Laboratory Testing of Mosquito Larvicides

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

                           X

• 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.