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Laboratory Testing of Mosquito Larvicides

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Laboratory Testing of Mosquito Larvicides

Mosquito larvicide testing refers to the thorough evaluation and analysis of insecticides/larvicides intended to kill mosquito larvae to reduce the transmission of vector-borne diseases.

Mosquito larvicides are chemical agents that disrupt the mosquito growth cycle by killing mosquitoes at their larvae stages. Larvicides are applied to mosquito breeding sites, such as stagnant water bodies before the larvae develop into adult mosquitoes. By targeting mosquitoes at an early stage of their life cycle, larvicides can prevent the emergence of large numbers of adult mosquitoes and thus mosquito-borne diseases. Larvicide treatment is considered an effective choice in reducing mosquito populations over time.

Introduction to Mosquito Larvicide Testing

Larval bioassays are conducted in controlled laboratory settings to assess the efficacy, safety, and appropriate dosage of larvicides. Laboratory evaluation of mosquito larvicide helps researchers and public health authorities determine the most effective products and concentrations for controlling mosquito larvae.

Mosquito Larvicides are classified into 3 categories –

  • Bacterial Larvicides – Bacterial larvicides are made from naturally soil-occurring strains of bacteria such as Bacillus thuringiensis israelensis (Bti) or Bacillus sphaericus (Bs). The former strain is toxic to mosquito larvae, blackflies, and fungus while the later is toxic to mosquitoes, ants, fruit flies, and other insects.
  • Insect growth regulators (IGRs) – Methopreneexternal icon and pyriproxyfenexternal
    examples of insect growth regulators. These IGRs disrupt the normal growth cycle of mosquitoes and prevent them from multiplying.
  • Oils and films – These larvicides act by forming physical barriers on the water surface, preventing mosquito larvae from breathing and feeding, ultimately leading to their death.

Mosquito Larvicide Testing

Mosquito Larvicide Testing is divided into these three stages –

Phase I Laboratory studies

  • Biopotency and activity
  • Diagnostic concentration and assessment of cross-resistance

Phase II Small-scale field trials

  • Efficacy under different ecological settings
  • Method and rate of application
  • Initial and residual activity
  • Effect on non-target organisms

Phase III Large-scale field trials

  • Efficacy and residual activity
  • Operational and community acceptance
  • Effect on non-target organisms

Phase I Laboratory studies

In laboratory studies, newly developed larvicide products/ formulations are assessed under controlled conditions. This testing phase is based on the determination of the inherent bio potency of the mosquito larvicides.

Mosquito larvae with precisely known or identified developmental stages (age or instar) are exposed for 24 h – 48 h or extended duration in water treated with the larvicide at various concentrations and then mortality is recorded. After exposure time, mortality is recorded to validate the biological activity of a mosquito larvicide.

For IGRs and other materials with delayed activity, mortality should be assessed until the mosquito strains emerge into adults. Using homogenous populations of mosquito larvae or a given instar is important.

The objectives of Phase I Laboratory studies

  • Establishing dose-response lines against susceptible vector species
  • Determining the lethal concentration (LC) of the larvicide for 50% and 90% mortality (LC50 and LC90) or for 50% and 90% inhibition of adult emergence (IE50 and IE90)
  • Establishing a diagnostic concentration for monitoring susceptibility to the mosquito larvicide in the field
  • Assessing cross-resistance with commonly used insecticides

Determination of biological activity

  • Larvicides are exposed to a wide range of test concentrations and a control to find out the activity range of the product samples.
  • After determining the mortality of larvae across a broad spectrum of concentrations, a more specific range of 45 concentrations ( resulting in 10% to 95% mortality within 24 or 48 hours, is employed to calculate the LC50 and LC90 values.
  • Groups of 25 larvae that are in their third or fourth developmental stage (instar stage) are transferred to small disposable test cups or vessels containing 100 – 200 ml of water using strainers, screen loops or droppers.
  • Small, unhealthy or damaged larvae are removed and replaced.
  • The depth of the water in the cups or vessels should be maintained between 5 cm and 10 cm because deeper levels may cause undue mortality.
  • To achieve the desired target dosage, the appropriate volume of dilution is added to either 100 ml or 200 ml of water in the cups, starting with the lowest concentration. Four or more replicates are used for each concentration. Simultaneously, an equivalent number of control tests are conducted using tap water, to which 1 ml of alcohol (or the organic solvent employed) is used.
  • Each test is performed three times on different days. For long exposures, larval food is added to each test cup, if high mortality is noted in control. The test containers are maintained at 25–28°C and preferably a photoperiod of 12 h of light followed by 12 h of darkness.
  • After 24 h exposure, larval mortality is recorded.
  • For slow-acting insecticides, 48 h reading is required. Moribund larvae (weak or in a dying state) are counted and added to dead larvae to calculate the percentage of mortality.
  • The results are recorded on the form provided which includes the LC50, LC90, and LC99 values, as well as information about the slope and heterogeneity analysis. This form can be used for three different tests, each having six concentrations along with four replicates for each concentration.
  • If the larvae pupate during the test period, this will invalidate the results of the test.
  • If more than 10% of the control larvae pupate during the experiment, the test should be repeated.

If the control mortality falls between the range of 5% and 20%, the mortalities of treated groups should be corrected according to Abbott’s formula

                         X  –  Y

    Mortality  =  ————  100


  • where X denotes the percentage survival in the untreated control 
  •  Y denotes the percentage survival in the treated sample

Data analysis

  • Data from all replicates are combined for analysis.
  • LC50 and LC90 values are calculated from a log dosage–probit mortality regression line either through computer software programs or estimation via log–probit paper.
  • Bioassays are carried out at least three times, using new solutions or suspensions and different batches of larvae each time.
  • Standard deviation or confidence intervals of the means of LC50 values are calculated and recorded (A test series is valid if the relative standard deviation (or the coefficient of 8
    variation) is less than 25% or if confidence limits of LC50 overlap (significant level at P < 0.05).
  • The strength of the chemical (larvicides) against the larvae of a particular vector and strain is compared with the LC50 or LC90 values of other insecticides.

Determination of diagnostic concentration

The diagnostic dose is calculated by multiplying the upper fiducial limit of LC99.9 (lethal concentration for target insects) with a factor of 2. This means that the diagnostic concentration is set at double the value of the estimated LC99.9.

Cross-resistance assessment

Cross-resistance assessment involves testing of sample larvicides against a small
number of distinct, multi-resistant mosquito strains. If cross-resistance is identified, the specific characteristics will be established by conducting tests on the larvicide against strains exhibiting distinct resistance mechanisms. Susceptible strains of certain mosquito species can be obtained from laboratory stocks. If there are no susceptible strains available, they must be collected from the field, assuming truly susceptible populations still exist. If such populations are no longer present, susceptible strains can be artificially induced through bioassays and assays targeting individual resistance mechanisms. This involves selecting between lines derived from individually mated females.

The identification of resistant strains requires the use of well-established assay techniques. The strains should be homozygous for one or more recognized resistance mechanisms. In cases where homozygosity cannot be attained, periodic selection becomes necessary to prevent susceptible strains from outcompeting the resistant ones. To ensure the integrity of the process, established reference strains should be regularly monitored through bioassays and biochemical and/or molecular assays. This monitoring allows for the assessment and correction of any alterations in resistance or underlying mechanisms through selective measures.

Phase II Small-scale field trials

Larvicide formulations that show efficacy against target vector strains are subjected to small-scale field trials. The objective of Phase II Small-scale field trials is to assess the efficacy of formulated larvicides on a small scale against target mosquitoes in sites that closely resemble the typical environments where mosquitoes naturally breed and thrive.

Phase III Large-scale field trials

In Phase III Large-scale field trials, the efficacy of the larvicides is determined in representative natural breeding habitats of the target species. The trial areas can be stagnant drains (cement lined and unlined), soakage pits, cesspits, cesspools, domestic service tanks collecting sewage water, pools, wetlands, irrigated fields, and so on.

Importance of mosquito larvicide testing

Mosquito larvicide testing is a critical step in the development and deployment of effective mosquito control strategies. The evaluation data obtained from mosquito larvicide bioassays helps Public Health Agencies and Mosquito Control Agencies to make informed decisions about the selection and application of appropriate larvicide products that can control the spread of mosquito-borne diseases. These assays also provide information about the safety and eco-toxicity of products on non-target organisms as well.

At MIS, we specialize in conducting entomology lab tests for products/formulations designed to be used in different fields, including agricultural, public health use (vector disease control), and industrial as well as household applications.

We also offer bespoke services meticulously crafted to meet the unique needs of clients’ products. Our focused approach towards customized solutions ensures that each project receives personalized attention, guaranteeing reproducible results and utmost satisfaction.

Additionally, our state-of-the-art laboratories are equipped with the latest technology, ensuring accurate and reliable results for your projects.

To know more about our entomology testing services, talk to our experts here.


Mosquito larvicide testing involves the comprehensive assessment and examination of insecticides or larvicides designed to eliminate mosquito larvae, aiming to decrease the spread of diseases transmitted by vectors.

Products that can be tested with the Mosquito Larvicide test include chemical larvicides, insect growth regulators, liquid, oils and films

The turnaround time for Mosquito Larvicide tests can vary depending on the specific protocol, the nature of the product, and lifecycle stages of the mosquitoes being tested. Generally, it may range from a few days to several weeks.

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