Impact of a spatial repellent intervention on Anopheles kdr insecticide resistance allele in Sumba, Indonesia.

BACKGROUND: The emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters. METHODS: This study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3-5 days against permethrin 21.5 µg/ml, deltamethrin 12.5 µg/ml, and transfluthrin 10 µg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene. RESULTS: A total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type-mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus. CONCLUSION: The presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted.

Population structure and invasion history of Aedes aegypti (Diptera: Culicidae) in Southeast Asia and Australasia.

The dengue mosquito, Aedes aegypti (Linnaeus, 1762), is a highly invasive and medically significant vector of dengue, yellow fever, chikungunya and Zika viruses, whose global spread can be attributed to increased globalization in the 15th through 20th century. Records of the invasion history of Ae. aegypti across Southeast Asia are sparse and there is little knowledge regarding the invasion routes that the species exploited to gain a foothold in the Indo-Pacific. Likewise, a broad and geographically thorough investigation of Ae. aegypti population genetics in the Indo-Pacific is lacking, despite this region being highly impacted by diseases transmitted by this species. We assess 11 nuclear microsatellites and mitochondrial COI sequences, coupled with widespread sampling through the Indo-Pacific region to characterise population structure at a broad geographic scale. We also perform a comprehensive literature search to collate documentation of the first known records of Ae. aegypti at various locations in the Indo-Pacific. We revealed additional spatial population genetic structure of Ae. aegypti in Southeast Asia, the Indo-Pacific and Australasia compared with previous studies and find differentiation between multiple Queensland and Torres Strait Islands populations. We also detected additional genetic breaks within Australia, Indonesia and Malaysia. Characterising the structure of previously unexplored populations through this region enhances the understanding of the population structure of Ae. aegypti in Australasia and Southeast Asia and may assist predictions of future mosquito movement, informing control strategies as well as assessing the risk of new invasion pathways.

Human behavior determinants of exposure to Anopheles vectors of malaria in Sumba, Indonesia.

Malaria vector control interventions in Sumba, Indonesia, have not been able to eliminate malaria. Human drivers of exposure to Anopheles bites were investigated as part of a larger clinical trial evaluating the impact of a spatial repellent product on malaria incidence. Human behavioral observations (HBOs) evaluating temporal and spatial presence, sleeping behaviors, and insecticide treated net (ITN) use, were collected parallel to entomological collections-indoor and outdoor human landing catches (HLCs), and house hold surveys. Data demonstrates that mosquito access to humans, enabled by structurally open houses, is evident by the similar entomological landing rates both inside and outside households. The presence of animals inside houses was associated with increased mosquito entry-however, the number of humans present inside houses was not related to increased mosquito landing. Analyzing mosquito landing rates with human behavior data enables the spatial and temporal estimation of exposure to Anopheles bites, accounting for intervention (ITN) presence and usage. Human behavior adjusted exposure to Anopheles bites was found to be highest in the early in the evening, but continued at lower levels throughout the night. Over the night, most exposure (53%) occurred when people were indoors and not under the protection of nets (asleep or awake) followed by exposure outside (44%). Characterized gaps in protection are outdoor exposure as well as exposure indoors-when awake, and when asleep and not using ITNs. Interestingly, in the primary trial, even though there was not a significant impact of the spatial repellent on vector biting rates by themselves (16%), when factoring in human behavior, there was approximately 28% less exposure in the intervention arm than in the placebo arm. The treated arm had less human behavior adjusted bites in all spaces evaluated though there was proportionally higher exposure indoors. This analysis points to the importance of using HBOs both towards understanding gaps in protection as well as how interventions are evaluated. To mitigate ongoing transmission, understanding context specific spatial and temporal exposure based on the interactions of vectors, humans and interventions would be vital for a directed evidence-based control or elimination strategy.

Comparison of Vector Trapping Methods for Outdoor Biting Malaria Vector Surveillance in Thailand and Vietnam.

The performances of the human-baited double net trap (HDNT) and the human-baited host decoy trap (HDT) methods were compared against the outdoor human landing catch (OHLC) method in Thailand and Vietnam. Two study sites were selected in each country: a rural village and a nearby forest setting. The three outdoor trap methods were rotated nightly between three set trapping positions, in a pre-assigned Latin square design. Volunteers were rotated following the trap rotation to avoid bias. The greatest number of adult mosquitoes was collected from the forest sites in both countries, showing Anopheles minimus (s.s.) Theobald (96.54%) and Anopheles dirus (s.s.) Peyton & Harrison (25.71%) as the primary malaria vectors in Thailand and Vietnam, respectively. At the Thai forest site, OHLC collected significantly more anopheline mosquitoes per trap night than HDNT and HDT, with mean ± standard error values of 14.17 ± 4.42, 4.83 ± 1.56, and 4.44 ± 1.45, respectively, whilst HDNT and HDT were significantly less productive at 0.34 times and 0.31 times, respectively, than OHLC in capturing anopheline mosquitoes. However, there were no significant differences among the three methods of trapping malaria vectors for the village site. At the Vietnamese forest site, HDNT achieved the highest performance in collecting Anopheline mosquitoes at 1.54 times compared to OHLC, but there was no significant difference between the two traps. The results suggested HDNT could be a possible alternative trap to OHLC in this area. Although HDT was less efficient at attracting Anopheline mosquitoes, it was highly efficient at trapping culicine mosquitoes.

Impact of a spatial repellent product on Anopheles and non-Anopheles mosquitoes in Sumba, Indonesia.

BACKGROUND: The East Nusa Tenggara province, Indonesia, contributed to 5% of malaria cases nationally in 2020, with other mosquito-borne diseases, such as dengue and filariasis also being endemic. Monitoring of spatial and temporal vector species compositions and bionomic traits is an efficient method for generating evidence towards intervention strategy optimization and meeting disease elimination goals. METHODS: The impact of a spatial repellent (SR) on human biting mosquitoes was evaluated as part of a parent cluster-randomized, double-blinded, placebo-controlled trial, in Sumba, East Nusa Tenggara. A 10-month (June 2015-March 2016) baseline study was followed by a 24-month intervention period (April 2016 to April 2018)-where half the clusters were randomly assigned either a passive transfluthrin emanator or a placebo control. RESULTS: Human-landing mosquito catches documented a reduction in landing rates related to the SR. Overall, there was a 16.4% reduction (21% indoors, and 11.3% outdoors) in human biting rates (HBR) for Anopheles. For Aedes, there was a 44.3% HBR reduction indoors and a 35.6% reduction outdoors. This reduction was 38.3% indoors and 39.1% outdoors for Armigeres, and 36.0% indoors and 32.3% outdoors for Culex species. Intervention impacts on the HBRs were not significant and are attributed to large inter-household and inter cluster variation. Anopheles flavirostris, Anopheles balabacensis and Anopheles maculatus individually impacted the overall malaria infections hazard rate with statistically significance. Though there was SR-based protection against malaria for all Anopheles species (except Anopheles sundaicus), only five (Anopheles aconitus, Anopheles kochi, Anopheles tessellatus, An. maculatus and An. sundaicus) demonstrated statistical significance. The SR numerically reduced Anopheles parity rates indoors and outdoors when compared to the placebo. CONCLUSION: Evidence demonstrating that Anopheles vectors bite both indoors and outdoors indicates that currently implemented indoor-based vector control tools may not be sufficient to eliminate malaria. The documented impact of the SR intervention on Aedes, Armigeres and Culex species points to its importance in combatting other vector borne diseases. Studies to determine the impact of spatial repellents on other mosquito-borne diseases is recommended.

An inventory of human night-biting mosquitoes and their bionomics in Sumba, Indonesia.

Mosquitoes are important vectors that transmit pathogens to human and other vertebrates. Each mosquito species has specific ecological requirements and bionomic traits that impact human exposure to mosquito bites, and hence disease transmission and vector control. A study of human biting mosquitoes and their bionomic characteristics was conducted in West Sumba and Southwest Sumba Districts, Nusa Tenggara Timur Province, Indonesia from May 2015 to April 2018. Biweekly human landing catches (HLC) of night biting mosquitoes both indoors and outdoors caught a total of 73,507 mosquito specimens (59.7% non-Anopheles, 40.3% Anopheles). A minimum of 22 Culicinae species belonging to four genera (Aedes, Armigeres, Culex, Mansonia), and 13 Anophelinae species were identified. Culex quinquefasciatus was the dominant Culicinae species, Anopheles aconitus was the principal Anopheles species inland, while An. sundaicus was dominant closer to the coast. The overall human biting rate (HBR) was 10.548 bites per person per night (bpn) indoors and 10.551 bpn outdoors. Mosquitoes biting rates were slightly higher indoors for all genera with the exception of Anopheles, where biting rates were slightly higher outdoors. Diurnal and crepuscular Aedes and Armigeres demonstrated declining biting rates throughout the night while Culex and Anopheles biting rates peaked before midnight and then declined. Both anopheline and non-anopheline populations did not have a significant association with temperature (p = 0.3 and 0.88 respectively), or rainfall (p = 0.13 and 0.57 respectively). The point distribution of HBR and seasonal variables did not have a linear correlation. Data demonstrated similar mosquito-human interactions occurring outdoors and indoors and during early parts of the night implying both indoor and outdoor disease transmission potential in the area-pointing to the need for interventions in both spaces. Integrated vector analysis frameworks may enable better surveillance, monitoring and evaluation strategies for multiple diseases.

Scientific achievements and reflections after 20 years of vector biology and control research at the Pu Teuy mosquito field research station, Thailand.

Additional vector control tools are needed to supplement current strategies to achieve malaria elimination and control of Aedes-borne diseases in many settings in Thailand and the Greater Mekong Sub-region. Within the next decade, the vector control community, Kasetsart University (KU), and the Ministry of Higher Education, Science, Research and Innovation must take full advantage of these tools that combine different active ingredients with different modes of action. Pu Teuy Mosquito Field Research Station (MFRS), Department of Entomology, Faculty of Agriculture, Kasetsart University (KU), Thailand was established in 2001 and has grown into a leading facility for performing high-quality vector biology and control studies and evaluation of public health insecticides that are operationally relevant. Several onsite mosquito research platforms have been established including experimental huts, a 40-m long semi-field screening enclosure, mosquito insectary, field-laboratory, and living quarters for students and researchers. Field research and assessments ranged from 'basic' investigations on mosquito biology, taxonomy and genetics to more 'applied' studies on responses of mosquitoes to insecticides including repellency, behavioural avoidance and toxicity. In the course of two decades, 51 peer-reviewed articles have been published, and 7 masters and 16 doctoral degrees in Entomology have been awarded to national and international students. Continued support of key national stakeholders will sustain MFRS as a Greater Mekong Subregion centre of excellence and a resource for both insecticide trials and entomological research.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes.

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect's avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85-97% escape) and Anopheles minimus Theobald (97-99%) at high concentrations (2.5-5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90-100% escape) and An. minimus (83-100%). Whereas moderate contact irritancy was observed against An. dirus (40-50% escape) and Ae. aegypti (51-59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Semi-field evaluation of novel chemical lures for Aedes aegypti, Culex quinquefasciatus, and Anopheles minimus (Diptera: Culicidae) in Thailand.

BACKGROUND: Entomological surveillance is an important means of assessing the efficacy of insect vector management programs and estimating disease transmission thresholds. Among baited traps, Biogents' BG-Sentinel (BGS) trap baited with BG-Lure is considered to have the most similar outcome to, and be a possible replacement for, human-landing catches for the epidemiologically relevant monitoring of adult Aedes aegypti and Culex quinquefasciatus. In contrast to the BGS trap, the Black Hole ultraviolet (UV) light trap, which is widely used to catch nocturnal flying insects, is not baited with synthetic human odor-mimicking lures. METHODS: We evaluated the L-lactic acid-based Kasetsart University (KU)-lures nos. 1-6 as novel candidate chemical lures for the diurnal species Ae. aegypti and the nocturnal species Cx. quinquefasciatus using two commercial traps (the BGS trap and the Black Hole UV light trap) in a semi-field screen (SFS) house. Firstly, we optimized the dose of each KU-lure in an SFS house (140 m3). Secondly, six different candidate KU-lures were screened by comparing their percent attraction using a single discriminating dose (0.5 g). Finally, we evaluated the synergism of the KU-lures selected in this way with commercially available traps. RESULTS: BGS traps baited with KU-lure no. 1 exhibited the greatest percent attraction for Ae. aegypti (29.5% ± 14.3%), whereas those baited with KU-lure no. 6 most strongly attracted Cx. quinquefasciatus (33.3% ± 10.7%). Interestingly, BGS traps treated with 10 g BG-Lure did not significantly attract more Ae. aegypti or Cx. quinquefasciatus than the untreated BGS traps. CO2 at a flow rate of 250 ml/min most strongly attracted both Ae. aegypti and Cx. quinquefasciatus (42.2% ± 14.2% and 75.1% ± 16.9%, respectively). BGS and Black Hole UV light traps with KU-lure no. 6 exhibited a stronger attraction for Cx. quinquefasciatus than untreated traps, and the percent attraction did not differ between the treated traps. CONCLUSIONS: Synergistic effects of KU-lures nos. 1 and 6 with the mosquito traps were demonstrated for both the diurnal and nocturnal species in the SFS house assays. However, further studies are urgently needed for the development of species-specific lures to increase trap efficacy in the field for local vector mosquitoes in Thailand.

Time of Test Periods Influence the Behavioral Responses of Anopheles minimus and Anopheles dirus (Diptera: Culicidae) to DEET.

Information on factors influencing the behavioral responses of mosquitoes to repellents is lacking and poorly understood, especially in the Anopheles species, night-biting mosquitoes. Our goal was to investigate the impact of different time periods on circadian activity and behavioral responses of two malaria vectors, Anopheles minimus and An. dirus, to 5% DEET using an excito-repellency test system. Each mosquito species was exposed to the repellent during the daytime (06.00-18.00) and nighttime (18.00-06.00), and time of observation was further divided into four 3-h intervals. Significant escape responses were observed between daytime and nighttime for An. minimus in both noncontact and contact tests. An. dirus showed statistical differences in contact irritancy escape response, whereas no significant difference was found in noncontact repellency tests. Both mosquito species showed more significantly higher escape responses when exposed to DEET during the afternoon and late in the night. This finding indicates that the time of testing may affect the behavioral responses of mosquitoes to repellents, especially in An. minimus and An. dirus. A better understanding of nocturnally active mosquito behavioral responses spanning from dusk to dawn would assist in optimizing product development, screening, and effective evaluation.

Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018.

Dengue is hyperendemic in most Southeast Asian countries including Thailand, where all four dengue virus serotypes (DENV-1 to -4) have circulated over different periods and regions. Despite dengue cases being annually reported in all regions of Thailand, there is limited data on the relationship of epidemic DENV infection between humans and mosquitoes, and about the dynamics of DENV during outbreaks in the northeastern region. The present study was conducted in this region to investigate the molecular epidemiology of DENV and explore the relationships of DENV infection in humans and in mosquitoes during 2016-2018. A total of 292 dengue suspected patients from 11 hospitals and 902 individual mosquitoes (at patient's houses and neighboring houses) were recruited and investigated for DENV serotypes infection using PCR. A total of 103 patients and 149 individual mosquitoes were DENV -positive. Among patients, the predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%) respectively, whereas in 2017, DENV-1, -3 and -4 had similar prevalence (38%). Additionally, only 19% of DENV infections in humans and mosquitoes at surrounding houses were serotypically matched, while 81% of infections were serotypically mismatched, suggesting that mosquitoes outside the residence may be an important factor of endemic dengue transmission. Phylogenetic analyses based on envelope gene sequences showed the genotype I of both DENV-1 and DENV-4, and co-circulation of the Cosmopolitan and Asian I genotypes of DENV-2. These strains were closely related to concurrent strains in other parts of Thailand and also similar to strains in previous epidemiological profiles in Thailand and elsewhere in Southeast Asia. These findings highlight genomic data of DENV in this region and suggest that people's movement in urban environments may result in mosquitoes far away from the residential area being key determinants of DENV epidemic dynamics.

Variable residual activity of K-Othrine® PolyZone and Actellic® 300 CS in semi-field and natural conditions in the Democratic Republic of the Congo.

BACKGROUND: Indoor Residual Spray (IRS) against vector mosquitoes is a primary means for combating malaria transmission. To combat increased patterns of resistance to chemicals against mosquito vectors, alternative candidate insecticide formulations should be screened. With mortality as the primary endpoint, the persistence of residual efficacy of a polymer-enhanced pyrethroid suspension concentrate containing deltamethrin (K-Othrine® PolyZone-KOPZ) applied at 25 mg active ingredient (ai)/m2 was compared with a microencapsulated organophosphate suspension formulation of pirimiphos-methyl (Actellic® 300CS-ACS) applied at 1 g ai/m2. METHODS: Following standard spray application, periodic contact bioassays were conducted for at least 38 weeks on four types of wall surfaces (unbaked clay, baked clay, cement, and painted cement) sprayed with either KOPZ or ACS in simulated semi-field conditions. Similarly, two types of existing walls in occupied houses (painted cement and baked clay) were sprayed and examined. A colonized strain of female Anopheles arabiensis mosquitoes were exposed to treated or untreated surfaces (controls) for 30 min. For each wall surface test period, 40 treatment mosquitoes (4 cones × 10) in semi-field and 90 (9 cones × 10) in 'natural' house conditions were used per wall. 30 mosquitoes (3 cones × 10) on a matching unsprayed surface served as the control. Insecticide, wall material, and sprayed location on wall (in houses) were compared by final mortality at 24 h. RESULTS: Insecticide, wall material, and sprayed location on wall surface produced significant difference for mean final mortality over time. In semi-field conditions, KOPZ produced a 72% mean mortality over a 38-week period, while ACS gave 65% (p < 0.001). Painted cement wall performed better than other wall surfaces throughout the study period (73% mean mortality). In the two occupied houses, KOPZ provided a mean mortality of 88%, significantly higher than ACS (p < 0.001). KOPZ provided an effective residual life (≥ 80% mortality) between 7.3 and 14 weeks on experimental walls and between 18.3 and 47.2 weeks in houses, while ACS persisted between 3 and 7.6 weeks under semi-field conditions and between 7.1 and 17.3 weeks in houses. Household painted cement walls provided a longer effective residual activity compared to baked clay for both formulations. Greater mortality was recorded at the top and middle sections of sprayed wall compared to the bottom portion near the floor. CONCLUSION: KOPZ provided longer residual activity on all surfaces compared to ACS. Painted cement walls provided better residual longevity for both insecticides compared to other surfaces. Insecticides also performed better in an occupied house environment compared to semi-field constructed walls. This study illustrates the importance of collecting field-based observations to determine appropriate product active ingredient formulations and timing for recurring IRS cycles.

Transmitted Light as Attractant with Mechanical Traps for Collecting Nocturnal Mosquitoes in Urban Bangkok, Thailand.

Mosquito surveillance is the cornerstone for determining abundance, species diversity, pathogen infection rates, and temporal and spatial distribution of different life stages in an area. Various methods are available for assessing adult mosquito populations, including mechanical trap devices using different forms of attractant cues (chemical and visual) to lure mosquitoes to the trap. So-called "light traps" use various electromagnetic wavelengths to produce a variety of visible spectral colors to attract adult mosquitoes. However, this type of trapping technology has not been widely used in Thailand. This study compared the efficacy of 4 light-emitting diodes (LEDs) (blue, green, yellow, and red) and 2 fluorescent (ultraviolet [UV] and white) lights for collecting mosquitoes in urban Bangkok. Using a Latin square experimental design, 6 light traps equipped with different lights were rotated between 6 trap site locations within the Kasetsart University (KU) campus. Each location received 6 replicate collections (6 consecutive trap-nights represented 1 replicate) over 36 collection nights for a total of 216 trap-nights. Traps were operated simultaneously (1800 to 0600 h), with captured mosquitoes removed at 3-h intervals. In total, 2,387 mosquitoes consisting of 11 species across 5 genera (Aedes, Anopheles, Armigeres, Culex, and Mansonia) were captured. Collectively, Culex species represented the predominant group sampled (2,252; 94.4%). The UV light source captured 1,544 (64.7%) of the total mosquitoes collected, followed by white 389 (16.3%), with the 4 LED sources collecting between 6.8% (blue) and 1.9% (yellow). Traps equipped with UV light were clearly the most effective for capturing nocturnally active mosquito species on the KU campus.

Evaluation of Mosquito Attractant Candidates Using a High-Throughput Screening System for Aedes aegypti (L.), Culex quinquefasciatus Say. and Anopheles minimus Theobald (Diptera: Culicidae).

Several types of olfactometers have been used to evaluate mosquito responses to agents that mimic natural volatiles that repel or attract. The Y-tube olfactometer has been widely used to study repellents and attractants, while the high-throughput screening system assay has only been used to study repellents. Whether the high-throughput screening system assay is suitable for evaluating attractants is unknown. We evaluated the responses to four lactic-acid-based mixtures and two non-lactic-acid-based chemical lure candidates using the high-throughput screening system (HITSS) for three mosquito species (laboratory strains and field populations of both Aedes aegypti (L.) and Culex quinquefasciatus Say.; laboratory strain of Anopheles minimus Theobald) under laboratory-controlled conditions. HITSS assay results showed that KU-lure #1 elicited the greatest percent attraction for pyrethroid-resistant and -susceptible Ae. aegypti. KU-lure #6 elicited the strongest attractive response for pyrethroid-susceptible and -resistant Cx. quinquefasciatus and pyrethroid-susceptible An. minimus. The response to the lures from each species was independent of the pyrethroid susceptibility status (Ae. aegypti, p = 0.825; Cx. quinquefasciatus, p = 0.056). However, a significant difference in attraction to KU-lure #6 was observed between diurnal and nocturnal mosquitoes (Cx. quinquefasciatus vs. Ae. aegypti, p = 0.014; An. minimus vs. Ae. aegypti, p = 0.001). The laboratory-level HITSS assay effectively selects potential lure candidates. Because the host-seeking behavior differs between mosquito species, further studies are needed to develop species-specific attractants. Additional studies in semi-field screen houses using commercial traps are necessary to evaluate the accuracy of these laboratory assay results.

Effect of Different Wall Surface Coverage With Deltamethrin-Treated Netting on the Reduction of Indoor-Biting Anopheles Mosquitoes (Diptera: Culicidae).

Indoor residual spray with deltamethrin remains the most common tool for reducing malaria transmission in Thailand. Deltamethrin is commonly used to spray the entire inner surfaces of the walls to prevent mosquitoes from resting. This study compared the mosquito landing responses on humans inside three experimental huts treated with deltamethrin at three different extents of wall coverage (25%, 50%, and full coverage), with one clean/untreated hut serving as a control. There were no significant differences between the numbers of Anopheles mosquitoes landing in the 50% and full coverage huts, whereas, in comparison to both of these, there was a significantly greater number landing in the 25% coverage hut. This study demonstrates that varying the percent coverage of indoor surfaces with deltamethrin-treated netting influences the blood-feeding success of wild Anopheles, and our findings suggest that it may be possible to reduce the extent of insecticide surface treatment while maintaining equivalent mosquito avoidance action to that seen in fully treated structures.

Serological biomarker for assessing human exposure to Aedes mosquito bites during a randomized vector control intervention trial in northeastern Thailand.

BACKGROUND: Aedes mosquitoes are vectors for several major arboviruses of public health concern including dengue viruses. The relationships between Aedes infestation and disease transmission are complex wherein the epidemiological dynamics can be difficult to discern because of a lack of robust and sensitive indicators for predicting transmission risk. This study investigates the use of anti-Aedes saliva antibodies as a serological biomarker for Aedes mosquito bites to assess small scale variations in adult Aedes density and dengue virus (DENV) transmission risk in northeastern Thailand. Individual characteristics, behaviors/occupation and socio-demographics, climatic and epidemiological risk factors associated with human-mosquito exposure are also addressed. METHODS: The study was conducted within a randomized clustered control trial in Roi Et and Khon Kaen provinces over a consecutive 19 months period. Thirty-six (36) clusters were selected, each of ten houses. Serological and entomological surveys were conducted in all houses every four months and monthly in three sentinel households per cluster between September 2017 and April 2019 for blood spot collections and recording concurrent immature and adult Aedes indices. Additionally, the human exposure to Aedes mosquito bites (i.e., Mosquito Exposure Index or MEI) was estimated by ELISA measuring levels of human antibody response to the specific Nterm-34 kDa salivary antigen. The relationships between the MEI, vector infestation indices (adult and immature stages) and vector DENV infection were evaluated using a two-level (house and individual levels) mixed model analysis with one-month lag autoregressive correlation. RESULTS: There was a strong positive relationship between the MEI and adult Aedes (indoor and outdoor) density. Individuals from households with a medium mosquito density (mean difference: 0.091, p<0.001) and households with a high mosquito density (mean difference: 0.131, p<0.001) had higher MEI's compared to individuals from households without Aedes. On a similar trend, individuals from households with a low, medium or high indoor Aedes densities (mean difference: 0.021, p<0.007, 0.053, p<0.0001 and 0.037, p<0.0001 for low, medium and high levels of infestation, respectively) had higher MEI than individuals from houses without indoor Aedes. The MEI was driven by individual characteristics, such as gender, age and occupation/behaviors, and varied according to climatic, seasonal factors and vector control intervention (p<0.05). Nevertheless, the study did not demonstrate a clear correlation between MEI and the presence of DENV-infected Aedes. CONCLUSION: This study represents an important step toward the validation of the specific IgG response to the Aedes salivary peptide Nterm-34kDa as a proxy measure for Aedes infestation levels and human-mosquito exposure risk in a dengue endemic setting. The use of the IgG response to the Nterm-34 kDa peptide as a viable diagnostic tool for estimating dengue transmission requires further investigations and validation in other geographical and transmission settings.

Dose-Response Assay for Synthetic Mosquito (Diptera: Culicidae) Attractant Using a High-Throughput Screening System.

Natural volatile host cues play a critical role for mosquito orientation and locating a blood source for egg production. Similar olfactory activation responses have allowed the use and development of artificial chemical attractants to lure mosquitoes to trapping devices. Using a pre-formulated commercial product mixture of different attractant chemicals, a high-throughput screening system (HITSS) is used to screen varying doses of chemical required to activate behavioral responses. Two strains of Aedes aegypti (L.): permethrin-susceptible (USDA) and -resistant (Pu Teuy) phenotypes and one Culex quinquefasciatus Say. (NIH) laboratory strain were tested. Overall, mosquitoes showed repellency between 1.0 g and to 10.0 g dose of each compound. However, by progressively reducing the dose, Cx. quinquefasciatus showed a greater positive percent attraction (88.9%) at 0.025 g, whereas the USDA and Pu Teuy Ae. aegypti produced optimum attractant activation at 0.005 g (72.6% and 58.9%, respectively) without significant difference within species (p > 0.05). In parallel control assays, Cx. quinquefasciatus was significantly attracted to 1 g of dry ice (carbon dioxide) (76%) more than Ae. aegypti (USDA) (12.2%). The HITSS was originally designed to measure three chemical actions to sublethal concentrations of chemicals by mosquitoes: toxicity and the two primary behavior avoidance responses (contact excitation and spatial repellency). These findings demonstrate that the HITSS assay, with only minor modifications, allows comparison screening of candidate compounds as potential attractants for anemotactic responses under laboratory-controlled conditions. Further investigations will be required to equate measurements obtained from controlled laboratory assays to more varied field conditions for attracting natural mosquito populations.

Residual Malaria Transmission in Select Countries of Asia-Pacific Region: Old Wine in a New Barrel.

BACKGROUND: Despite substantial reductions in malaria burden and improvement in case management, malaria remains a major public health challenge in the Asia-Pacific region. Residual malaria transmission (RMT) is the fraction of total transmission that persists after achievement of full operational coverage with effective insecticide-treated bed nets (ITNs)/long-lasting insecticidal nets (LLINs) and/or indoor residual spray interventions. There is a critical need to standardize and share best practices for entomological, anthropological, and product development investigative protocols to meet the challenges of RMT and elimination goals. METHODS: A systematic review was conducted to describe when and where RMT is occurring, while specifically targeting ownership and usage of ITN/LLINs, indoor residual spray application, insecticide susceptibility of vectors, and human and vector biting behavior, with a focus on nighttime activities. RESULTS: Sixty-six publications from 1995 to present met the inclusion criteria for closer review. Associations between local vector control coverage and use with behaviors of human and mosquito vectors varied by locality and circumstance. Consequently, the magnitude of RMT is insufficiently studied and analyzed with sparse estimates of individual exposure in communities, insufficient or incomplete observations of ITN/LLIN use, and the local human population movement into and from high-risk areas. CONCLUSIONS: This review identified significant gaps or deficiencies that require urgent attention, namely, developing standardized procedures and methods to estimate risk exposure beyond the peridomestic setting, analytical approaches to measure key human-vector interactions, and seasonal location-specific agricultural or forest use calendars, and establishing the collection of longitudinal human and vector data close in time and location.

Correction: Anopheles sundaicus complex and the presence of Anopheles epiroticus in Indonesia.

[This corrects the article DOI: 10.1371/journal.pntd.0008385.].