WHO Cone Bioassay Test:
A Complete Practical Guide

1. What Is the WHO Cone Bioassay? Definition & Importance

The WHO cone bioassay is a standardized laboratory and field method, developed under the World Health Organization Pesticide Evaluation Scheme (WHOPES), used to evaluate the biological activity of insecticide-treated surfaces. It is the reference test for assessing long-lasting insecticidal nets (LLINs), insecticide-treated nets (ITNs), indoor residual spraying (IRS) on walls and ceilings, and other treated vector control materials. The assay works by confining a small, defined number of mosquitoes inside a plastic conical chamber placed against the treated surface for a fixed exposure period, and then recording knockdown and mortality over defined time points.

The test derives its name from the shape of the exposure chamber — a truncated plastic cone, open at the narrow end (which is sealed against the treated surface) and closed at the wide end with a mesh-covered opening through which mosquitoes are introduced and later removed. Because the cone restricts mosquitoes to tarsal and body contact with the treated surface only, it isolates the effect of the insecticide from other variables such as flight behavior or feeding response.

Cone bioassays are important because they provide a simple, low-cost, reproducible, and field-deployable method of measuring the residual efficacy of an insecticide treatment. Unlike laboratory chemical assays that only measure the concentration of active ingredient present on a surface, the cone bioassay measures actual biological effect on the target organism — mosquito knockdown and death — which is ultimately what determines a product's usefulness for disease prevention.

The method is central to WHO's prequalification process for vector control products, to national malaria and dengue control programmes that need to decide when nets should be replaced or walls re-sprayed, and to manufacturers who must demonstrate that their products meet WHO efficacy thresholds before, during, and after specified periods of use (including simulated washing for nets or weathering for IRS).

2. Why and When Cone Bioassays Are Performed

Cone bioassays are conducted across a wide range of research, regulatory, and programmatic contexts. Understanding when the test is applied helps clarify why the procedure is designed the way it is.

Product Development and WHO Prequalification

Manufacturers seeking WHO prequalification for a new LLIN or IRS insecticide formulation must submit cone bioassay data demonstrating that the product meets minimum knockdown and mortality thresholds, both when new and after standardized washing (for nets) or over time (for IRS). This is typically the first stage of efficacy evaluation, prior to experimental hut trials and community-level effectiveness studies.

Manufacturing Quality Control and Batch Release

LLIN manufacturers routinely use cone bioassays as part of batch release testing to confirm that insecticide incorporation or coating in a production run meets specification before the product is shipped.

Wash Resistance and Durability Studies

Because LLINs are designed to remain effective through repeated washing over 2–3 years of household use, WHO guidelines specify standardized washing regimens (e.g., 20 washes using a defined detergent, temperature, and agitation protocol) with cone bioassays performed after each wash interval to track the decline — or retention — of insecticidal activity.

Residual Efficacy Monitoring of Indoor Residual Spraying

After a wall or ceiling surface is sprayed as part of an IRS campaign, cone bioassays are performed at regular intervals (commonly 1, 3, 6, 9, and 12 months post-spray) to determine how long the insecticide remains effective against mosquitoes, which directly informs the timing of the next spray round.

Insecticide Resistance Monitoring

Comparing knockdown and mortality of a laboratory-susceptible reference strain against a field-collected mosquito population on the same treated surface helps quantify phenotypic resistance and its operational impact on product performance.

Comparative and Research Studies

Researchers use cone bioassays to compare different insecticide classes, next-generation nets (such as PBO-synergist nets or dual active-ingredient nets), and alternative wall substrates, generating evidence for policy decisions and procurement guidance.

3. Principle of the Test

The WHO cone bioassay is a contact exposure assay. A plastic cone is sealed against the treated surface so that its narrow open end forms an airtight boundary with the material being tested. A small, standardized number of mosquitoes — usually five — is introduced into the cone through a mesh-covered opening at the wide end, using an aspirator. The mosquitoes are confined within the cone for a fixed exposure period, during which they make tarsal (leg) and general body contact with the treated surface as they rest, walk, or attempt to fly within the chamber.

After the exposure period, mosquitoes are removed and transferred to clean holding cups, where knockdown is recorded at set intervals and mortality is assessed after a holding period. Because exposure is standardized in terms of mosquito number, contact duration, temperature, and humidity, differences in knockdown and mortality between test and control surfaces can be attributed to the insecticidal activity of the treatment rather than to extraneous variables.

4. Materials & Equipment Required

A properly equipped cone bioassay setup ensures standardized, reproducible results. The following items are required for routine testing.

5. Mosquito Selection, Handling & Pre-Test Care

The quality and physiological uniformity of test mosquitoes directly affects the reliability of cone bioassay results. Poorly conditioned or damaged mosquitoes can produce false knockdown or mortality readings unrelated to the insecticide being tested.

Mosquito Requirements

• Sex: Female mosquitoes only — the relevant target for disease transmission and host-seeking behavior.
• Age: 3–5 days old, allowing full cuticular hardening while remaining within a standardized physiological window.
• Parity: Nulliparous (have not yet laid eggs), which standardizes physiological condition and avoids variability introduced by blood-feeding or oviposition history.
• Feeding status: Sugar-fed until approximately one hour before testing, then sugar-starved immediately prior to exposure so that feeding behavior does not interfere with the assay.
• Condition: Only healthy, active, undamaged insects should be used — mosquitoes with missing legs, damaged wings, or visibly reduced activity should be excluded, as pre-existing injury can be mistaken for insecticide-induced knockdown.
• Strain: Use a WHO-recommended insecticide-susceptible reference strain for standard efficacy testing, or field-collected/F1 populations when the objective is resistance monitoring.

Handling and Care Before Release Into Test Chambers

Careful handling in the hours before testing is essential to avoid introducing stress-related mortality that could be misattributed to the treated surface.

• Gentle transfer only: Move mosquitoes using an aspirator rather than by hand at every stage — from rearing cage to holding cup, and from holding cup into the cone.
• Minimize handling frequency: Each additional transfer increases the risk of physical damage; batch mosquitoes into holding cups in the exact numbers required for each cone in advance.
• Avoid prolonged chilling or CO₂ immobilization: Brief cold knock-down or CO₂ may be used to ease transfer, but mosquitoes must be allowed at least 30–60 minutes to fully recover normal activity before exposure; residual sedation can be misread as insecticide-induced knockdown.
• Acclimatize before testing: Hold mosquitoes in the test room for approximately one hour before exposure so they equilibrate to the ambient temperature and humidity of the testing environment.
• Protect from desiccation: Keep a moistened cotton pad or sugar-soaked pad on holding cups at all times except during the brief pre-test starvation window, and avoid placing cups in direct sunlight or drafts.
• Reduce visual and physical disturbance: Keep holding cups covered and undisturbed in a quiet area; excessive vibration, light, or noise can elevate baseline activity and stress levels.
• Discard unsuitable individuals: Before loading a cone, visually inspect the batch and replace any mosquito that appears sluggish, injured, or abnormally positioned.
• Match testing time to design intent: Where possible, schedule testing to coincide with the mosquito species' natural activity period (for example, many Anopheles species are most active around dusk and at night), since activity level can influence contact rate with the treated surface.

6. Types of Test Surfaces Used

The WHO cone bioassay is applied to a variety of treated surfaces depending on the vector control intervention being evaluated.

Long-Lasting Insecticidal Nets (LLINs) and Treated Fabrics

Netting material is the most common surface tested. Nets vary by fiber type (polyester, polyethylene), construction (multifilament or monofilament), and insecticide incorporation method (coated or incorporated into the fiber during extrusion). Testing typically samples multiple net brands and treatment technologies, including PBO-synergist nets and dual active-ingredient nets.

Indoor Residual Spraying (IRS) Substrates

For IRS efficacy monitoring, cone bioassays are performed directly on sprayed wall or ceiling surfaces, or on portable standardized substrate panels prepared to represent common construction materials, including:

• Cement / concrete — representing urban and permanent housing structures.
• Mud plaster — representing traditional rural housing walls, one of the most common substrates in malaria-endemic settings.
• Wood / plywood — representing wooden housing panels or ceiling boards.
• Whitewashed or lime-washed surfaces — representing walls treated with traditional lime coatings that can affect insecticide persistence.
• Ceramic tile or painted surfaces — representing modern finished interior walls in some settings.

Treated Test Papers

Standardized filter papers impregnated with a known concentration of insecticide in a defined carrier oil are used as reference or calibration surfaces, particularly in insecticide susceptibility and resistance studies. While these are more commonly used in the WHO tube test, they are sometimes mounted behind cones as a controlled reference surface when comparing against field-treated net or wall samples.

7. Preparing Test Materials & Surfaces

Net Sample Preparation

• Cut treated fabric samples to a standard size, typically 25 × 25 cm.
• Sample from multiple representative positions on the net (e.g., roof panel and each of the four sides) rather than a single location, since insecticide distribution can vary across a net.
• Avoid cutting directly on seams, hems, or edges, which may have atypical insecticide concentration.
• Bring refrigerated or cold-stored samples to room temperature before testing to avoid condensation or handling stress.
• Label each sample piece with net identity, position, wash number (if applicable), and replicate number.

IRS Substrate Panel Preparation

• Prepare substrate panels (cement, mud plaster, wood, etc.) to standard WHO dimensions before spraying, ensuring a smooth, representative, and consistent surface finish.
• Cure or dry panels fully according to material type (mud plaster panels typically require several days of drying) before insecticide application.
• Apply the insecticide formulation at the target dosage using a calibrated sprayer, matching field application conditions as closely as possible.
• Allow the sprayed deposit to dry completely — generally at least 24 hours — before the first bioassay is conducted.
• Store panels under conditions representative of the monitoring objective: indoors under ambient conditions if assessing typical household residual life, or under controlled/accelerated weathering if simulating extended exposure.

Treated Test Paper Preparation

• Impregnate filter papers with a precise, pre-determined concentration of insecticide dissolved in the WHO-specified carrier oil (e.g., silicone oil or specified alternative).
• Dry impregnated papers fully and store away from light and heat until use to prevent degradation.
• Always prepare a matching control paper treated only with carrier oil, with no insecticide.

8. Test Conditions

Environmental conditions must be tightly controlled and continuously monitored throughout testing, since temperature and humidity directly influence both mosquito activity and insecticide performance.

• Temperature: 27 ± 2°C
• Relative humidity: 80 ± 20%
• Lighting: Maintain consistent, non-glaring ambient lighting; avoid direct sunlight falling on cones or holding cups.
• Timing: Where feasible, conduct testing during the target species' normal activity period to reflect realistic contact behavior.
• Airflow: Avoid direct drafts from fans, air conditioning vents, or open windows near the test setup, as airflow can affect both mosquito behavior and volatile insecticide loss.

A calibrated thermo-hygrometer should be placed near the test area and readings logged at the start and end of every exposure session.

9. Mounting & Securing the Cones

Correct mounting is critical: any gap between the cone rim and the treated surface allows mosquitoes to escape and invalidates the exposure. The technique for securing the cone depends on the surface type.

10. Exposure Procedure

1. Introduce five female mosquitoes into each secured cone using an aspirator, inserting them through the mesh-covered opening at the cone's wide end.
2. Seal the opening immediately after introduction to prevent escape.
3. Expose mosquitoes to the treated surface for the standard duration — 3 minutes for net/fabric bioassays, or 30 minutes for IRS wall-surface bioassays, per WHO guidance for the relevant surface type.
4. At the end of the exposure period, remove mosquitoes from the cone using the aspirator, handling them gently to avoid physical damage.
5. Transfer the exposed mosquitoes into labelled holding cups, keeping cones and their respective mosquito batches clearly matched to sample identity and replicate number.
6. Provide access to a cotton pad soaked in 10% sugar solution on the holding cup mesh throughout the holding period.

11. Observation Schedule

Systematic, time-bound observation is central to generating valid, comparable bioassay data.

A mosquito is scored as knocked down if it is unable to stand or fly in a coordinated manner. A mosquito is scored as dead if it shows no movement when the holding cup is gently prodded or tapped. For newer dual active-ingredient nets containing slow-acting compounds such as chlorfenapyr, WHO guidance recommends extending the mortality holding period beyond the standard 24 hours — commonly to 72 hours — because the mode of action of these compounds produces delayed mortality that would otherwise be underestimated.

Additional endpoints, such as fertility, fecundity, or blood-feeding inhibition, may also be assessed depending on the specific objectives of the study.

12. Controls

Every test run must include appropriate controls to distinguish insecticide-induced effects from background mortality or handling stress.

• Negative control: Untreated polyester or polyethylene netting (matching the base fabric of the test net), or an unsprayed section of the same substrate material for IRS studies. This should be handled identically to test samples throughout preparation, mounting, and exposure.
• Positive control: A WHO-recommended insecticide-treated reference material appropriate to the insecticide class under study, confirming that the test mosquito batch is responsive and that test conditions are capable of producing an expected effect.

Where the objective is resistance monitoring, it is also useful to run the same treated surface against both a known-susceptible reference strain and the field or F1 population being investigated, in parallel, under identical conditions.

13. Test Acceptance Criteria & Abbott's Correction

WHO guidance sets clear thresholds for control mortality to ensure that results reflect the treated surface's activity rather than background mortality:

• Negative control mortality should not exceed 10% at 24 hours for the test to be considered valid.
• If control mortality falls between 5% and 10%, apply Abbott's correction to adjust test mortality for background mortality.
• If control mortality exceeds 10%, the test should be discarded and repeated.

Example: Test mortality = 92%, control mortality = 6% → Corrected mortality = [(92 − 6) ÷ (100 − 6)] × 100 = 91.5%

14. Data Recording

For each sample tested, the following should be recorded on a standardized data sheet:

• Mosquito strain and source (colony name or field collection site)
• Sample identification (net brand/lot, wash number, wall position, or paper batch)
• Number of cones tested and cone/position identifiers
• Exposure time and start/end time of each replicate
• Knockdown counts at each observation interval, including KD60
• Mortality count at 24 hours (and extended holding if applicable)
• Test temperature and relative humidity (start and end of session)
• Date, location, and operator name
• Control results (negative and positive) for the same session

WHO guidance recommends testing multiple positions per net (commonly the roof plus all four sides) and running a sufficient number of replicate cones and mosquitoes per position to support valid statistical analysis — as a general benchmark, aim for a minimum of around 50 mosquitoes tested per net position across replicates. Maintaining consistent, complete records across all replicates allows for pooled analysis and comparison across time points, wash numbers, or field sites.

15. Good Laboratory Practices

• Use separate aspirators for treated and control samples to prevent cross-contamination of insecticide residue.
• Clean cones and frames thoroughly between tests, and dedicate separate equipment sets to different insecticide classes where possible.
• Keep testing conditions (temperature, humidity, lighting, timing) consistent throughout a study to allow valid comparison across sessions.
• Perform sufficient replicates to support reliable statistical analysis, and avoid drawing conclusions from single-cone results.
• Conduct all assays using standardized, written standard operating procedures (SOPs) to improve reproducibility across operators and sites.
• Calibrate thermo-hygrometers, timers, and sprayers used in substrate preparation on a regular schedule.
• Maintain chain-of-custody and batch records for insecticide-treated materials, particularly when results will support regulatory submissions.
• Handle insecticide-treated materials with gloves, and prepare or store treated papers and substrates in a well-ventilated, dedicated area away from untreated colony-rearing rooms.

16. LabItems WHO Bioassay Equipment

For standardized WHO cone bioassays, LabItems offers:

• WHO Cone Test Frame — securely mounts treated fabric samples at the recommended testing angle.
• WHO Bioassay Cone (Conical Chamber) — manufactured to WHO specifications for uniform mosquito exposure.

Designed for medical entomology laboratories, universities, vector control programmes, and insecticide efficacy studies, these products facilitate reliable and reproducible WHO cone bioassays.

17. References