Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes
August 7th, 2018
For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.
This method can help to answer key questions in the field of mosquito-borne diseases, such as whether or not a mosquito species is a competant vector for a certain virus. The main advantage of this method is that a large amount of mosquitoes can be analyzed simultaneously, without the use of laboratory animals like mice. Generally, individuals new to this method will struggle because it needs fine motor skills that can only be acquired through practice.
Visual demonstration of this technique is critical, as the key steps are difficult to learn;they require experience with the handling of mosquito and viruses, and also fine motor skills. To begin, dilute the virus stock. Then, mix expired human blood with fructose, filtrated bovine serum, and the virus stock.
Freeze 140 microliters of bloodmeal mix for further analysis via TCID50. Next, put two 50-microliter droplets of the bloodmeal mix in a plastic vial, and allow the mosquitoes to feed for two hours. After anesthetizing the mosquitoes, count the fully-engorged individuals, and transfer them to a new vial containing a cotton pad soaked in fructose.
Then, maintain the mosquitoes at 27 degrees Celsius and 80%humidity for 14 or 21 days. Replenish the fructose-saturated cotton pads every 72 hours with 1.5 milliliters of fructose. First, seed 20, 000 Vero cells per well in a 96-well plate with supplemented growth medium.
To prepare the salivation device, place a squared glass plate onto the bench at a 30-degree angle. Then, adhere double-sided tape to the top of the glass plate. Next, roll modeling clay until it is 0.5 centimeters in diameter, and attach it to the plate horizontally one centimeter away from the double-sided tape.
Cut the tips off of the requisite number of 10-microliter filter tips, and fill each tip with 10 microliters of PBS. Then, place the tips on the modeling clay, and secure them with gentle pressure. Next, immobilize the mosquitoes by removing their legs and wings.
Fix the mosquitoes to the tape above the filter tips. Gently place the proboscis of each mosquito into a filter tip. After 30 minutes, remove the filter tips, and discard the clay and tape.
Transfer the collected saliva to reaction tubes containing 10 microliters of PBS. Mix the saliva and PBS gently, and transfer the mixture to the wells of a 96-well plate. Incubate the plate for seven days at 37 degrees Celsius with 5%carbon dioxide.
Using a light microscope, check the cells for the presence of cytopathic effect. Once a well is positive for cytopathic effect, use a pipette to harvest 140 microliters of the supernatant. Then, transfer the supernatant to a reaction tube.
Next, mix 140 microliters of the supernatant with 560 microliters of AVL buffer, and incubate for 10 minutes at room temperature. Finally, add 560 microliters of ethanol, and invert the sample to mix. Using this protocol, several mosquito species were tested for Zika-Virus infection and transmission under various climatic conditions.
Under 27-degree-Celsius incubation conditions all Aedes species displayed Zika-Virus-positive saliva. Conversely, none of the saliva samples from the Culex taxa contained evidence of the virus. Once mastered, this technique can be performed for 50 mosquitoes in one hour if performed properly.
After watching this video you should have a good understanding of how to perform forced-salivation experiments. Don't forget that working with infected mosquitoes can be very dangerous, so special safety measures for the handling of mosquitoes under BSA-3 conditions should always be taken while performing any experiment.
Overview
This article discusses a method for analyzing the vector competence of mosquitoes, specifically Aedes albopictus and various Culex taxa, for Zika virus transmission. The forced salivation technique allows for the simultaneous analysis of multiple mosquitoes without the use of laboratory animals.
Key Study Components
Area of Science
- Vector biology
- Virology
- Mosquito-borne diseases
Background
- Understanding vector competence is crucial for controlling mosquito-borne viruses.
- Forced salivation is a novel method for assessing mosquito transmission potential.
- Previous methods often required laboratory animals, which this technique aims to eliminate.
- Fine motor skills are essential for this method, necessitating practice and experience.
Purpose of Study
- To evaluate the vector competence of different mosquito species for Zika virus.
- To demonstrate a method that allows for high-throughput analysis of mosquito saliva.
- To provide a visual guide for researchers new to the technique.
Methods Used
- Preparation of a bloodmeal mix for mosquito feeding.
- Forced salivation technique to collect saliva from mosquitoes.
- Cell culture methods to assess the presence of Zika virus.
- Incubation and analysis of samples for cytopathic effects.
Main Results
- Aedes species showed positive saliva for Zika virus under controlled conditions.
- Culex taxa did not exhibit evidence of Zika virus in their saliva.
- The method allows for testing of up to 50 mosquitoes in one hour once mastered.
- Visual demonstrations significantly aid in learning the technique.
Conclusions
- Forced salivation is an effective method for studying mosquito-borne virus transmission.
- Training and practice are essential for successful implementation of the technique.
- Safety measures must be adhered to when handling infected mosquitoes.
Frequently Asked Questions
Executive Industry Relevance
Forced salivation assays enable high-throughput evaluation of mosquito vector competence without animal models, supporting early-stage target validation in antiviral discovery. The method provides quantitative, replication-competent virus detection from individual insects, informing go/no-go decisions for prophylactic or therapeutic candidates against arboviruses. Its scalability and reproducibility enhance predictive confidence in preclinical de-risking of transmission-blocking interventions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by measuring virus transmission potential in individual disease vectors.
- Operational Value: Enables biological de-risking of targets through direct assessment of vector competence under controlled conditions.
- Predictive Value: Supports portfolio triage by identifying non-competent vector species, reducing false-positive leads in arbovirus programs.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream antiviral screening by confirming virus susceptibility in vector models.
- Operational Value: Standardizes saliva collection and virus detection, ensuring reproducible quantitative outputs across large mosquito cohorts.
- Scalability: Facilitates platform reuse for high-throughput evaluation of transmission-blocking compounds or vaccines.
Translational & Preclinical Research
- Scientific Value: Aligns with disease-relevant systems by testing vector competence under epidemiologically relevant temperatures (18–27°C).
- Operational Value: Ensures continuity from discovery to preclinical validation via consistent saliva-based virus readouts.
- Risk Mitigation: Informs advancement decisions by establishing temperature-dependent transmission thresholds for lead candidates.
Pipeline & Workflow Integration
The forced salivation method fits within the discovery continuum from target validation through lead identification to preclinical evaluation, particularly for arbovirus-directed interventions.
- Discovery Biology: Supports hypothesis testing and pathway clarification by quantifying virus presence in mosquito saliva as a functional readout of transmission potential.
- Screening: Delivers assay readiness and reproducibility through standardized saliva collection and TCID50-equivalent virus detection in Vero cells.
- Analytics: Generates quantitative dependent variable measurements (viral titer in saliva) enabling comparison across species, strains, and environmental conditions.
- Translational Research: Connects to preclinical continuity via temperature-stratified transmission data that mirrors field-relevant transmission windows.
- Enterprise Reuse: Functions as a reusable capability for screening multiple arboviruses (e.g., ZIKV, DENV, CHIKV) across vector species without reoptimization.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation, reduction of mechanistic ambiguity in transmission biology.
- Operational Value: Standardization, reproducibility, and scalability across entomology and virology teams.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk in antiviral programs.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions based on empirical vector competence data.
Implementation Considerations
- Requires expertise in mosquito handling, virology, and microsurgical techniques under BSL-3 containment.
- Dependent on specialized instrumentation including salivation devices, incubators with precise humidity control, and microscopy for cytopathic effect scoring.
- Necessitates cross-team standardization of bloodmeal preparation, incubation timelines, and saliva collection intervals.
- Adaptation considerations include species-specific proboscis size, feeding behavior, and salivary gland morphology across vector models.
- Practical limitations include the need for extensive training to achieve proficiency and the method’s dependence on viable virus recovery from saliva.
