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

The mosquito genera considered of primary medical and veterinary importance are confined to Aedes, Anopheles, Culex, and Mansonia, and of these, only a relatively few species are regarded as a vectors of pathogens, primarily viruses, protozoa, and nematodes (Hay et al. 2004, Bhatt et al. 2013, Kollars et al. 2016). With a few exceptions (e.g., effective vaccines), primary prevention to reduce transmission risk remains dependent on various vector control tools supported by well-organized mosquito surveillance and control programs (Roberts et al. 1997, Chareonviriyaphap et al. 2004, Grieco et al. 2007). Routine monitoring of mosquito populations serves as a crucial indicator for estimating disease risk, directing timely control measures, and assessing program performance.

For female mosquitoes with obligatory bloodfeeding, the human landing collection (HLC) method is considered the “gold” standard for obtaining indoor and outdoor measurements of anopheline and culicine mosquitoes attracted to humans, thus providing an estimate of biting activity for a variety of purposes (Muirhead-Thomson 1968, Kweka and Mahande 2009, Dusfour et al. 2010, Duo-Quan et al. 2012). However, HLC is very labor intensive, requiring trained and motivated collectors, extensive supervision, and resources generally absent or unsustainable for routine sampling in operational settings. Moreover, using unprotected humans and animals as attractants for host-seeking mosquitoes has safety and ethical concerns for the welfare of the host. Alternatively, animal (e.g., cow or buffalo)–baited collections using single- or double-net trap designs have limitations due to strong host specificity (i.e., selection bias) by some mosquitoes, thereby diminishing representative sampling of species diversity (Suwonkerd et al. 2002, 2004). Many types of trapping devices and sampling methods for adult female mosquitoes have been developed, in particular mechanical traps using some form of olfactory (e.g., carbon dioxide) or visually stimulating illumination (light source) as potential attractant (i.e., positive phototaxis) (Silver 2008). Mechanisms and environmental interactions governing mosquito visual responses and flight paths (e.g., orientation towards a target for long- and short-range attraction flights) are controlled primarily by visual or olfactory cues (Bidlingmayer 1994). Visual orientation alone to targets of interest is appetitive flight (endogenous-driven need), whereas attraction flights are “goal-oriented,” involving an appropriate stimulus, either visual or olfactory or both.

The use of light traps to capture nocturnally active mosquitoes has a long history and continues to be one of the main methods for monitoring populations of mosquito vectors devoid of live bait cues (Mulhern 1934, Headlee 1937, Sudia and Chamberlain 1962, Muirhead-Thomson 1991, Silver 2008). Subsequent to those early developments, various light sources have been used in traps to enhance mosquito collection efficiency (Service 1970, Chandler et al. 1975). In the adult mosquito, vision spectral sensitivity is influenced by electromagnetic wavelength perception (Peach et al. 2019). Investigations on electromagnetic sensitivity of mosquito compound eyes indicate that mosquitoes are capable of wavelength-discriminative behavioral responses (e.g., attraction to an oviposition site) to transmitted and reflected light of different wavelengths (Clements 1999).

Discriminative behavior is defined as any particular behavior (e.g., negative or positive phototaxis–orientation) toward or away from a light source associated with the spectral composition of incident light. It can be elicited not only by light of a given wavelength (color), but also by certain intensities of light. Artificial light sources differ in intensity and spectral distribution of light produced (Jagger 1967, Smith 1977). These differences are important when selecting lights used in mosquito traps or behavioral studies. For example, fluorescent lights vary according to the phosphors used and typically have a broad emission spectrum but are relatively low in the red region, whereas light-emitting diodes (LEDs) differ by types of diodes and emit a narrow light bandwidth from low to high wavelengths (Schubert et al. 2000). The physical parameters of mosquito eyes influence visual perception and behavioral responses to light, shapes, patterns, movement, etc. (Allan 1994). Consequently, the intensity of illumination is an important consideration in mosquito perception and attraction to light sources. Perception at a certain distance varies with intensity of the light source, varying inversely with the square of the distance from the source.

Various light traps have been developed and used for mosquito sampling and surveillance, but most have been used for experimental comparison purposes only (Silver 2008, Sriwichai et al. 2015, Mwanga et al. 2019). The Centers for Disease Control and Prevention (CDC) miniature light trap and similar devices have been extensively used and evaluated (Silver 2008). In some instances, light traps have been shown comparable to HLC for capturing indoor species when using protected humans as attractant cues (Mathenge et al. 2005), while others have not (Overgaard et al. 2012). Of the different types of light sources ranging from incandescent, fluorescent to LED lighting systems, LED lights have been utilized to attract various insects and pests (Bishop et al. 2004, Bentley et al. 2009, Yoon et al. 2012, Zheng et al. 2014, González et al. 2016). Advantages of LED lights are low power consumption, cool operating temperatures, durability, and monochromatic light production (Hoel et al. 2007). The LED lights are available in various wavelengths depending on need (Schubert et al. 2000, Singh et al. 2015). For mosquitoes, Costa-Neta et al. (2017) found that LED blue (470 nm) was more effective than LED green (520 nm) for attracting anophelines in Brazil. Burkett et al. (1998) reported no significant difference among 4 LED light traps (red, orange, yellow, and green) for catching Anopheles mosquitoes in Florida (USA). Comparisons of LED lights with ultraviolet (UV) fluorescent lights in trap systems have shown that LED light traps displayed greater trapping efficacy for mosquitoes (Kim et al. 2017). Conversely, traps equipped with UV-LEDs and colored LEDs were less performing than that of incandescent bulbs for collecting anophelines (Ponlawat et al. 2017). However, no study on the use of LED lights for collecting urban nocturnally active mosquitoes has been studied in Thailand. Therefore, we compared the efficiency of different light sources: 4 LED-equipped traps emitting blue, green, yellow, and red wavelengths and 2 fluorescent light sources, UV and white, for attracting and capturing mosquito species present in a varied urban environment in Bangkok, Thailand.

The study was conducted at the Kasetsart University (KU) main campus, Bangkhen (13°50′32.96″N, 100°34′2.98″E), Chatuchak District, in northern Bangkok, Thailand. The campus covers approximately 135.7 ha (335 acres) and is located inside a heavily urbanized area of the greater Bangkok metropolis. The campus grounds consist of educational and dormitory buildings, internal roadways, and numerous open drainage lines. The area also has many trees, planted vegetation, and permanent ponds throughout the campus. Six fixed locations within the campus were randomly selected using a grid system for light trap placement, and geo-coordinates of each location are provided (Fig. 1).

The mosquito trap (Black Hole™ Mosquito Trap; Bio-Trap Inc., Seoul, Korea) used in this study was purchased in Thailand (Pan Science Co., Ltd., Bangkok). The trap is made of durable black plastic material equipped with an electrical fan and a UV light source (2 4-W fluorescent UV bulbs) powered by an alternating current 220–240V electrical system. Each trap is 25 × 25 × 32 cm in size and weighs 1.2 kg. Before testing, 5 traps were modified by replacement of the factory-equipped light source with 1 of 4 LED bulbs (blue, green, yellow, and red), or a fluorescent white bulb (Eve Lighting Co., Ltd., Bangkok, Thailand). Six identical traps were each equipped with different illumination at the following wavelength emission band range: fluorescent UV (peak at 354–468 nm, light intensity 63,913 arbitrary units [a.u.]), and visible light from fluorescent white (peak at 277–400 and above 750 nm, light intensity 53,791 a.u.), LED blue (peak at 416–428 nm, light intensity 63,294 a.u.), LED green (peak at 553–567 nm, light intensity 61,040 a.u.), LED yellow (peak at 575–589 nm, light intensity 61,576 a.u.), and LED red (peak at 740–755 nm, light intensity 62,154 a.u.). The LED bulbs had a 6-W power requirement and the fluorescent white bulb, 5-W. Each trap was hung with the objects, approximately 1.5 m above the ground.

Between February and August 2019, nocturnally active mosquitoes were captured from the 6 light traps operated continuously from 1800 to 0600 h. Approximately every 3 h (2100, 2400, 0300, and 0600 h), all captured mosquitoes were removed by hand from each trap using a mouth aspirator, transferred to labeled holding cups for each time interval, processed in the laboratory, and enumerated by species and sex under a dissecting microscope. Mosquitoes were killed in the freezer (−20°C) at least 30 min and placed individually in petri dish under a stereomicroscope (Carl Zeiss™ Stemi™, Göttingen, Germany) DV4 Series at 40–50× magnification. All mosquitoes were morphologically identified using illustrated keys for adult mosquitoes (Rattanarithikul et al. 2005a, 2005b, 2006a, 2006b, 2007). Mosquito larvae/pupae were sampled once around each respective trap location. The habitats for sampling mosquito larvae were close to the trap location, within a radius of 100 m. Search for mosquito larvae were conducted using a dipping collection method during the study period. Immature mosquitoes were reared to adults and identified using referenced keys.

A 6 × 6 Latin square design rotation was used between different light traps and fixed locations. The initial distribution of the collection sites was randomly selected using an overlay grid system of the KU campus, dividing the area into 6 sections. Each section was further divided into 6 smaller grids and numbered from which a location was selected based on a blind draw. After location selection, traps were rotated in a sequential pattern each collection period to avoid sampling bias. Pretrial trapping at the locations was excluded in the experiment. Each light trap had either an LED or fluorescent light source (no blank trap). Each trapping trial was conducted during 6 consecutive nights, representing 1 replicate. The experiment required 6 replicates, comprising 36 collection nights for a total of 216 trap-nights.

The efficiency of traps was evaluated by using generalized linear model with negative binomial regression and log link function. Total number of collected mosquitoes per trap-night was the response variable, while type of light sources and collection periods were set as key factors. In addition, location of setting trap was considered as covariate for the model. The goodness-of-fit model was validated by considering the deviance value. The mean deviance close to 1 was the optimal model. Result from the test of model effects was interpreted as significant when P < 0.05. The parameter (key factor or covariate) that was a statistically significant predictor of the number of collected mosquitoes resulted in the regression coefficients (B), standard errors, P-values, and 95% confidence intervals for the coefficients by Wald Chi Square. Efficacy of mosquito traps was analyzed based on the value of incident rate ratios (IRR), which provided the incident rate for the reference group to the other variables. The statistical software package, SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY), was used for the analysis.

In this study, 6 mosquito light traps with different spectral ranges were compared for numbers of collected mosquitoes and species diversity. A total of 2,387 mosquitoes were trapped over the 36 collection nights (mean, 11 mosquitoes/trap-night). The fluorescent UV light caught 1,544 mosquitoes (64.7%), whereas the fluorescent white bulb trapped 389 mosquitoes (16.3%). Blue, green, yellow, and red LEDs collected 161 (6.8%), 108 (4.5%), 46 (1.9%), and 139 (5.8%), respectively (Fig. 2).

To evaluate the factors that contributed to the efficiency of traps for attracting and capturing mosquito species, deviance from goodness-of-fit test at 0.404, and Pearson chi square at 567.038 indicated that the negative binomial regression was suitable for data (the Omnibus test; P = 0.000). Based on Table 1, type of light sources and collection periods were the significant predictors that influence the number of mosquitoes captured per trap (P < 0.05), while locations were not significant variables to predict the model (P = 0.840). The output of IRR indicated that the predicted count for mosquito captures of fluorescent UV is 3.20 times compared with the fluorescent white bulb as the reference (IRR = 1). Likewise, the lower of IRR were predicted for LED blue, LED red, LED green, and LED yellow at 0.58, 0.53, 0.48, and 0.36 times to the fluorescent white bulb, respectively. In addition, the best collection period for mosquito collection was predicted for 0300–0600 h (IRR = 1), followed by 2400–0300 h (IRR = 0.91), 2100–2400 h (IRR = 0.79), and 1800–2100 h (IRR = 0.76) (Table 1).

The number of mosquitoes captured after attraction to the different light sources at the 6 locations was compared. Traps at location 4 collected 867 mosquitoes, representing 36.3% of all combined locations, whereas location 6 produced the least (202; 8.5%) (Fig. 3). Eleven species within 5 genera were identified morphologically (Table 2). By genera, Culex (2,252; 94.4%) was the predominant genus collected for 36 nighttime collections (1800–0600 h), followed by Aedes (118; 4.9%), Anopheles (7; 0.2%), Armigeres (1; <0.1%), and Mansonia (9; 0.4%). Some damaged specimens (49 mosquitoes) could not be correctly identified. Culex mosquitoes included Cx. quinquefasciatus Say, Cx. gelidus Theobald, Cx. brevipalpis (Giles), Cx. fuscana Wiedemann, Cx. fuscocephala Theobald, and Cx. vishnui Theobald. Culex quinquefasciatus was consistently the predominant mosquito species, amounting to 75.05% (1,869) of the collection. Two species of Aedes were identified: Ae. aegypti (L.) (8; 0.3%) and Ae. albopictus (Skuse) (84; 3.5%). Only one Anopheles species was identified (An. vagus Doenitz), along with 6 specimens in the subgenus Anopheles that could not be identified to species due to poor condition. Two species of Mansonia, Ma. indiana Edwards (3) and Ma. uniformis (Theobald) (4), were collected. A single specimen of Armigeres was damaged to the extent preventing definitive identification, but presumptively considered Ar. subalbatus (Coquillett). Overall, 1,544 (64.7%) mosquitoes were collected by the trap with the fluorescent UV bulb. Aedes mosquitoes were captured in the highest number from the LED blue–equipped trap, followed by UV light trap. The overall sex ratio of all captured mosquitoes was approximately 1:1 (males representing 45.6%).

The majority of mosquitoes were captured after midnight (1,351; 57%): up to 0300 h (659; 28%) and last quarter to 0600 h (692; 29%) (Table 3). During the first half of the night, 1,036 were caught: 529 (22%) between 1800 and 2100 h, and 507 (21%) between 2100 and 2400 h (Fig. 4). Culex quinquefasciatus showed activity from dusk to dawn, with a distinct peak between 0300 and 0600 h. Other species displayed different activity patterns; for example, Ae. albopictus had a major peak at twilight (Fig. 4).

Larval collections in the vicinity of trap placement identified 5 species within 3 genera (Culex, Aedes, Toxorhynchites) comprising Cx. quinquefasciatus (331; 57.5%), Cx. brevipalpis (89; 15.5%), Cx. fuscana (5; 0.9%), Ae. aegypti (103; 17.9%), Ae. albopictus (46; 8.0%), and Toxorhynchites sp. (2; 0.4%) (Fig. 5).

Four different LED wavelength ranges and 2 fluorescent (UV and white) lighting systems combined with a mechanical trapping device were evaluated for the collection of night-active mosquitoes within the KU campus grounds from February to August 2019. The goal was to identify the best single light attractant for enhanced efficiency in sampling local mosquito biodiversity and species of interest.

The mechanical trap equipped with fluorescent UV lighting was the most effective for mosquito surveillance, whereas both fluorescent lights outperformed all 4 LED lights. Culex quinquefasciatus represented the predominant mosquito species (78.3%) in the overall collections, regardless of light trap light configuration, trap location, or collection date. This species is a very common, cosmopolitan, urban, nighttime biting mosquito and is generally active during the entire evening depending on the availability of vertebrate hosts (Pipitgool et al. 1998, Uttah et al. 2013, Chen et al. 2014, Bhattacharya and Basu 2016).

The KU campus is located in a heavily urbanized area of Bangkok. The university grounds provide many suitable larval habitats for this species, ranging from large bodies of open stagnant/slow-moving water, numerous stormwater drainage lines, and various artificial containers infused with varying degrees of organic matter to higher levels of pollution. Large numbers of Cx. quinquefasciatus were collected (>50% of all mosquito species identified) at each respective trap location. Trap distribution influenced mosquito numbers, with more collected in locations 4 and 5, possibly due to both sites having more suitable aquatic habitats compared with other locations, as well as having relatively greater human activity. Culex gelidus was the second-most common species (12.6%) identified in traps. This species is of particular interest as it is a natural vector of Japanese encephalitis virus (JEV) between host birds and humans (Tiawsirisup and Nuchprayoon 2010). The campus is also home to resident and visiting avian species (Family Ardeidae; egrets, herons, and bitterns), potential reservoirs of JEV. Moreover, the campus is normally congested with human activity both day and nighttime; thus, virus transmission is a possibility.

Data of larval samplings around trap location showed that Culex larvae were the most predominant genus, accounting for 73.9%. In addition, 94.4% of Culex adults were captured by light traps. In contrast, Aedes accounted for 25.9% (larvae) and 4.9% (adults) of the collections. This phenomenon may be due to the different feeding habits of both species. Aedes is a diurnal species, whereas Culex is nocturnal feeder and the latter was reported to be strongly attracted to light source as compared with the other mosquito species (Chen et al. 2014, Chaiphongpachara et al. 2018). This is in agreement with our study in which higher numbers of Culex were collected using light traps. None of Anopheles, Armigeres, and Mansonia larvae were found during the larval survey.

The New Jersey light trap, developed in the 1930s, was the first mosquito trap to be used and is still regarded as valuable in mosquito surveillance (Mulhern 1934, Reinert 1989, Muirhead-Thomson 1991). Several types of mosquito trapping devices have since been developed and utilized for mosquito surveillance (Sudia and Chamberlain 1962, Kline 2006, Silver 2008, Ritchie et al. 2013). Today, the CDC (or similar design) light trap baited with CO₂ (or other semiochemicals) and the BG trap (Biogents AG, Regensburg, Germany) baited with BG-lure with a combination of olfactory attractants are available (Silver 2008, Li et al. 2016). Although other studies have used CDC-style light traps to capture mosquitoes in Thailand (Ponlawat et al. 2017), no study has evaluated the attractive efficacy of LED illumination compared with fluorescent UV light. Additionally, collection of outdoor active mosquitoes is limited in terms of effectiveness of trapping devices. The KU study found the fluorescent UV light trap produced the greatest yield for attracting mosquitoes, followed by fluorescent white light. Among the 4 LEDs, yellow demonstrated the least efficacy in trapping nocturnal mosquitoes. Previous studies have shown the fluorescent UV light wavelength to be an effective attractant for capturing mosquitoes (Service 1970, Wilton and Fay 1972, Lee et al. 2009, Li et al. 2015). Operationally, the Black Hole trap was found to be an acceptable device for mosquito collections. Only 2% of captured specimens were damaged to the point of preventing definitive species identification. However, one limitation was the requirement for a direct line electrical source (plug-in) compared with other traps powered by batteries, thus limiting placement options.

Chen et al. (1990) observed wild Cx. pipiens L., a species closely related to Cx. quinquefasciatus (Miller et al. 1996), responded differently to light sources depending on the wavelengths of the light source. Attraction was greatest between 333 and 405 nm, near UV light emission (Chen et al. 1990). Previous publications have reported UV-A results in high relative efficacy for attracting and trapping nocturnal mosquitoes (Kim et al. 2017, Mwanga et al. 2019). Furthermore, electroretinogram observations have shown the highest responses of Cx. pipiens to 335 nm, corresponding to the range of UV-A (315–400 nm) (Peach et al. 2019).

This study demonstrated that the type of light source influenced the numbers of mosquitoes caught each night. Visible electromagnetic wavelengths between 350 and 700 nm generally attract most insects, although responses may differ significantly over a species range and between species (Land 1997). Insect photoreceptors are involved in wavelength discrimination (Allan 1994, Clements 1999). For example, in Korea, wavelengths between 360 and 435 nm produced by a fluorescent UV trap attracted more Aedes compared with Anopheles or Culex species (Kim et al. 2017). Muir et al. (1992) reported UV and yellow-green wavelength ranges as more sensitive to Ae. aegypti photoreceptors. Breyev (1963) reported more Anopheles hyrcanus (Pallas) and Anopheles maculipennis Meigen collected from light traps emitting radiation between 300 and 400 nm compared with other wavelengths. Notwithstanding, this study did not include UV-LEDs or incandescent light to trap mosquitoes, both of which have been reported to attract mosquitoes in relatively high numbers compared with other light systems (Kim et al. 2017, Ponlawat et al. 2017). Other attraction cues, like synthetic human olfactory attractants, thermal (infrared) emissions, and carbon dioxide, should be integrated into a mechanical trapping device (Burkett et al. 1998, Ponlawat et al. 2017, Mwanga et al. 2019) to increase catch size.

One of the primary goals for understanding mosquito biology and ecology is measuring mosquito populations and species dynamics that help facilitate the design and implementation of appropriate prevention and control strategies. An in-depth evaluation and analysis of a mechanical light trapping system for attracting nocturnally active mosquitoes can provide important direction for conducting mosquito surveillance. This study is the first attempt to assess various light sources as mosquito attractants in a densely populated urban area of Bangkok while also obtaining information on mosquito species present in KU (Bangkhen Campus). Studies continue in the same location (KU campus) to evaluate the attractive responses by mosquitoes to different UV wavelengths and trapping systems.

The authors thank Chatchawal Wongchoosuk for his advice on measuring the LED light sources. The study was financially supported by the Thailand Research Fund through the International Research Network (IRN58W0003).