Sleeping on the floor decreases insecticide treated bed net use and increases risk of malaria in children under 5 years of age in Mbita District, Kenya.

Children who sleep on the floor are less likely to use long-lasting insecticidal nets (LLINs); however, the relationship between sleeping location and Plasmodium falciparum infection has not been investigated sufficiently. This study revealed whether sleeping location (bed vs floor) is associated with P. falciparum infection among children 7-59 months old. More than 60% of children slept on the floor. Younger children were significantly more likely to sleep in beds [odds ratio, OR 2.31 (95% confidence interval (CI) 2.02-2.67)]. Nearly 70% of children slept under LLINs the previous night. LLIN use among children who slept on the floor was significantly less than ones sleeping in beds [OR 0.49 (95% CI 0.35-0.68)]. The polymerase chain reaction (PCR) based P. falciparum infection rate and slide based infection rate were 65.2 and 29.7%, respectively. Both infections were significantly higher among children slept on the floor [OR1.51 (95% CI 1.08-2.10) for PCR base, OR 1.62 (95% CI 1.14-2.30) for slide base] while net availability was not significant. Sleeping location was also significant for slide based infection with fever (⩾ 37.5 °C) [2.03 (95% CI 1.14-3.84)] and high parasitemia cases (parasite ⩾ 2500 µL(-1)) [2.07 (95% CI 1.03-4.50)]. The results suggest that sleeping location has a direct bearing on the effectiveness of LLINs.

Reconsideration of importance of the point mutation L982W in the voltage-sensitive sodium channel of the pyrethroid resistant Aedes aegypti (L.)(Diptera: Culicidae) in Vietnam.

Pyrethroid resistance in Aedes aegypti is widespread in southern Vietnam because the photostable 2nd generation pyrethroids have been used in large amounts over extensive areas for malaria and dengue vector control. In our previous report in 2009, F1534C, one of the point mutations in the voltage-sensitive sodium channel (VSSC) in Ae. aegypti, was widespread at high frequency in south and central area. However, no significant correlation between the frequency of F1534C and pyrethroid susceptibility was detected primarily because the F1534C mutation frequency in the southern highland area was very low, despite that the bioassay indicated high pyrethroid resistance. The point mutation in the VSSC, L982W, which was not the target mutation in our previous study, was recently determined to be an important mutation causing high-pyrethroid resistance in Vietnamese Ae. aegypti. In the present study, a re-investigation of L982W in the mosquito samples collected in 2006-2008 revealed a greater distribution of this mutation (allelic percentage 59.2%) than F1534C (21.7%) and the greater proportion of homozygous L982W as compared to F1534C provided a plausible answer to the question concerning the unknown resistance factor in the southern highland area. L982W frequencies were uniformly higher in the southern part of Vietnam, including the highland area with a significantly high positive correlation with pyrethroid resistance in Ae. aegypti.

Effects of Indoor Environmental Factors and House Structures on Vaporization of Active Ingredient from Spatial Repellent Devices in Rural Houses in Malawi.

The use of a metofluthrin-impregnated spatial repellent device (MSRD) is a new and effective method for preventing mosquito blood feeding. Indoor environmental factors such as room temperature and ventilation rate are thought to be important for MSRD activity. Measurements of room temperature and vaporization of metofluthrin from MSRD in typical rural metal-roof and thatched-roof houses in southeastern Malawi were conducted. The relationship between house structure and the number of collected Anopheline mosquitoes with and without MSRD treatment was also investigated. The difference between daytime and nighttime room temperature was significantly higher in metal-roof houses than in thatched-roof houses. The vaporization of metofluthrin from the MSRD was not accelerated by the high room temperature, but by the high indoor air flow by ventilation. The number of mosquito collections was significantly higher in thatched-roof houses than in metal-roof houses. MSRD-treated thatched-roof houses have a higher probability of mosquito infestation, but the vaporization of metofluthrin is also higher because of indoor air flow, resulting in a reduction in mosquito numbers. Metal-roof houses with closed eaves reduce the probability of mosquito invasion, and a longer predicted effectiveness occurs with MSRD because of the controlled release of metofluthrin through lower indoor air flow.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

The efficacy and physical condition of olyset insecticide-treated nets after 5 years use in rural Lao PDR.

We assessed the insecticidal efficacy of Olyset nets after 5 years of use in rural villages of Lao PDR and evaluated the relationship between the physical condition of the nets and their insecticidal effect. Our results showed that most of the Olyset nets remained effective after 5 years of use; however, there was no significant relationship between the physical condition of the nets (ie, presence of holes, level of stains) and the insecticidal effect. The presence of large holes in polyester nets compared to the Olyset nets suggest the Olyset nets are stronger; however, nearly half of Olyset nets had small holes or had been previously repaired. Interestingly, the insecticide concentration and knockdown (KD) rate for 3 stored nets was low compared to the other nets routinely used in the house. To maintain the effectiveness of Olyset nets in rural villages of Lao PDR and other areas, residents should be advised to repair and store the nets appropriately and avoid exposure to high temperatures and direct sunlight for long periods.

Searching for a sign of exotic Aedes albopictus (Culicidae) introduction in major international seaports on Kyushu Island, Japan.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, has spread around the world. The migration was mainly mediated by maritime transportations. This species is known as an efficient vector for arboviruses, and it was responsible for the recent dengue outbreak in Tokyo, Japan. As the vector competence varies among geographical populations, and insecticide resistant populations have emerged, it is important to reveal their movements. The present study uses molecular techniques to search for a sign of introduction of an exotic population in three major international seaports on Kyushu Island. METHODOLOGY/PRINCIPAL FINDINGS: Adults of Ae. albopictus were sampled around the international seaports of Fukuoka, Kitakyushu, and Nagasaki. Pairwise fixation indexes were estimated between the sampled populations based on 13 microsatellite markers. There was no clear genetic differentiation between distant and port populations in Kitakyushu and Nagasaki. However, the analysis found one distinct group near the container terminal in Fukuoka, which handles international freight containers mainly from adjacent countries. DNA samples were also obtained from Goto, Tsushima, Honshu, Ryukyu, Thailand, and the Philippines; and a cluster analysis and discriminant analysis revealed that the distinct group in Fukuoka did not belong to these groups. Combined with the results of phylogenetic analysis based on CO1, these results implied that this group originated from one Asian temperate region outside of Japan. Neutrality test and mismatch distribution analysis suggested that the establishment of this group was not recent. CONCLUSIONS/SIGNIFICANCE: The present study found a sign of Ae. albopictus introduction from a temperate region of Asia through maritime freight container transportation. The genetically distinct group found in Fukuoka likely originated from a temperate region outside of Japan. Maritime container transportation may introduce to Japan mosquitoes with greater vector competence/insecticide resistance. This is the first study to describe the spatial population structure of Ae. albopictus in Japan using molecular techniques.

Efficacy and safety of a dimethicone lotion in patients with pyrethroid-resistant head lice in an epidemic area, Okinawa, Japan.

Pyrethroid insecticides are the only pesticides approved for the treatment of head lice (pediculosis capitis) infestations in Japan. However, in Okinawa Prefecture, 96% of head lice are resistant to pyrethroids. Here, we conducted a clinical trial to assess the safety and efficacy of a dimethicone preparation against head lice infestations in Okinawa Prefecture. Dimethicone-containing lotion was applied over the entire scalp three times over a 7-day period. Lice bodies (nymphs/adults) and eggs (nits) were counted before (day 0) and after treatment (day 8); a subset of eggs was collected to estimate viability based on hatch rate. Efficacy was evaluated based on improvement (reduction) in head lice counts post-treatment with respect to baseline. Safety was evaluated based on subjects' scalp condition and adverse event incidence. Utility was a composite end-point combining efficacy and safety. In total, 35 subjects were enrolled. Efficacy and safety were evaluated in 23 and 35 subjects, respectively. No side-effects of note were reported during the treatment period. The dimethicone lotion resulted in a utility rating of "marginally useful" or higher in over 80% of the study population, signifying the formulation to be both safe and effective. The dimethicone lotion was also a potent ovicide; 99.4% of eggs collected after treatment failed to hatch when incubated. Eradication of head lice remained successful for at least 4 weeks after the final topical dimethicone application in 25 of the 28 subjects reached by telephone survey. Lice bodies and eggs were genotyped to analyze the prevalence of three knockdown resistance (kdr)-type mutations within the voltage-sensitive sodium channel known to confer pyrethroid resistance. One or more kdr mutations were confirmed in 30 of the 32 subjects from whom specimens were collected (93.8%). Dimethicone was confirmed to be both safe and effective in treating pyrethroid-resistant head lice.

New mosquito repellency bioassay for evaluation of repellents and pyrethroids using an attractive blood-feeding device.

BACKGROUND: With the increasing threat of the worldwide spread of mosquito-borne infectious diseases, consumer interest in anti-mosquito textiles that protect against mosquito bites is also increasing. Accordingly, repellent- or insecticide-treated textiles are gaining popularity. The standardization of commercial textile products is, therefore, indispensable for an authentic and objective evaluation of these products. Here we report a textile testing method using an artificial blood-feeding system that does not involve human volunteers or live animals, which aligns with the policy of protecting human and animal welfare. METHODS: The attractive blood-feeding device (ABFD) was designed using the Hemotek® membrane feeding system. The repellency of DEET, icaridin and permethrin was assayed using unfed female adults of Aedes albopictus (Skuse) under two different test conditions, namely choice and no-choice tests. The choice test consisted of two feeding units, one chemically treated and untreated, that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the overall blood-feeding rates recorded. The no-choice test consisted of two feeding units treated with the same chemical that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the blood-feeding rates were recorded. A control test was conducted using two feeding units, both sides of which were untreated. RESULTS: In the choice test, high repellency (> 95% inhibition of resting on the treated surface) of 1% DEET and 2% icaridin was observed, whereas 2% permethrin was not an effective repellent. Also, high blood-feeding inhibition (> 95%) was observed for 2% DEET and 2% icaridin. In the no-choice test, high repellency was observed for 1% DEET and 2% icaridin, whereas the repellency of 2% permethrin was low. Also, high blood-feeding inhibition was observed for 2% DEET, 4% icaridin and 2% permethrin. CONCLUSIONS: The accuracy and reproducibility of the developed method demonstrate that the ABFD may be widely used for fundamental experiments in the field of mosquito physiology, for the development of new repellent chemicals and in evaluation studies of mosquito repellent products, such as anti-mosquito textiles. The further development of the membrane and feeding unit systems will enable a more practical evaluation of mosquito repellents and blood-feeding inhibitors, such as pyrethroids.

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial.

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were -2.5 (95% CI: -6.4 to -0.6) and -1.3 (95% CI: -2.0 to -0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs.

Sleeping arrangement and house structure affect bed net use in villages along Lake Victoria.

BACKGROUND: Although insecticide-treated bed nets are effective tools, use often does not follow ownership. House structure and space arrangements may make the attempt to use bed nets difficult, especially for school age children. The objectives of this study were to explore whether an individual's sleeping arrangements and house structure affect bed net use in villages along Lake Victoria in western Kenya. METHODS: Sleeping arrangements of residents were directly observed for use of a bed net, use of a bed, and location. House size, number and types of rooms, bed availability, and residents' ages were estimated. The family heads and mothers were asked about the reason for not using bed nets. Individual bed net use was examined against age and sleeping arrangement. Net use at the household level was examined against four variables: bed availability, bed net availability, house size, and number of rooms. RESULTS: Bed net use by children between five and 15 years of age was lower than that among the other age classes. However, age was dropped from the final model, and sleeping arrangement was significantly associated with net use. Net use was significantly associated with bed availability, number of rooms and their interaction. CONCLUSION: Net use was affected by sleeping arrangement and availability of suitable locations for hanging nets, in addition to net availability. Most residents had likely not realized that sleeping arrangement was a factor in net use. The ease of hanging a net is particularly important for children.

Predators of Anopheles gambiae sensu lato (Diptera: Culicidae) larvae in wetlands, western Kenya: confirmation by polymerase chain reaction method.

Polymerase chain reaction analysis was performed to determine whether mosquito predators in wetland habitats feed on Anopheles gambiae sensu lato (s.l.) larvae. Aquatic mosquito predators were collected from six wetlands near Lake Victoria in Mbita, Western Kenya. This study revealed that the whole positive rate of An. gambiae s.l. from 330 predators was 54.2%. The order of positive rate was the highest in Odonata (70.2%), followed by Hemiptera (62.8%), Amphibia (41.7%), and Coleoptera (18%). This study demonstrates that the polymerase chain reaction method can determine whether aquatic mosquito predators feed on An. gambiae s.l. larvae if the predators have undigested An. gambiae s.l. in their midgut or stomach.

Geographic distribution of Aedes aegypti and Aedes albopictus collected from used tires in Vietnam.

The spatial distribution of Aedes aegypti and Aedes albopictus in environmental and geographical zones, e.g., urban-rural, coastal-mountainous, and north-south, was investigated throughout Vietnam. Immature stages were collected from used tires along roads. The effects of regions, seasons, and the degree of urbanization on the density and the frequency were statistically analyzed. Aedes aegypti predominated in the southern and central regions, while Ae. albopictus predominated in the northern region, which may be related to climatic conditions (temperature and rainfall). Larval collection from used tires may be suitable to assess rapidly the current distribution of dengue mosquitoes for estimating health risks and implementing vector control measures.

Distribution and pyrethroid resistance status of Aedes aegypti and Aedes albopictus populations and possible phylogenetic reasons for the recent invasion of Aedes aegypti in Nepal.

BACKGROUND: When the first systematic list of mosquitoes in Nepal was reported in 1990, there was no description of Aedes aegypti (L.), while Aedes albopictus (Skuse) has been included in the Stegomyia subgroup since the 1950s. The first record of Ae. aegypti in Nepal was reported in 2009, suggesting some coincidence between the invasion of this species and the first record of dengue fever in Nepal in 2006. RESULTS: We performed a field survey of the distribution and insecticide susceptibility of Ae. aegypti and Ae. albopictus in Nepal in 2017 and 2018. Mosquito larvae were collected from used tires located along the streets of Kathmandu, Bharatpur and Pokhara, and a simplified bioassay was used to assess the susceptibility of the larvae to pyrethroid insecticides using d-allethrin. The presence or absence of point mutations in the voltage-gated sodium channel was also detected by direct sequencing. V1016G was detected at a high frequency and a strong correlation was observed between the frequencies of V1016G and susceptibility indices in Ae. aegypti populations. F1534C was also detected at a relatively low frequency. In Ae. albopictus populations, susceptibilities to d-allethrin were high and no point mutations were detected. Analysis of the cytochrome c oxidase subunit 1 (cox1) gene was performed for assessing genetic diversity and the existence of two strains were identified in Ae. aegypti populations. One consisted of 9 globally-distributed haplotypes while the other was derived from an African haplotype. CONCLUSIONS: The high pyrethroid resistance, high V1016G frequency, and relatively low quantity of insecticides used to control dengue vectors in Nepal may have resulted in only weak selection pressure favoring insecticide resistance and could support the hypothesis that this species has recently been introduced from neighboring Asian countries where pyrethroid resistance is relatively widespread.

Effect of Metofluthrin-Impregnated Spatial Repellent Devices Combined with New Long-Lasting Insecticidal Nets (Olyset® Plus) on Pyrethroid-Resistant Malaria Vectors and Malaria Prevalence: Field Trial in South-Eastern Malawi.

Long-lasting insecticidal nets (LLINs) experience some operational problems that reduce their effectiveness, such as limited spaces for hanging, biting of mosquitoes outdoors, a shift of key biting time from midnight to dawn or dusk, and development of pyrethroid resistance in mosquitoes. The concept of spatial repellency may be a countermeasure to overcome the above issues. The effect of the combined use of metofluthrin-impregnated spatial repellent devices (MSRDs) and LLINs (Olyset® Plus) on malaria prevalence and vector mosquitoes were examined in malaria endemic villages in south-eastern Malawi. The intervention reduced the infection rate in children as well as the number of pyrethroid-resistant vector mosquitoes. To achieve effective malaria control, continued intervention using MSRDs with 2 strips per 10 m2 at 3-month intervals to reduce the density of malaria mosquitoes is recommended.

A preliminary study on designing a cluster randomized control trial of two new mosquito nets to prevent malaria parasite infection.

BACKGROUND: Although long-lasting insecticidal nets (LLINs) are the most effective tool for preventing malaria parasite transmission, the nets have some limitations. For example, the increase of LLIN use has induced the rapid expansion of mosquito insecticide resistance. More than two persons often share one net, which increases the infection risk. To overcome these problems, two new mosquito nets were developed, one incorporating piperonyl butoxide and another covering ceilings and open eaves. We designed a cluster randomized controlled trial (cRCT) to evaluate these nets based on the information provided in the present preliminary study. RESULTS: Nearly 75% of the anopheline population in the study area in western Kenya was Anopheles gambiae s. l., and the remaining was Anopheles funestus s. l. More female anophelines were recorded in the western part of the study area. The number of anophelines increased with rainfall. We planned to have 80% power to detect a 50% reduction in female anophelines between the control group and each intervention group. The between-cluster coefficient of variance was 0.192. As the number of clusters was limited to 4 due to the size of the study area, the estimated cluster size was 7 spray catches with an alpha of 0.05. Of 1619 children tested, 626 (48%) were Plasmodium falciparum positive using a rapid diagnostic test (RDT). The prevalence was higher in the northwestern part of the study area. The number of children who slept under bed nets was 929 (71%). The P. falciparum RDT-positive prevalence (RDTpfPR) of net users was 45%, and that of non-users was 55% (OR 0.73; 95% CI 0.56, 0.95). Using 45% RDTpfPR of net users, we expected each intervention to reduce prevalence by 50%. The intracluster correlation coefficient was 0.053. With 80% power and an alpha of 0.05, the estimated cluster size was 116 children. Based on the distribution of children, we modified the boundaries of the clusters and established 300-m buffer zones along the boundaries to minimize a spillover effect. CONCLUSIONS: The cRCT study design is feasible. As the number of clusters is limited, we will apply a two-stage procedure with the baseline data to evaluate each intervention.

Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors.

The impact of insecticide resistance in malaria vectors is poorly understood and quantified. Here a series of geospatial datasets for insecticide resistance in malaria vectors are provided, so that trends in resistance in time and space can be quantified, and the impact of resistance found in wild populations on malaria transmission in Africa can be assessed. Specifically, data have been collated and geopositioned for the prevalence of insecticide resistance, as measured by standard bioassays, in representative samples of individual species or species complexes. Data are provided for the Anopheles gambiae species complex, the Anopheles funestus subgroup, and for nine individual vector species. Data are also given for common genetic markers of resistance to support analyses of whether these markers can improve the ability to monitor resistance in low resource settings. Allele frequencies for known resistance-associated markers in the Voltage-gated sodium channel (Vgsc) are provided. In total, eight analysis-ready, standardised, geopositioned datasets encompassing over 20,000 African mosquito collections between 1957 and 2017 are released.

Comparative evaluation of fecundity and survivorship of six copepod (Copepoda: Cyclopidae) species, in relation to selection of candidate biological control agents against Aedes aegypti.

The fecundity and survival of 6 copepod species were assessed under laboratory conditions in order to choose the best candidates to control the aquatic stages of dengue mosquitoes in the field. Females of all the 6 species (Mesocyclops aspericornis, Mesocyclops pehpeiensis, Mesocyclops woutersi, Mesocyclops thermocyclopoides, Mesocyclops ogunnus, and Megacyclops viridis) mated more than once. Multiple mating resulted in increased egg production. The reproductive ability and longevity varied among the species, and M. aspericornis had the highest values. The lowest values were observed in M. thermocyclopoides. Multiple mating of males of M. aspericornis was also observed. The paternal fecundity decreased with each additional mating. There was no difference in the paternal fecundity between the males that mated at low and high female frequencies. The sperm stored in the M. aspericornis females remained viable for 30 days after storage under moist conditions at 25 degrees C or 15 degrees C. This feature in M. aspericornis represents an additional positive factor indicating that this species is a good biological agent for controlling mosquito larvae, especially in domestic water containers that may dry intermittently.

Field evaluation of spatial repellency of metofluthrin-impregnated plastic strips against Anopheles gambiae complex in Bagamoyo, coastal Tanzania.

Metofluthrin is a newly synthesized pyrethroid possessing high knockdown and lethal activity against mosquitoes. Studies of metofluthrin-impregnated plastic strips have been performed with dengue vectors. This study reports the efficacy of the new prototypes of metofluthrin-impregnated plastic strips against malaria vectors, Anopheles gambiae complex, in the Kongo villages of Bagamoyo district in coastal Tanzania. The study, using 20 houses, half intervention, half control, was conducted for a 124-day period. Pyrethrum spray sheets and Centers for Disease Control and Prevention light traps were used to sample mosquito population indices. The mosquito density indices of the intervention houses were observed to be significantly lower than those of the control houses when pyrethrum spray sheet collection was used (F = 4.61, 1 df, P = 0.038; 98.7% reduction of total mosquito collection compared with that for the controls). These low indices were observed despite the large openings found in Bagamoyo houses, which were predicted to have a considerable negative effect on the spatial repellency of metofluthrin. Based on the present results, the pyrethrum spray sheet collection was the better of the 2 collection methods.

Comparative laboratory study on the reaction of Aedes aegypti and Aedes albopictus to different attractive cues in a mosquito trap.

The behavioral responses of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults to several attractive cues, as reactions to mosquito traps, are compared in the laboratory, and differences in the primary attractive factors for both species are discussed. Target-attacking frequency of unfed Ae. aegypti females was >30 times that of unfed Ae. albopictus females under simulated conditions. Changes in the percentage of trapped mosquitoes under several attractive conditions using commercial mosquito traps showed that Ae. aegypti mosquitoes were trapped 2-3 times faster than Ae. albopictus. For Ae. aegypti, the combination of a visual cue + CO, alone enhanced attractiveness, whereas both a visual cue + CO, as well as a visual cue + octenol enhanced Ae. albopictus. The combination of at least three factors, such as a visual cue, CO2, and a chemical cue is thought to be valuable for trapping and estimating the relative adult population sizes of Ae. aegypti and Ae. albopictus