444-4.040 ppb) groups effectively inhibited adult Ae. aegypti emergence. Methoprene and fenoxycarb significantly reduced blood feeding capacity. Egg production was comparable among strains while methoprene, pyriproxyfen and diflubenzuron induced egg production. Egg retention was detected in females fed on diflubenzuron. Methoprene, fenoxycarb, diflubenzuron, and teflubenzuron reduced egg hatching rates in mosquito field strains compared to laboratory strain. Male mosquitoes fed on fenoxycarb showed significantly lower sperm production compared to other treatments. CONCLUSION: Juvenile hormone analogues and chitin synthesis inhibitors affecting CHS1 groups showed excellent results in adult emergence inhibition in this study. They also disrupted reproductive systems in both adult males and females. This study suggested that they can be used as an alternative larvicide in mosquito control programs.

Entomological Risk Assessment for Dengue Virus Transmission during 2016-2020 in Kamphaeng Phet, Thailand.

Individual houses with high risks of dengue virus (DENV) transmission might be a source of virus transmission within the neighborhood. We conducted an entomological risk assessment for DENV transmission at the household level, comprising family cohort members residing in the same location, to assess the risk for dengue virus transmitted by mosquito vectors. The studies were conducted in Kamphaeng Phet Province, Thailand, during 2016-2020. Entomological investigations were performed in 35 cohort families on day 1 and day 14 after receiving dengue case reports. DENV was found in 22 Aedes samples (4.9%) out of 451 tested samples. A significantly higher DENV infection rate was detected in vectors collected on day 1 (6.64%) compared to those collected on day 14 (1.82%). Annual vector surveillance was carried out in 732 houses, with 1002 traps catching 3653 Aedes females. The majority of the 13,228 water containers examined were made from plastic and clay, with used tires serving as a primary container, with 59.55% larval abundance. Larval indices, as indicators of dengue epidemics and to evaluate disease and vector control approaches, were calculated. As a result, high values of larval indices indicated the considerably high risk of dengue transmission in these communities.

Sand fly identification and screening for Leishmania spp. in six provinces of Thailand.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania spp. At least 27 species of sand flies have been recorded in Thailand. Although human leishmaniasis cases in Thailand are mainly imported, autochthonous leishmaniasis has been increasingly reported in several regions of the country since 1999. Few studies have detected Leishmania infection in wild-caught sand flies, although these studies were carried out only in those areas reporting human leishmaniasis cases. The aim of this study was therefore to identity sand fly species and to investigate Leishmania infection across six provinces of Thailand. METHODS: Species of wild-caught sand flies were initially identified based on morphological characters. However, problems identifying cryptic species complexes necessitated molecular identification using DNA barcoding in parallel with identification based on morphological characters. The wild-caught sand flies were pooled and the DNA isolated prior to the detection of Leishmania infection by a TaqMan real-time PCR assay. RESULTS: A total of 4498 sand flies (1158 males and 3340 females) were caught by trapping in six provinces in four regions of Thailand. The sand flies were morphologically classified into eight species belonging to three genera (Sergentomyia, Phlebotomus and Idiophlebotomus). Sergentomyia iyengari was found at all collection sites and was the dominant species at most of these, followed in frequency by Sergentomyia barraudi and Phlebotomus stantoni, respectively. DNA barcodes generated from 68 sand flies allowed sorting into 14 distinct species with 25 operational taxonomic units, indicating a higher diversity (by 75%) than that based on morphological identification. Twelve barcoding sequences could not be assigned to any species for which cytochrome c oxidase subunit I sequences are available. All tested sand flies were negative for Leishmania DNA. CONCLUSIONS: Our results confirm the presence of several sand fly species in different provinces of Thailand, highlighting the importance of using DNA barcoding as a tool to study sand fly species diversity. While all female sand flies tested in this study were negative for Leishmania, the circulation of Leishmania spp. in the investigated areas cannot be ruled out.

Field evaluation of four commercial light traps, trap placement, and effect of carbon dioxide on phlebotomine sand fly collection in northern Thailand.

Several light trap devices have been invented and developed to assess the abundance of sand flies. Traps available in the market have different designs and attractant combinations to catch sand fly vectors. We evaluated the efficacy of four commercial light traps and determined the effect of trap placement and carbon dioxide (CO2) on sand fly collection in northern Thailand. Trap evaluations were conducted at two natural caves located in Chiang Rai province, Thailand. In the first part of the study, the efficacies of four trap types including the Centers for Disease Control miniature light trap (CDC LT), Encephalitis Vector Survey trap (EVS), CDC Updraft Blacklight trap (CDC UB), and Laika trap (LK) were evaluated and compared using a Latin square experimental design. The second half of the study evaluated the influence of trap placement and CO2 on sand fly collection. Additionally, CDC LT were placed inside, outside, and at the entrance of caves to compare the number of sand flies collected. For the trap efficacy experiment, a total of 11,876 phlebotomine sand flies were collected over 32 trap-nights. Results demonstrated that CDC LT, CDC UB, and LK collected significantly more sand flies than EVS (P > 0.05). However, there were no significant differences between the numbers of sand flies collected by CDC LT, CDC UB, and LK. A total of 6,698 sand flies were collected from the trap placement and CO2 experiment over 72 trap-nights. Results showed that CO2 did not influence the numbers of sand flies captured (P < 0.05), whereas trap placement at the entrance of the caves resulted in collection of significantly more sand flies than traps placed inside and outside of the caves. We found the CDC LT, CDC UB, and LK without CO2 captured the greatest amount of sand flies. This was particularly observed when traps were placed at the entrance of a cave, perhaps because of the greater passage of stimuli caused by wind flow at the entrance of the cave. The light traps in this study can be used effectively to collect sand fly vectors in northern Thailand.

Too "sexy" for the field? Paired measures of laboratory and semi-field performance highlight variability in the apparent mating fitness of Aedes aegypti transgenic strains.

BACKGROUND: Evaluating and improving mating success and competitive ability of laboratory-reared transgenic mosquito strains will enhance the effectiveness of proposed disease-control strategies that involve deployment of transgenic strains. Two components of the mosquito rearing process, larval diet quantity and aquatic environment - which are linked to physiological and behavioural differences in adults - are both relatively easy to manipulate. In mosquitoes, as for many other arthropod species, the quality of the juvenile habitat is strongly associated with adult fitness characteristics, such as longevity and fecundity. However, the influence of larval conditioning on mating performance is poorly understood. Here, we investigated the combined effects of larval diet amount and environmental water source on adult male mating success in a genetically modified strain of Aedes aegypti mosquitoes in competition with wild-type conspecifics. Importantly, this research was conducted in a field setting using low generation laboratory and wild-type lines. RESULTS: By controlling larval diet (high and low) and rearing water source (field-collected and laboratory water), we generated four treatment lines of a genetically modified strain of Ae. aegypti tagged with fluorescent sperm. Laboratory reared mosquitoes were then competed against a low generation wild-type colony in a series of laboratory and semi-field mating experiments. While neither food quantity nor larval aquatic environment were found to affect male mating fitness, the transgenic lines consistently outperformed wild-types in laboratory competition assays, an advantage that was not conferred to semi-field tests. CONCLUSIONS: Using a model transgenic system, our results indicate that differences in the experimental conditions of laboratory- and field-based measures of mating success can lead to variation in the perceived performance ability of modified strains if they are only tested in certain environments. While there are many potential sources of variation between laboratory and field lines, laboratory adaptation - which may occur over relatively few generations in this species - may directly impact mating ability depending on the context in which it is measured. We suggest that colony-hybridization with field material can potentially be used to mitigate these effects in a field setting. Release programs utilising mass-produced modified laboratory strains should incorporate comparative assessments of quality in candidate lines.

A Comparative Study of Mosquito and Sand Fly (Diptera: Psychodidae: Phlebotominae) Sampling Using Dry Ice and Chemically Generated Carbon Dioxide From Three Different Prototype CO2 Generators.

A comparative study was conducted to test the efficiency of Centers for Disease Control and Prevention (CDC) light traps baited with either dry ice or carbon dioxide (CO2) produced from one of three different sources in collecting mosquitoes (Diptera: Culicidae) in Thailand. Treatments consisted of dry ice pellets, CO2 gas produced from one of three prototype CO2 generator systems (TDA, CUBE, Moustiq-Air Med-e-Cell - MEC), and a CDC light trap without a CO2 source. The best performing prototype from Thailand was then tested in collecting sand flies (Diptera: Psychodidae: Phlebotominae) in Greece. A total of 12,798 mosquitoes and 8,329 sand flies were sampled during the experimentation. The most prevalent mosquito species collected in Thailand were: Culex vishnui Theobald > Anopheles minimus Theobald > Culex tritaeniorhynchus Giles > Anopheles sawadwongporni Rattanarithikul & Green. By far the most prevalent sand fly species collected in Thessaloniki was Phlebotomus perfiliewi Parrot followed by Phlebotomus tobbi Adler and Theodor and Phlebotomus simici Nitzulescu. In general, the TDA treatment was the only treatment with no significant difference from the dry ice-treatment in mean trap catches. Although dry ice-baited traps caught higher numbers of mosquitoes and sand flies than the TDA-baited traps, there was no difference in the number of species collected. Results indicate that the traps baited with the TDA CO2 generator were as attractive as traps supplied with dry ice and, therefore, the TDA CO2 generator is a suitable alternative to dry ice as a source of carbon dioxide for use with adult mosquito and sand fly traps.

Ultra-Low Volume Application of Spinosad (Natular 2EC) Larvicide as a Residual in a Tropical Environment Against Aedes and Anopheles Species.

We investigated the efficacy of a liquid larvicide, Natular 2EC® (spinosad), applied with ultra-low volume sprayer as a residual application during the dry season in southeastern Thailand against 4 medically important species-Aedes aegypti, Ae. albopictus, Anopheles dirus, and An. minimus. We found that this larvicide could be applied as a residual to dry areas known to collect water and potentially still be effective after rains or irrigation, which could increase the flexibility and efficiency of an integrated vector management program targeting these species. This investigation also demonstrated, for the 1st time, efficacy of spinosad against An. minimus and An. dirus.

Field evaluation of two commercial mosquito traps baited with different attractants and colored lights for malaria vector surveillance in Thailand.

BACKGROUND: Sampling for adult mosquito populations is a means of evaluating the efficacy of vector control operations. The goal of this study was to evaluate and identify the most efficacious mosquito traps and combinations of attractants for malaria vector surveillance along the Thai-Myanmar border. METHODS: In the first part of the study, the BG-Sentinel™ Trap (BGS Trap) and Centers for Disease Control and Prevention miniature light trap (CDC LT) baited with different attractants (BG-lure® and CO2) were evaluated using a Latin square experimental design. The six configurations were BGS Trap with BG-lure, BGS Trap with BG-lure plus CO2, BGS Trap with CO2, CDC LT with BG-lure, CDC LT with BG lure plus CO2, and CDC LT with CO2. The second half of the study evaluated the impact of light color on malaria vector collections. Colors included the incandescent bulb, ultraviolet (UV) light-emitting diode (LED), green light stick, red light stick, green LED, and red LED. RESULTS: A total of 8638 mosquitoes consisting of 42 species were captured over 708 trap-nights. The trap types, attractants, and colored lights affected numbers of female anopheline and Anopheles minimus collected (GLM, P < 0.01). Results revealed that BGS Trap captured many anophelines but was significantly less than the CDC LT. The CDC LT, when baited with BG-lure plus CO2 captured the greatest number of anopheline females with a catch rate significantly higher than the CDC LT baited with BG-lure or CO2 alone (P < 0.05). The number of anopheline females collected from the CDC LT baited with CO2 was greater than the CDC LT baited with BG-lure (646 vs 409 females). None of the alternative lights evaluated exceeded the performance of the incandescent light bulb in terms of the numbers of anopheline and An. minimus collected. CONCLUSION: We conclude that the CDC LT augmented with an incandescent light shows high potential for malaria vector surveillance when baited with CO2 and the BG-lure in combination and can be effectively used as the new gold standard technique for collecting malaria vectors in Thailand.

Semi-Field Evaluation of Metofluthrin-Impregnated Nets on Host-Seeking Aedes aegypti and Anopheles dirus.

The efficacy of a metofluthrin-impregnated net (MIN) known as the "Mushikonazu" on the house entry behavior of female Aedes aegypti and Anopheles dirus mosquitoes was evaluated using a semi-field 50-m tunnel setup. While the MIN is labeled for the control of chironomids and moth flies, this study determined the feasibility of using the device, given its current construction and metofluthrin formulation, as a spatial repellent against mosquitoes. Sentinel and cone bioassays were used to determine the insecticidal effect of the MIN. A spatial activity index (SAI) was calculated to evaluate responses of the mosquitoes. For the spatial repellent evaluation against Ae. aegypti, the overall mean of SAI was slightly less than 0 at wk 1 after the MIN application and then decreased for the last 4 wk showing a preference to treatment tent. For An. dirus, the mean SAI at wk 1 was positive, indicating a presumed repellent effect of the MIN against An. dirus. For the subsequent 4 wk, the SAI was negative, indicating a preference for the MIN. Results suggested that the MIN may not be a promising approach to repel Ae. aegypti and An. dirus under field conditions in Thailand. However, it remains probable that the MIN may be effective as a spatial repellent if modifications are made to the metofluthrin concentration or formulation and/or the construction of the device.

No evidence for local adaptation of dengue viruses to mosquito vector populations in Thailand.

Despite their epidemiological importance, the evolutionary forces that shape the spatial structure of dengue virus genetic diversity are not fully understood. Fine-scale genetic structure of mosquito vector populations and evidence for genotype × genotype interactions between dengue viruses and their mosquito vectors are consistent with the hypothesis that the geographical distribution of dengue virus genetic diversity may reflect viral adaptation to local mosquito populations. To test this hypothesis, we measured vector competence in all sympatric and allopatric combinations of 14 low-passage dengue virus isolates and two wild-type populations of Aedes aegypti mosquitoes sampled in Bangkok and Kamphaeng Phet, two sites located about 300 km apart in Thailand. Despite significant genotype × genotype interactions, we found no evidence for superior vector competence in sympatric versus allopatric vector-virus combinations. Viral phylogenetic analysis revealed no geographical clustering of the 14 isolates, suggesting that high levels of viral migration (gene flow) in Thailand may counteract spatially heterogeneous natural selection. We conclude that it is unlikely that vector-mediated selection is a major driver of dengue virus adaptive evolution at the regional scale that we examined. Dengue virus local adaptation to mosquito vector populations could happen, however, in places or times that we did not test, or at a different geographical scale.

Differential susceptibility of two field aedes aegypti populations to a low infectious dose of dengue virus.

BACKGROUND: The infectious dose required to infect mosquito vectors when they take a blood meal from a viremic person is a critical parameter underlying the probability of dengue virus (DENV) transmission. Because experimental vector competence studies typically examine the proportion of mosquitoes that become infected at intermediate or high DENV infectious doses in the blood meal, the minimum blood meal titer required to infect mosquitoes is poorly documented. Understanding the factors influencing the lower infectiousness threshold is epidemiologically significant because it determines the transmission potential of humans with a low DENV viremia, possibly including inapparent infections, and during the onset and resolution of the viremic period of acutely infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: We compared the susceptibility of two field-derived Aedes aegypti populations from Kamphaeng Phet, Thailand when they were orally exposed to low titers of six DENV-2 isolates derived from the serum of naturally infected humans living in the same region. The infectious dose, time-point post-blood feeding, viral isolate and mosquito population, were significant predictors of the proportion of mosquitoes that became infected. Importantly, the dose-response profile differed significantly between the two Ae. aegypti populations. Although both mosquito populations had a similar 50% oral infectious dose (OID50), the slope of the dose-response was shallower in one population, resulting in a markedly higher susceptibility at low blood meal titers. CONCLUSIONS/SIGNIFICANCE: Our results indicate that mosquitoes in nature vary in their infectious dose-response to DENV. Thus, different mosquito populations have a differential ability to acquire DENV infection at low viremia levels. Future studies on human-to-mosquito DENV transmission should not be limited to OID50 values, but rather they should be expanded to account for the shape of the dose-response profile across a range of virus titers.

Development and evaluation of a pyriproxyfen-treated device to control the dengue vector, Aedes aegypti (L.) (Diptera:Culicidae).

The resurgence of dengue fever and the chikungunya epidemic make the control of Aedes aegypti mosquitoes, the vectors of these diseases, critically important. We developed and evaluated an Ae. aegypti control device that is visually-attractive to mosquitoes. This pyriproxyfen-treated device was evaluated for its impact on Ae. aegypti egg production and population dynamics in dengue-endemic areas in Thailand. The device consists of a "high rise" shaped ovitrap/ resting station covered with black cotton cloth. The device is easily collapsible and transportable. Ae. aegypti are generally drawn towards darker, shadier areas making this device physically attractive as a resting station to mosquitoes of all physiological stages. The results show this device suppressed Ae. aegypti populations after it was introduced into a village. The observed effect was primarily the result of the Ae. aegypti exposure to pyriproxyfen shortly after adult emergence or after taking a blood meal resulting in decreased egg production. We believe the device may be further improved physically and the formulation should be replaced to provide even better efficacy for controlling Ae. aegypti mosquito, populations.

Dengue-1 virus clade replacement in Thailand associated with enhanced mosquito transmission.

Dengue viruses (DENV) are characterized by extensive genetic diversity and can be organized in multiple, genetically distinct lineages that arise and die out on a regular basis in regions where dengue is endemic. A fundamental question for understanding DENV evolution is the relative extent to which stochastic processes (genetic drift) and natural selection acting on fitness differences among lineages contribute to lineage diversity and turnover. Here, we used a set of recently collected and archived low-passage DENV-1 isolates from Thailand to examine the role of mosquito vector-virus interactions in DENV evolution. By comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to be transmitted by a present-day Aedes aegypti population from Thailand, we found that a major clade replacement event in the mid-1990s was associated with virus isolates exhibiting increased titers in the vector's hemocoel, which is predicted to result in a higher probability of transmission. This finding is consistent with the hypothesis that selection for enhanced transmission by mosquitoes is a possible mechanism underlying major DENV clade replacement events. There was significant variation in transmission potential among isolates within each clade, indicating that in addition to vector-driven selection, other evolutionary forces act to maintain viral genetic diversity. We conclude that occasional adaptive processes involving the mosquito vector can drive major DENV lineage replacement events.