Grass Infusions in Autocidal Gravid Ovitraps to Lure Aedes albopictus

Aedes albopictus (Skuse) is a known vector of dengue, chikungunya, Zika, and other pathogens (Parry et al. 2021). There is a lack of effective vaccines to control these pathogens, therefore most current strategies focus on surveillance and vector control approaches to diminish the arboviral disease impact on human settlements, especially in the Americas. Among novel advances in vector surveillance and control tools, autocidal gravid ovitraps (AGOs) (Mackay et al. 2013) using hay infusions of Cynodon nlemfuensis Vanderyst have been effective in attracting gravid Ae. aegypti (L.) females in Puerto Rico (Barrera et al. 2019). Nonetheless, the use of hay infusions to attract and control the other important dengue vector, Ae. albopictus (Skuse), has been negligible. In these situations, to lure Ae. albopictus females, attempts in laboratory settings with limited success have been conducted including organic infusions of Cynodon dactylon (L.) and Quercus spp. (Zhang and Lei 2008, Dixson et al. 2020). Here we report a Mexican study to improve the efficacy of AGOs traps to collect Ae. albopictus matched with 4 different grass infusions under lab conditions. Additionally, we also tested these infusions under semifield and field conditions as attractants for Ae. albopictus gravid females.

Aedes albopictus were generated from a colony originally collected from Tapachula, Chiapas, Mexico, bred in the insectary facilities of the Centro de Biotecnología Genómica from Instituto Politécnico Nacional in Reynosa, Mexico (García-Munguía et al. 2011). Males and females were allowed to mate in 50 × 50 × 50 cm cages with 10% sucrose ad libitum; females 4–6 days old were blood fed on an immobilized rabbit for 40 min, and 3 days before the experiments unfed females were removed from the cage, leaving gravid females to be used for the bioassays.

For infusions using Megathyrsus maximus (Jacq.), C. dactylon, Cenchrus purpureus (Schumach), and Pennisetum ciliare (L.), dehydrated green leaves collected from an experimental station in Las Huastecas, Tamaulipas, Mexico (22°34′2.93″N, 98°9′53.64″W) were prepared as reported by Reiter et al. (1991). Then a 10% infusion concentration in unchlorinated water was prepared for use in the bioassays. For each bioassay, 4 replicates of twenty 7-to-9-day-old gravid females each were tested in mosquito cages (50 × 50 × 50 cm), containing 2 black cups with 100 ml of either unchlorinated water as control or grass infusion placed diagonally in the corner and lined up with a strip of AGO replacement glue board (4 × 21.5 cm). The position of each cup was switched between replicates; after 24 h, the cups were removed from the cage and the captured females in each cup counted.

Identical infusions were assayed to lure Ae. albopictus released in a 3.35 m long × 2.74 m wide × 1.98 m high greenhouse during August–November 2022 in Reynosa, Tamaulipas, Mexico (26°04′09.2″N, 98°18′46.8″W). The greenhouse, which was empty, received natural light and maintained an average temperature of 26.66 ± 3.59°C, with relative humidity 69.79% ± 9.51%.

Four replicates using a group of 100 blood-fed females for each of the 4 grass infusions and controls were assayed. Each replicate had 2 AGOs containing either 10 liters of unchlorinated water (control) or grass infusion placed diagonally 4.3 m apart from opposing greenhouse corners. The position of AGOs was switched between replicates to account for location bias. Aedes albopictus females were released in each replicate between 0800 h and 0830 h at the center of the greenhouse, approximately 2.15 m away from AGOs. After 48 h, the AGO traps were retrieved, and the mosquitoes captured on the glue boards were counted for 4 replicates.

Field experimental studies were conducted from March 29 to July 7, 2023, in the residential neighborhood of Pedro José Méndez (26°01′04.0″N, 98°16′29.3″W) in Reynosa. The site was selected due to the highly observed populations of Ae. albopictus during monitoring studies in the spring, summer, and fall of 2021 and 2022.

We selected C. dactylon and M. maximus as the most effective infusions for Ae. albopictus under lab and semifield conditions. Experimental design included individual or combinations of infusions in a 50:50 mixture as follows: 1) C. dactylon, 2) M. maximus, 3) hay, 4) tap water, 5) C. dactylon + M. maximus, 6) M. maximus + hay, and 7) C. dactylon + hay. Positive control was an infusion of Medicago sativa.

Under field conditions, 14 trap positions were selected in the study area, with collection sites located at least 30 m apart. Two replicates were conducted concurrently each week by setting 7 at the beginning of the week and 7 more later in the week. Trap positions were reset every 7 days. Sticky glue boards were removed, and mosquitoes were counted and identified visually to gender and species level. To evaluate the attractiveness of the infusions by trap positions, traps and baits were rotated between each site. The grass infusions were replaced and evaluated after 7 wk, following the study of Santana et al. (2006).

Data obtained from lab and semifield conditions were tested for normality (Shapiro-Wilk test) and equality of variance (Bartlett’s test). One-way ANOVA followed by post hoc Student-Newman-Keuls (SNK) analysis was conducted to compare the mean (+SE) of gravid Ae. albopictus females caught on glue board in ovitraps or AGOs across replicate/infusion treatment and control groups.

For the field experiment, the difference in the number of captured mosquitoes across infusions and controls was compared using a Generalized Linear Mixed Model (GLMM) with the GLIMMIX procedure, with a negative binomial distribution to account for nonnormality and overdispersed data. All statistical analyses were performed in SAS OnDemand for Academics (SAS 2021) and α = 0.05 for statistical differences.

Under lab conditions, there was a significantly higher number (F_{(1, 6)} = 15.47, P = 0.0077) of Ae. albopictus counted in the cups containing C. dactylon (mean of 11.75 ± 0.62 SE) compared to the control (8.25 ± 0.62 SE). However, no significant differences were observed between the other infusions: M. maximus (mean of 11.0 ± 1.08 SE; F_{(1, 6)} = 1.71, P = 0.2383), C. purpureus (mean of 11.50 ± 1.19 SE; F_{(1, 6)} = 3.18, P = 0.1250), P. ciliare (mean of 11.0 ± 0.70 SE; F_{(1, 6)} = 4.00, P = 0.0924), and controls (9.0 ± 1.08, 8.5 ± 1.19, 9 ± 0.70 SE, respectively; Fig. 1A).

Under semifield conditions, both C. dactylon (F_{(1, 6)} = 460.15, P < 0.0001) and M. maximus (F_{(1, 6)} = 248.47, P < 0.0001) infusions lured Ae. albopictus gravid females, showing a higher number (mean of 74.25 ± 2.05 SE and 70.25 ± 2.01 SE, respectively) of attracted mosquitoes than that of controls (20 ± 1.47 SE and 26.25 ± 1.93 SE, respectively). No variation between the infusions of C. purpureus (mean of 44 ± 2.94 SE; F_{(1, 6)} = 2.25, P = 0.1841), P. ciliare (mean of 48.25 ± 2.56 SE; F_{(1, 6)} = 0.40, P = 0.5525), and controls (50.75 ± 3.40 SE and 45.75 ± 3.03, respectively) was noted (Fig. 1B).

In the field experiment, when assessing the attractant effect of organic infusions with AGOs, a total of 308 Ae. Aegypti, 181 Culex quinquefasciatus Say, and 22 Ae. Albopictus were collected over 14 wk (Table 1). None of the 6 infusions, whether tested individually or in a mixture, resulted in a significantly higher collection of female mosquitoes than that of the control water. Furthermore, there was no significant difference in the number of either Ae. Aegypti females (GLIMMIX: F_6,187 = 0.84, P = 0.5405), Ae. Albopictus (GLIMMIX: F_6,187 = 1.05, P = 0.3965), or Culex quinquefasciatus (GLIMMIX: F_6,187 = 0.84, P = 0.5437) or the total of mosquitoes (GLIMMIX: F_6,187 = 0.59, P = 0.7341) captured among all the infusions tested.

Diverse grass infusions have been assessed against vector mosquitoes, including Ae. aegypti and Ae. albopictus under both laboratory and field conditions (Dormont et al. 2021). These studies have primarily focused on evaluating the oviposition response of these species to grass infusions as an indicator of their attractiveness (Santana et al. 2006). However, some authors have suggested egg count as an indirect indicator for measuring attraction of female Aedes spp. because of their skip-oviposition behavior (Mulatier et al. 2022). Instead, we employed sticky-screen glue boards from ovitraps and AGOs as a direct indicator of attraction of Ae. albopictus gravid females to the infusions.

Our results from the lab and semifield bioassays demonstrate the relative attraction of Ae. albopictus females to C. dactylon and M. maximus infusions, with more females caught in traps baited with these infusions than in control traps. These findings support previous results indicating Ae. albopictus females being attracted to infusions of C. dactylon (Zhang and Lei 2008), and M. maximus (Santana et al. 2006, Santos et al. 2010), in lab bioassays. However, none of these infusions lured Ae. albopictus females when evaluated under field conditions in earlier studies (Burkett-Cadena and Mullen 2007, Brisco et al. 2023).

Some limitations in this study should be mentioned. First, the low mosquito densities can be attributed to abiotic factors, particularly the low precipitation in Tamaulipas documented in 2023 when the field study was conducted. Without enough precipitation, mosquitoes lack proper habitats for laying eggs, and their aquatic larvae struggle to thrive and grow because of the competence between larvae and pupae at higher population densities (Morin et al. 2015). Additionally, in the summer, temperatures can reach 40–42°C, and temperatures above 35°C lead to a decline in Aedes mosquito populations (Jia et al. 2019). Therefore, our study was limited to only 14 wk, from March to the beginning of July, preceding the onset of the period of high summer temperatures.

Further field studies are needed at different seasons, localities, and collection sites to confirm the efficacy of these grass infusions to lure Ae. albopictus using AGOs, aiding public health agencies in surveillance and control efforts for this dengue vector, with the ultimate goal of developing an AGO for Ae. albopictus.

The study was funded by the Instituto Politécnico Nacional SIP-IPN 20181120, 20201972, 20222157, 20221576, 20226932, and 20230712. We thank Cuauhtemoc Villarreal-Treviño (Instituto Nacional de Salud Pública) for providing the Aedes albopictus colony. We also acknowledge Abner Alejandro Soto-Hernández and Eber Eduardo Soto-Hernández for keeping the Ae. albopictus colony ongoing.