Designing a simple bioassay to assess repellents against Aedes aegypti (Diptera: Culicidae): investigating stimuli sources and exposure duration on mosquito behavior

Projetando um bioensaio simples para avaliar repelentes contra Aedes aegypti (Diptera: Culicidae): investigando fontes de estímulos e duração da exposição no comportamento do mosquito

Abstract

Repellents are effective in preventing mosquito contact with the skin and in vitro test modules are widely used in the research of potential repellents against mosquito bites. This study evaluated different stimuli and exposure times to develop a new cost-effective bioassay protocol to test repellents against Aedes aegypti. The test module was designed to present different source of stimuli, i.e. human blood, artificial blood meal (SkitoSnack), and human forearm (standard stimulus) and exposure times, i.e., 2, 5, and 10 min. Ten females were introduced in the module, i.e., 480 mL polypropylene adapted cage, and the number of landings and engorgements on stimuli sources was recorded. Subsequently, the Complete Protection Time (CPT) of DEET was evaluated. A. aegypti mosquitoes consistently landed on the module, with an average of 4.3 mosquitoes in the first min, 5.3 in the second min, and 6.5 in the third min, after which the number decreased after eight min. Similarly, engorgement increased from 3.0 mosquitoes after two min to 7.7 mosquitoes after ten min, across all tested conditions, regardless of the stimulus source. However, prolonged exposure to the stimuli led to an increase in both landings and feedings. The CPT of DEET when using alternative stimulus sources (≈ 48 min) differed from that of the human source (75 min), yet both demonstrated similar antifeeding outcomes. Additionally, both SkitoSnack and blood, when offered through an artificial feeder with a 5-min exposure time, are effective for use in experiments with our module for repellent testing.

Keywords:
artificial feeding; membrane assay; repellent; in vitro

Resumo

Os repelentes são eficazes na prevenção do contato do mosquito com a pele e os módulos de teste in vitro são amplamente utilizados na pesquisa de potenciais repelentes contra picadas de mosquito. Este estudo avaliou diferentes estímulos e tempos de exposição para desenvolver um novo protocolo de bioensaio de baixo custo para testar repelentes contra A. aegypti. O módulo de teste foi projetado para apresentar diferentes fontes de estímulos, ou seja, sangue humano, refeição de sangue artificial (SkitoSnack) e antebraço humano (estímulo padrão) e tempos de exposição, i.e, 2, 5 e 10 min. Dez fêmeas foram introduzidas no módulo, i.e, uma gaiola adaptada de polipropileno de 480 mL, e o número de pousos e ingurgitamentos nas fontes de estímulo foi registrado. Posteriormente, o Tempo Completo de Proteção (TCP) do DEET foi avaliado. Os mosquitos A. aegypti consistentemente pousaram no módulo, com uma média de 4,3 mosquitos no primeiro min, 5,3 no segundo min e 6,5 no terceiro min, após o que o número diminuiu após oito min. Da mesma forma, para ingurgitamento que aumentou de 3,0 mosquitos após dois min para 7,7 mosquitos após dez min, em todas as condições testadas, independentemente da fonte de estímulo. No entanto, a exposição prolongada aos estímulos levou a um aumento tanto nos pousos, quanto no ingurgitamento. O TCP do DEET ao usar fontes de estímulo alternativas (≈ 48 min) diferiu daquele da fonte humana (75 min), mas ambos demonstraram resultados antialimentação semelhantes. Além disso, tanto o SkitoSnack quanto o sangue, quando oferecidos por meio de um alimentador artificial com um tempo de exposição de 5 min, são eficazes para uso em experimentos com nosso módulo para teste repelente.

Palavras-chave:
alimentação artificial; ensaio com membrana; repelente; in vitro

1. Introduction

The term “repellent” refers to substances that modify various behavioral patterns, such as host-seeking and host acceptance, thereby reducing the risk of pathogen transmission (Mulatier et al., 2022). Deletre et al. (2016) proposed classifying repellents based on behavioral responses, distinguishing them into true repellents, odor-masking agents, contact irritants, antifeedants, and visual-masking agents. Consequently, each type requires specific testing methods. Conventional arm-cage assays (WHO, 2009) and various in vitro tests (Ali et al., 2017; Morimoto et al., 2021) evaluate attraction inhibition or odor masking by measuring reductions in host-seeking behavior, typically indicated by fewer landings on the host or stimulus source. These assays also assess antifeedant effects, where contact with an antifeedant inhibits feeding, as evidenced by a lower number of engorged mosquitoes.

Although the repellency of various substances against mosquitoes is often tested using humans as the stimulus source in conventional arm-cage assays (WHO, 2009), some in vitro tests such as the modified 6-celled Klun & Debboun module design (Klun et al., 2005) and Hemotek membrane feeding system (Cockcroft et al., 1998) have been shown to produce comparable results. These in vitro methods are particularly useful for preliminary screening of potential repellent compounds, as they eliminate the need to expose human subjects to untested substances and mitigate the inherent physical and chemical variability among volunteers (Cockcroft et al., 1998; De Obaldia et al., 2022).

In vitro test modules are widely employed in mosquito repellent research, offering a rapid and cost-effective assessment approach. However, their designs vary considerably, ranging from simple devices (Bar-Zeev and Smith, 1959; Klun et al., 2005; Jahn et al., 2010) to more complex experimental apparatuses (Ali et al., 2017; Morimoto et al., 2021). During routine artificial blood feeding of mosquitoes in our lab—using a modified method from Siria et al. (2018) and after unsuccessful attempts to construct a KD module (Klun et al., 2005), we developed a simple, affordable alternative. We then evaluated the effects of different stimulus sources—human forearm (standard stimulus), human blood, and an artificial blood meal (SkitoSnack; Gonzales et al., 2018) as well as varying mosquito exposure times (2, 5, and 10 min) on A. aegypti Linnaeus, 1762 landing and engorgement behavior. These response variables were analyzed to establish a bioassay protocol that eliminates direct human skin contact, facilitating the preliminary screening of potential mosquito repellents. Finally, we assessed the Complete Protection Time (CPT) of DEET (N,N-Diethyl-3-methylbenzamide) using the different stimulus sources.

2. Material and Methods

2.1. Ethical aspects

All procedures for collecting human blood for blood feeding and carrying out all tests were submitted to the Research Ethics Council of the Federal University of Rondônia (CAAE: 63761422.6.0000.5300), as well as the use of forearms after the laboratory volunteers' agreement by the Free and Informed Consent Form.

2.2. Mosquito rearing

The experiments were conducted using A. aegypti females from the colony maintained at the Laboratório de Bioecologia de Insetos (LaBEIn), Universidade Federal de Rondônia (UNIR), Porto Velho, Brazil. This colony originated from wild females collected on the university campus and was confirmed virus-free through molecular analysis.

Briefly, three-day-old colony females were starved for 48 h before being fed human blood via an artificial feeder (Siria et al., 2018). After 72 h, females were placed inside screened plastic containers (19 × 18 cm) containing plastic cups (7 × 5 cm) lined with filter paper strips as oviposition substrates, along with 100 mL of dechlorinated, filtered water.

Paper strips with eggs were transferred to plastic trays (30 × 22 × 8 cm) filled with one liter of dechlorinated filtered water to induce larval hatching. First- and second-instar larvae were fed ad libitum with finely ground fish food (TetraMin® Tropical Flakes). Third-instar larvae (L3) were transferred to screened plastic containers (17 cm high, 12 cm in diameter) filled with one liter of dechlorinated, filtered water and fed Reptolife® reptile food pellets until adult emergence. Adults were transferred to new cages and fed daily with 10% sucrose soaked in a cotton pad, maintained in controlled laboratory conditions of temperature between 26-27ºC, humidity 70-80% and 12 h photoperiod

2.3. Module design and construction

The mosquito repellent test module was designed using a 480 mL polypropylene container (11.5 x 10 x 7.5 cm), with a snap-on lid (Plasvale®). We drilled two holes using a hole saw: (1) on one of the sides (1.56 cm diameter) used to insert and remove of mosquitoes, sealed with EVA foam to prevent escaping and (2) on the top cover of the container (3.54 cm diameter), for mosquitoes to access the polyester screen – organza (12 cm²), placed on an EVA foam edge to avoid screen/container contact (Figure 1).

Figure 1
Module design for in vitro repellent assays with A. aegypti, (A) module size and side hole; (B) side hole sealed with EVA foam; (C) upper hole; and (D) upper hole with EVA foam support and impregnated screen.

For decontamination, the modules were washed with a mixture of neutral detergent and 10% hypochlorite, rinsed with water up to three times so that the entire mixture was completely removed and left to dry for 24 h. After drying, 70% alcohol was sprayed over its entire surface.

2.4. Mosquito selection for experiments

Nulliparous A. aegypti females, aged 5 to 7 days, were fasted for 36 h before the experiment, based on gut-emptying times observed in previous studies. Prior to testing, their host-seeking status was assessed by introducing the researcher's forearm—previously washed with neutral detergent and rinsed with 70% ethanol—into a cage (4.1 L) containing 50-100 mosquitoes. The number of females that landed and/or probed was recorded over 30 s.

To proceed with the experiment, a minimum of 10 landings and/or probings was required (WHO, 2009).

2.5. Evaluation of different stimuli sources and exposure times on mosquito landing and engorgement

Three stimulus sources were evaluated: (i) a human forearm (standard stimulus – WHO, 2009), (ii) human blood, and (iii) SkitoSnack, an artificial blood meal (Gonzales et al., 2018). Four human volunteers (two males and two females, aged 23-25 years) with no or minimal allergic reactions to mosquito bites were selected for testing. Before the test, each volunteer's forearm was washed with water and neutral soap, followed by 70% alcohol.

Human blood was obtained from the same volunteers after they completed an Informed Consent Form (ICF). SkitoSnack was prepared using bovine serum albumin, bovine hemoglobin, chicken egg yolk, glucose, and ATP, diluted in a previously prepared solution containing distilled water, sodium chloride (NaCl), magnesium chloride (MgCl₂), potassium chloride (KCl), calcium chloride (CaCl₂), and sodium bicarbonate (NaHCO₃) (Gonzales et al., 2018). All stimulus sources, except the forearm, were offered using an artificial feeder warmed to 38°C, adapted from Siria et al. (2018).

Prior to the experiments, the module and feeders were prepared, and 10 female mosquitoes were introduced into the module using a handheld aspirator. The mosquitoes were initially confined with a paper cover for 5 min, after which the cover was removed, and a polyester screen and the stimulus source were placed on top of the module (Figure 2). Each experiment was performed in duplicate and repeated five times, using female A. aegypti from different generations. Laboratory conditions were controlled, maintaining a temperature of 27-29 °C and humidity of 60-75%.

Figure 2
Bioassay module setup with A. aegypti females and a stimulus source, using an artificial feeder modified from Siria et al. (2018).

During experiments with different stimulus sources, the number of mosquito landings per min and the number of engorged mosquitoes were recorded at different time intervals (2, 5, and 10 min) in independent tests. After each test, mosquitoes were frozen and discarded.

2.6. Complete Protection Time (CPT) of DEET

Experiments were conducted using ten female mosquitoes fasted for 36 h, with different stimulus sources as previously described. Each experiment was repeated twice with different mosquito generations. Five human volunteers (two males and three females, aged 23-30 years) with no or minimal allergic reactions to mosquito bites were selected for testing.

A DEET stock solution (522.8 nmol/µL) was prepared by diluting 10 µL of 97% DEET (Sigma Aldrich) in 90 µL of absolute ethanol (Anidrol Produtos para Laboratórios Ltda). Five microliters of this stock solution were further diluted in 20 µL of ethanol and applied to a 12 cm² polyester screen, resulting in a final concentration of 200 nmol/cm². The polyester screen was dried for 10 min before being placed on top of the module.

The Complete Protection Time (CPT) was determined as the time elapsed between the moment the impregnated screen was exposed and the first mosquito landing (WHO, 2009). Additionally, we evaluated the mean number of mosquitoes that fed after CPT for each stimulus source 225 min post-exposure (the maximum time required to reach >80% engorgement in the control group).

During the experiment, the water temperature in the feeders was monitored every 10 min and replaced whenever it fell below 38 °C. The forearm stimulus was kept on top of the module continuously for 2 h, followed by 5 min exposures every 30 min until the experiment concluded.

2.7. Statistical analysis

The number of landings in different exposure times and stimuli sources was analyzed using repeated measures Two-Way Anova for (time x source) and comparisons were made using the Sidak test. The engorgement in each time interval using different stimuli source, the complete protection time (CPT), the number and time for landings after the CPT were analyzed by One-Way Anova and the comparisons were made by the Tukey test. A simple regression (engorged mosquitoes and time) was used to compare the stimuli sources was used. Analyzes were performed using Graphic Prism 10 software (GraphPad Inc).

3. Results

3.1. Effect of exposure time and stimulus source in mosquito’s landings and engorgement

The mean number of mosquito landings varied for different exposure times but was not affected by the stimuli sources used (p>0.05). The mean number of mosquitoes that landed in the 1^st min (4.3) increased significantly in the 2^nd min (5.3) (F=7.80; p=0.0071). Mosquito landing increased up to the 3^rd min (6.5) during the 5 min exposure (F=22.32; p<0.0001) but decreased after the 8^th min during the exposure time of 10 min (F=15.82; p<0.0001) (Figure 3).

Figure 3
Number of A. aegypti females’ landings in different exposure times (A) two minutes, (B) five minutes, (C) ten minutes and stimuli sources. Legend: Different letters indicate significant differences (P<0.05) in the number of landings in relation to the periods. NS=Not significant. Two-Way ANOVA (stimulus x time). Each experiment was performed in duplicate and repeated five times, using female A. aegypti from different generations.

Mosquito engorgement increased as the exposure time increased (F=6.57; p=0.0018) regardless the stimulus source used as no significant difference was observed in slopes (F=1.44; p=0.2410) (Figure 4) ranging from 3.0 during 2 min to 7.7 mosquitoes after 10 min. The number of engorged mosquitos did not differ among different stimuli sources for 2 min exposure (F=0.54; p=0.587) but fewer mosquitos engorged SkitoSnack (5.6) compared to other sources (7.3-7.6) tested when exposed for 10 min (p=0.035 and p=0.0067) (Figure 5).

Figure 4
Number of A. aegypti females engorged in different exposure times (A) two minutes, (B) five minutes, (C) ten minutes and stimuli sources. Legend: NS=not significant (P>0.05). Different letters indicate significant differences (P<0.05) in engorgement in relation to different stimuli. Different numbers indicate significant differences between the exposure times. One-Way ANOVA. Each experiment was performed in duplicate and repeated five times, using female A. aegypti from different generations.

Figure 5
Correlation between the number of engorged A. aegypti mosquitoes and exposure time across different stimuli sources. Legend: SkitoSnack: Y = 0.3597*X + 2.462; Blood: Y = 0.5469*X + 2.417 and Forearm: Y = 0.5199*X + 2.937.

3.2. Complete Protection Time (CPT) of DEET using different stimuli sources

The CPT of DEET using SkitoSnack and blood as stimuli sources was 32% lower compared to the forearm (75 min) (Figure 6A). Despite that, after the total observation time (225 min) of the experiment with DEET, the mean number of engorged mosquitoes (≈ 3.0) did not differ between the stimuli sources used but mosquito engorgement in the control was 64% higher compared to DEET treatment (F=399.1; p<0.0001) (Figure 6B).

Figure 6
Complete Protection Time (CPT) of DEET to A. aegypti (A) and Number of engorged mosquitoes exposed to different stimuli sources (B). Legend: Different letters indicate significant differences (P<0.05) between the different stimuli for each variable. CPT= Complete Protection Time (time elapsed from the application of the repellent and the landing of the first mosquito). NS= Not significant (P>0.05). Each experiment was repeated twice with different mosquito generations. Five human volunteers (two males and three females, aged 23-30 years).

4. Discussion

In this study, we developed a simple and cost-effective module to evaluate both mosquito landings, as a measure of attraction inhibition, and engorgement, to assess the anti-feeding effect of test compounds (see Deletre et al., 2016 for review).

Our findings align with previous studies demonstrating that heated human blood, circulated with hot water in a module, can effectively replace humans as an attraction source for A. aegypti and Anopheles stephensi Liston, 1901 (Klun et al., 2005). The authors reported that the proportion of mosquitoes prevented from biting in in vitro assays using DEET (0.81-0.86) was similar to results obtained with human subjects (0.98-0.68), respectively. Additionally, the proportion of bites observed in in vitro and in vivo controls (0.45 and 0.47, respectively) closely matched those recorded in our module after five min of exposure.

Similarly, Cockcroft et al. (1998), although not providing data on negative controls, also found that DEET-containing products had comparable effective doses when tested on A. aegypti using a collagen membrane technique (Hemotek) versus human arm tests, both observed over a five-min period.

Although some in vitro tests suggest that heated blood can effectively replace humans in mosquito feeding modules, the use and accessibility of blood samples raise ethical and biosafety concerns, often requiring additional time, specific laboratory conditions, and specialized personnel. To address these challenges, we also tested SkitoSnack (Gonzales et al., 2018) as a blood substitute for A. aegypti.

Blood substitutes have been evaluated in in vitro repellency tests with A. aegypti (Klun et al., 2008; Huang et al., 2015) and An. stephensi (Klun et al., 2008), but they were not compared directly to humans as a stimulus source. In our study, engorgement results using SkitoSnack were comparable to those reported for CPDA-1 (Klun et al., 2008) and the cocktail meal (Huang et al., 2015) at similar exposure times. However, due to their simpler and more cost-effective formulations, these alternatives may represent viable options after direct comparison with human-based stimuli. Despite this, SkitoSnack resulted in lower engorgement compared to human blood and human forearm in longer exposure time, i.e., 10 min. Although SkitoSnack meets the basic nutritional needs of A. aegypti as a substitute for vertebrate blood (Gonzales et al., 2018), its composition is much simpler compared to other stimuli, such as human blood or a human forearm. This simpler composition may limit the presence of certain chemical cues found in natural blood sources, e.g. Allan et al. (2006) argued that attractants present in natural blood sources using membrane assays places in olfactometers lead to greater attraction of A. aegypti when compared to water control.

In our investigation to determine the shortest exposure time yielding the highest number of mosquito landings and engorgement, a 5 min exposure provided the best results for both variables. This duration should be used in our bioassay for testing potential repellents. Previous in vitro repellency tests with A. aegypti have used exposure times of 3 min (Klun et al., 2005), 5 min (Adnan et al., 2020), and 10 min (Huang et al., 2015). However, we found no studies directly comparing different exposure times to optimize mosquito landing and engorgement outcomes. Despite this, the probing and engorgement observed in our study after 5 and 10 min of exposure were consistent with those of previous studies. In contrast, exposure times in in vitro tests tend to be significantly longer than those used in arm-in-cage tests, which follow the WHO (2009) protocol of 30 s. However, this shorter exposure time does not allow for the assessment of anti-feeding effects of test compounds.

During DEET tests for CPT evaluation, we used a fixed dose of 200 nmol/cm², based on preliminary tests. This is equivalent to 38.3 µg/cm² (3.8%). However, in vitro experimental conditions, such as application method and study design, significantly influence the outcomes. For example, Adnan et al. (2020) reported a CPT of 3 h (180 min) for 2.5% DEET applied to nets covering feeders in a bioassay with A. aegypti—a duration significantly longer than the CPT observed in our study. Conversely, using the same mosquito species, Jahn et al. (2010) found a mean CPT of 53.3 min for 25% DEET (1315 nmol/cm²) applied directly to the membrane in the feeder, which was similar to our results using only 200 nmol/cm² of DEET.

Feaster et al. (2009) examined DEET repellency in A. aegypti and Anopheles albimanus Wiedemann, 1821, both in vitro and on human subjects. For An. albimanus, they observed a CPT of 4 h (240 min) with 5% DEET (263.2 nmol/cm²) in human-based tests, which was considerably longer than their in vitro results—a pattern also observed in our study using the human forearm as a stimulus source for A. aegypti.

However, caution is needed when comparing in vitro and human-based tests, as we observed significant CPT variability among volunteers—118 min compared to 44 min with blood and 58 min with SkitoSnack. This variability may have biased CPT estimates when using humans as a stimulus source, likely due to the small sample size and individual differences in mosquito attractiveness (De Obaldia et al., 2022). Despite this, no significant differences in anti-feeding effects were detected between the various stimulus sources treated with DEET, even after 255 min of testing. Interestingly, the estimate duration of DEET repellency can be reduced to less than 40% after 4 h of application at much higher concentrations (0.2 mg/cm²) (Goodyer and Schofield, 2018) compared to those used in the present study (38 µg/cm²). This highlights that the anti-feeding effect can persist even as repellency decreases.

Although we observed similar trends in A. aegypti engorgement over time with different stimulus sources, quantitative differences were evident. Klun et al. (2005) reported a similar reduction in Ae. aegypti bites with increasing repellent concentrations in both in vitro and human tests. However, the distinction between compounds was more pronounced in in vivo tests. Despite this, the in vitro bioassay presented here provide a controlled, ethical, and cost-effective approach (De Obaldia et al., 2022; Klun et al., 2005) for evaluating mosquito repellents prior to in vivo studies, which remain essential for final confirmation.

5. Conclusions

A. aegypti mosquitoes consistently landed on and fed from the tested module across all conditions, regardless of the stimulus source. However, prolonged exposure to the stimuli led to an increase in both landings and feedings. The CPT of DEET when using alternative stimulus sources differed from that of the human source, yet both demonstrated similar antifeeding outcomes. Additionally, both SkitoSnack and blood, when offered through an artificial feeder with a 5-min exposure time, are effective for use in experiments with our module for repellent testing.

Acknowledgements

We thank our colleagues from the Laboratory of Insect Bioecology (LaBEIn) for their support during the rearing of the mosquitoes and during experiment set up. We also thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship and the Instituto Nacional de Ciência e Tecnologia de Epidemiologia da Amazônia Ocidental (INCT-EPIAMO) for providing financial support for this research.

Data Availability Statement

The entire data set that supports the results of this study was published in the article itself.

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Edited by

• Editor:
  Ana Paula Peron

Publication Dates

• Publication in this collection
  26 Sept 2025
• Date of issue
  2025

History

• Received
  02 Jan 2025
• Accepted
  02 July 2025