Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.
New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever, the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.
Mosquitoes transmit the causative agents of infectious diseases that impact human health globally1. The three most important mosquito genera affecting human health are Aedes, Anopheles, and Culex. Species of Aedes mosquitoes vector the arboviruses that cause Zika, dengue, yellow fever and chikungunya. Anopheles species vector malaria parasites and Culex species transmit West Nile virus and filarial nematodes2. The World Health Organization (WHO) has called for the eradication of ten Neglected Tropical Diseases (NTD) by 20203 and identified mosquito control as the most viable strategy. Insecticides are powerful tools to control mosquitoes and reduce disease transmission4,5. However, the emergence of insecticide-resistant mosquito populations threatens the continued control of many diseases6,7,8. The Innovative Vector Control Consortium (IVCC) has launched the first dedicated effort to develop replacement insecticides for mosquito control9. New insecticides with novel modes of action (MoA) are needed to meet WHO goals and achieve disease control. The discovery and development of new insecticide classes with novel MoAs require whole organism assays for rapid, high-throughput analysis of chemistries and in comparison to industry standards10.
Multiple assays are available to evaluate the repellency and toxicity of insecticides to larvae and adult mosquitoes, and some are suitable for the evaluation of novel formulated or unformulated chemistries. The WHO protocol11 is a contact assay used to test products against fourth instar larvae. Published studies also describe a variety of larval assays used to test the toxicity of small molecule chemistries to larvae of Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus10,12,13,14,15,16. The Centers for Disease Control and Prevention (CDC) bottle bioassay is used to assess insecticide resistance in field caught populations of adult mosquitoes via evaluation of lethal concentration (LC) and lethal time (LT) in comparison to a standard diagnostic dose of a commercial insecticide17. The WHO susceptibility test offers another approach to evaluate insecticide resistance in adult mosquitoes whereby mosquitoes are exposed to insecticide-impregnated paper inside glass vials for 1 h and mortality is assessed at 24 h18. Other assays spray formulations inside sealed cages and record mosquito knockdown and death at desired timepoints19. Topical application to the mosquito thorax has been used to evaluate potency of different pesticides20. The Liverpool Insect Testing Establishment (LITE)21 employs Standard Operating Procedures to evaluate the bioactivity of chemistries to adult mosquitoes. The LITE adult topical assay applies a small volume of test product to the thorax of adult mosquitoes with mortality scored at 48 h. The assay enables quantification of the dose received per individual mosquito21. The LITE tarsal assay evaluates the toxicity of test product to adult mosquitoes resting on a chemically treated surface and is used to identify chemistries with potential for development as indoor residual sprays (IRS) or use in insecticide treated bed-nets (ITNs) to control Anopheles mosquitoes.
The WHO and CDC assays have limitations for testing novel, unformulated chemistries, and the goal of the CDC bottle bioassay is evaluation of susceptibility over short assay periods (two hours) rather than toxicity22,23. In addition, assays for evaluating the toxicity of chemistries delivered in the blood meal and potential for development of systemic-acting products (i.e., endectocides) are lacking. Here, we describe assays for testing the mosquitocidal activity of novel chemistries that are soluble in water or one of several organic solvents against the larvae and adults of Aedes, Anopheles and Culex. First, we demonstrate a larval assay previously described by our group10,12,13,16, and performed here as (1) a screen to rapidly evaluate multiple chemistries at single point dose, and (2) a dose response assay to determine the lethal concentration (e.g., LC50, LC75 or LC90,) of a single chemistry. Next, we describe two adult assays for determination of lethal dose (LD). The first of these is an adaptation of the LITE protocol for testing of topically applied, novel chemistries against adult mosquitoes in comparison to a positive control. The second is a feeding assay for the evaluation of chemistries delivered systemically via the blood meal.
NOTE: All strains and reagents required for the work described in the following protocols, and suppliers are listed in the Table of Materials.
1. Culture of Mosquito Larvae and Adults
NOTE: Insecticide susceptible strains of mosquitoes are available from the Malaria Research and Reagent Reference Repository. Recommended strains are as follows: Aedes aegypti Liverpool (LVP) strain, Anopheles gambiae Kisumu (KISUMU1) strain and Culex quinquefasciatus Johannesburg (JHB) strain.
2. Larval Contact Assay (Single Point Dose or Dose Response Assay)
NOTE: The assay can be conducted with chemistries that are soluble in water or dimethyl sulfoxide (DMSO), provided that the final concentration of DMSO in test wells does not exceed 1%. Alternative solvents may be an option, but it is essential to first confirm that the final concentration does not cause more than 10% mortality at 72 h post exposure. The assay may be performed as a single point dose or dose response assay. If performing the latter, it is recommended to test a range of concentrations (minimum of five), spanning the expected LC50.
3. Adult Topical Assay (Single Point Dose or Dose Response Assay)
NOTE: The adult topical assay is conducted using acetone as a solvent. Alternative solvents may be an option, but it is essential to first confirm that the final concentration does not cause more than 10% mortality at 48 h post exposure. The assay may be performed as a single point dose or dose response assay and is used to determine lethal dose (LD). If performing the latter, it is recommended to test a range of concentrations (minimum of five), spanning the expected LD50.
4. Adult Blood-Feeding Assay (Single Point Dose or Dose Response Assay)
Figure 6 shows the percent mortality of L3 Ae. aegypti following exposure to five doses of the dopamine receptor antagonist, amitriptyline in comparison to the water only (negative) control at 24, 48 and 72 h. In this example, the data collected at each of the time-points display a typical logarithmic curve and reveal a dose-dependent effect of amitriptyline on larval mortality. The LC50 value decreases over the time course of the experiment and can be calculated using the 72 h data (i.e., midpoint of the logarithmic curve).
Figure 7 shows the percent mortality of 3 – 5-day-old adult females Ae. aegypti following exposure to a 400 µM dose (10 µg/µL) of the dopamine receptor antagonist, amitriptyline and in comparison to the synthetic pyrethroid, bifenthrin-positive control (technical grade; 500 pg/µL), acetone-only negative control and untreated mosquitoes (blank). In this example, both amitriptyline and bifenthrin cause significant adult mortality relative to the negative control. Note that adult mortality decreases over time, presumably due to metabolic detoxification of test chemistries.
Figure 8 shows the percent mortality (left y-axis) of 3-5 day old adult female Ae. aegypti fed a blood meal treated with one of four doses of the dopamine receptor antagonist, amitriptyline, and the fecundity (right y-axis; average egg count/female) for the first gonotrophic cycle post exposure in comparison to control mosquitoes (blood-fed only; negative control). The data reveal that there is no significant effect of amitriptyline on adult mortality relative to the control but demonstrate that amitriptyline significantly increases fecundity at the highest dose (400 µM).
Figure 1. Set-up of the larval dose response assay. Image showing the set-up of the larval dose response assay performed using Aedes aegypti L3 larvae in 24-well plate.
Figure 2. Schematic showing procedure for set up of the (A) single point dose or (B) dose response larval assay in 24-well plate. Following transfer of five L3 larvae to wells, excess water is gently removed via pipette and replaced with fresh, sterile ddH2O. Next, stock and serial dilutions of the test chemistry are prepared and added to a final volume of 1 mL/well. Please click here to view a larger version of this figure.
Figure 3. Culture of adult mosquitoes. Image showing 20 L plastic bucket used for culture of adult mosquitoes and use of an aspirator to remove mosquitoes.
Figure 4. Adult topical assay performed using 4-5 day old female Aedes aegypti. Following treatment with chemistry or solvent, adult mosquitoes are placed in paper cups and transferred to a test chamber for the duration of the assay.
Figure 5. Stock and serial dilutions. Schematic diagram showing the procedure for preparation of (A) stock and (B) serial test solutions for the adult topical assay. Please click here to view a larger version of this figure.
Figure 6. Representative data from a larval contact dose response assay. Representative data from a larval contact dose response assay showing percent mortality of Aedes aegypti L3 larvae following exposure to the dopamine receptor antagonist, amitriptyline at 24, 48 and 72 h. Each data point represents mean in µM ± SEM. Data represent n = 3 biological replicates.
Figure 7. Representative data from an adult topical dose response assay. Representative data from an adult topical dose response assay showing toxicity of the dopamine receptor antagonist, amitriptyline (10 µg/µL) to 3-5 day old female Aedes aegypti relative to the synthetic pyrethroid, bifenthrin (500 pg/µL) positive control) and negative control (acetone vehicle only) at 2, 24 and 48 h. Data represent n = 3 biological replicates. Error bars indicate SEM.
Figure 8. Representative data from an adult ingestion assay. Representative data from an adult ingestion assay showing toxicity of the dopamine receptor antagonist, amitriptyline at 100 µM, 200 µM and 400 µM dose to 3-5 day old adult female Aedes aegypti and impact on fecundity (expressed as the average total egg count per female over the first gonotrophic cycle). Data represent n = 3 biological replicates ± SEM.
A. Single point dose assays with three chemistries | |||||
화학 | Concentration (400 uM) | ||||
Amitriptyline | 80 mM Stock: 2 mg of amitriptyline X 98% (purity of powder) X 0.001 g/1 mg X 1 mol /313.86 g X 1 L/0.08 mol X 106 µL/L = 78.06 µL of water. | ||||
Serial Dilutions: Dilute stock 1:2 with ddH2O and then 1:4 to achieve 10 mM. Add 40 µL of 10 mM stock to well containing 960 µL of ddH20 to achieve a final concentration of 400 µM/well. Prepare serial dilutions of 10 mM stock 1:2 to achieve 5 mM, 2.5 mM, 1.25 mM and 0.625 mM stocks. | |||||
Cis-(z)-flupenthixol | 80 mM Stock: 2 mg of cis(z)flupenthixol X 98% (purity of powder) X 0.001 g/1 mg X 1 mol/507.44 g cis(z)flupenthixol X 1 L/0.08 mol X 106 µL/L = 48.28 µL of water. | ||||
Serial Dilutions: As above. | |||||
Amitraz | 80 mM Stock: 2 mg of Amitraz X 0.001 g/1 mg X 1 mol/293.4 g of amitraz X 1 L/0.08 mol X 106 µL/1 L = 85.21 µL of DMSO (note: Amitraz is only soluble in DMSO). | ||||
Serial Dilutions: As above. | |||||
B. Dose response assay | |||||
화학 | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 |
400 µM | 200 µM | 100 µM | 50 µM | 25 µM | |
e.g., Amitriptyline, cis-(Z)-flupenthixol or amitraz | 40 µL | 40 µL | 40 µL | 40 µL | 40 µL |
10 mM stock | 5 mM stock | 2.5 mM stock | 1.25 mM stock | 0.625 mM Stock |
Table 1. Example calculations for Aedes aegypti L3 larval (A) single point dose assay and (B) dose response assay.
Table 2. Example score sheet used to record data from the mosquito larval dose response assay. Control, water-only or water with 1% DMSO or other solvent only. Typical doses used to test small molecule antagonist chemistries such as amitriptyline are 25 µM, 50 µM, 100 µM, 200 µM and 400 µM.
Mosquito Adult (3-5 day old) Topical Assay | ||||||
Date initiated: | ||||||
Investigator: | ||||||
Species/Strain: | ||||||
Assay Type: Single point dose/dose response | ||||||
A. Mortality (No. dead mosquitoes) | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
0.5 | ||||||
1 | ||||||
1.5 | ||||||
2 | ||||||
2.5 | ||||||
B. Percent Mortality | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
0.5 | ||||||
1 | ||||||
1.5 | ||||||
2 | ||||||
2.5 |
Table 3. Example score sheet used to record data from the adult topical dose response assay.
Mosquito Adult (3-5 day old) Ingestion Assay | ||||||
Date initiated: | ||||||
Investigator: | ||||||
Species/Strain: | ||||||
Assay Type: Single point dose/dose response | ||||||
A. Number Fed Mosquitoes | ||||||
Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control | |
Fed | ||||||
Not Fed | ||||||
Total | ||||||
% Fed | ||||||
% Not Fed | ||||||
B. Mortality (No. dead mosquitoes) | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
2 | ||||||
24 | ||||||
48 | ||||||
72 | ||||||
C. Percent Mortality | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
2 | ||||||
24 | ||||||
48 | ||||||
72 |
Table 4. Example score sheet used to record data from the adult blood-feeding assay. Typical doses used to test small molecule antagonist chemistries such as amitriptyline are 50 µM, 100 µM, 200 µM and 400 µM.
Insecticides are powerful tools to combat mosquito-borne diseases such as malaria25, dengue and Zika26. Extensive insecticide use has produced mosquito populations that are resistant to chemical control, leading to the development of insecticide resistance, which is considered the single biggest threat to continued disease control. The last decade has seen a dramatic increase in populations of Anopheles spp. resistant to synthetic pyrethroids (SPs) used in bed nets in Africa7. Populations of Aedes albopictus, a vector of dengue and Zika, have been reported in Florida and New Jersey that are resistant to organophosphates6. Similarly, resistance to DDT and pyrethroids has been documented in populations of Anopheles and Aedes spp. in Colombia27,28. There is urgent need to develop new larvicides and adulticides that bind to diverse orthosteric or allosteric sites on known molecular targets or that disrupt new targets (i.e., novel MoA chemistries)29,30 and have minimal environmental impact. There is also increasing recognition of the potential around endectocides for mosquito control31,32,33.
To facilitate the rapid development of such products, we describe a series of protocols designed to evaluate the toxicity of small molecule chemistries to mosquito larvae and adults via two delivery routes – contact and ingestion. Protocols such as the WHO susceptibility test and the CDC bottle assay are in common use for evaluating the toxicity and repellency of existing insecticidal formulations1,2. However, these assays have limitations for analyses of unformulated novel chemical entities (NCEs) and are not amenable to testing large numbers of chemistries in moderate or high throughput.
Here we describe a standard larval assay to rapidly evaluate NCEs in single point dose that can scale for high throughput analyses. We also describe an adaption of an assay developed by the LITE21 to evaluate the contact toxicity of small molecule chemistries to adult mosquitoes. Finally, we describe an ingestion assay to evaluate toxicity of NCEs delivered via the blood meal to adult female mosquitoes and explore endectocide potential. These larval and adult assays can be performed at a range of doses in order to determine LC and LD values, respectively, and both larval and adult contact assays can be performed in comparison to one or more existing chemical formulations. The adult topical assay also enables the calculation of LD values per individual mosquito. The protocols can be performed using species of Aedes, Anopheles and Culex16,29 and could be modified to accommodate the specific biological characteristics of a species, if desired. In our experience, mosquitocidal chemistries typically exhibit broad activity across these three genera16,29, but there is value to assessment of species selectivity.
The assays are a starting point to rapidly screen multiple chemistries for insecticidal activity. Those chemistries that exhibit toxicity in one or more assays could be selected for further analyses via secondary and tertiary assays. Examples include the LITE tarsal assay and sugar feeding assays34,35. The selection of additional assays is typically determined by considerations around anticipated market and application, with results providing insights to possible product delivery modes. In the representative results shown here, we explore the toxicity of amitriptyline, an antagonist of mammalian and invertebrate dopamine receptors that has been evaluated for potential as a novel MoA insecticide10,12,13,16. Amitriptyline exhibited toxicity to larvae and adults of Ae. aegytpi in the μg range suggesting larvicidal and adulticidal activity, and providing rationale for an exploration of an amitriptyline-based chemical series to identify analogues with high potency. No significant effect on mortality or fecundity was observed in Ae. aegypti exposed to amitriptyline in the blood meal. While the stability of the chemistry in defibrinated blood and effective dose received per mosquito is not known, notable limitations of the assay, the data suggest that amitriptyline and other GPCR-active compounds may have little potential as endectocides, at least in unformulated state.
Precautions to limit biological variation between assays are essential. All assays should be performed under standard conditions of temperature and humidity, preferably in an insect growth chamber, in order to ensure reproducibility. It is imperative to standardize procedures for scoring phenotypic end-points as these can be highly subjective, leading to significant variation in data recorded by different laboratory personnel. The larval assay is best suited to evaluation of chemistries that are soluble in water, although the assay can be performed using organic solvents such as DMSO provided that the final concentration is less than 1% per well. We recognize several limitations to the adult topical assay. First, it is necessary to work rapidly when preparing serial dilutions and delivering doses as the carrier (acetone) is highly volatile and evaporation may produce variation in the amount of chemistry delivered. Second, care should be taken to limit the period of anesthesia as this can contribute to mortality. It should also be noted that solvents such as acetone can produce a rapid "knock-down" effect in the first several hours of the topical assay, which should not be confused with effects due to the test chemistry. We note that the volume of the mosquito blood meal varies between individuals, complicating an assessment of the dose received per adult in the adult ingestion assay. Finally, assay data should not be considered in cases where the mortality in the negative control exceeds 10%.
Products that act synergistically are increasingly recognized for their potential to extend the utility of existing products and provide control of insecticide resistant pest populations15,16,36. Larval assays have been used to examine synergism between the formamidine pesticide, amitraz and a variety of chemistries that disrupt invertebrate octopamine receptors14,15. The larval assay and adult topical assay described are suitable for evaluating synergistic or additive effects between combinations of test chemistries. We note that synergists such as piperonyl butoxide (PBO) can be incorporated into each of the three assays described here.
The above-described assays provide a standardized experimental series for evaluation of unformulated, small molecule drugs against aquatic and terrestrial mosquito life stages. These assays are specifically designed to evaluate toxicity and offer several advantages over existing assays in common use. Importantly, the assays are amenable to automation and can be performed at industrial scale to enable evaluation of thousands of test compounds. Lastly, as the field considers the development of products that act via new MoAs, including via disruption of novel molecular targets and biochemical pathways, it will be essential to score multiple phenotypic endpoints in addition to those such as mortality and paralysis. The above assays permit such investigations, thus providing a step towards tomorrow's highly innovative insecticidal technologies.
The authors have nothing to disclose.
The authors thank H. Ranson, Liverpool School of Tropical Medicine for assistance with assay development and provision of the LITE protocol, and M. Scharf, Purdue University for advice regarding assay design.
Insecticide susceptible mosquito strains | Malaria Research and Reagent Reference Repository | https://www.beiresources.org/MR4Home.aspx; Recommended strains: Aedes aegypti Liverpool (LVP) strain, Anopheles gambiae Kisumu (KISUMU1) strain and Culex quinquefasciatus Johannesburg (JHB) strain | |
Acetone | Mallinckrodt Chemical | CAS 67-64-1 | Use for dilutions and control |
Amitraz | Sigma-Aldrich | CAS 33089-61-1 | Requires dilution in DMSO |
Amitriptyline | Sigma-Aldrich | CAS: 549-18-8 | Can be diluted in acetone |
Bifenthrin | Sigma-Aldrich | CAS: 51529-01-2 | Synthetic pyrethorid used as positive control |
Cis-(Z)-flupenthixol | Sigma-Aldrich | CAS: 51529-01-2 | Pharmacologically tested as disruptor of dop-like receptors |
Defibrinated rabbit blood | Hemostat | DRB0100 | Blood source for adapted mosquitoes to artificial feeding |
Dimethylsulphoxide (DMSO) | Sigma-Aldrich | MKBF2985 | Organic solvent used to disolve amitraz |
Hemotek membrane feeding system | Hemotek Ltd | Serial no. 1303 | Used for feeding mosquitoes |
Micro-applicator | Burkard Manufacturing Co. | – | Tool needed for topical application experiments |
24-well cell culture plate with lid | Corning Incorporated | 3526 | – |
Advantage rubber bands | Alliance Rubber Co. | – | Used to seal the paper cups |
Glass syringe (1 ml) | Becton Dickinson and Co. | 512004 | Needed for the micro-applications. Glass is better than plastic |
Disposable scintillation vials (20 ml) | Fisher Scientific | 74505-20 | Glass vials prevent evaporation |
Tulle fabric, white | Walmart | – | – |
Paper cups | Dixie Consumer Products LLC. | PF15675/13D | Used to keep adult mosquitoes in adult topical assays |
Petri dishes (150 mm) | Corning Life Sciences | – | Used to maintain the mosquitoes "slept" on cold without direct contact with ice |
Transfer pipettes | Fisher Scientific | 13-711-7M | Used to sort larvae |
Stereo microscope | Olympus | SZ6045 | Used to score larval assays and perform micro-applications |
Tweezers (fine) | Fontax | – | Used to handle adult mosquitoes |