Temephos–Bti mixture shows laboratory synergy and field larvicidal activity

A mixture of the organophosphate insecticide temephos and the mosquito larvicide Bacillus thuringiensis israelensis (Bti) was synergistic against Asian tiger mosquito larvae in laboratory bioassays and showed larvicidal activity in a three-month residential field trial in Zhejiang Province, China. Researchers tested three mass ratios of temephos to Bti against susceptible early fourth-instar Aedes albopictus larvae. All three mixtures exceeded the study’s threshold for synergy, while the 1:5 formulation produced the highest co-toxicity coefficient, 150.009.

When the 1:5 mixture was applied to naturally infested water containers in one village near Shaoxing, larval density fell by 100% at every monitored point under an every-two-weeks regimen and by 87.03% to 96.80% under a less frequent, higher-concentration regimen. Both treatment groups had significantly lower larval densities than untreated controls, although the difference between schedules was not statistically significant. The study addresses two practical limitations in mosquito larval control.

Temephos has been widely used for decades, but sustained use has contributed to resistance in Aedes populations, including populations in Zhejiang Province. Bti provides a biological alternative with target specificity, limited environmental persistence and low toxicity to non-target organisms, but its field efficacy can be lower against older larvae, particularly fourth-instar mosquitoes. Earlier compatibility research had identified temephos as a promising partner for Bti, but the optimal ratio for A. albopictus and the practical performance of the mixture in field containers had not been established.

The laboratory phase used early fourth-instar larvae from a susceptible laboratory colony maintained at the Zhejiang Provincial Center for Disease Control and Prevention and originally sourced from the Chinese Center for Disease Control and Prevention. Bioassays were conducted at 26 ± 1 °C and a relative humidity of 70 ± 5%. Each assay placed 30 larvae in 50 mL of water within a 150 mL test vessel.

Mortality was recorded after 24 hours, every concentration was tested in triplicate, and distilled water served as the control; control mortality was 0%. The researchers used log-probit analysis to estimate median lethal concentrations, or LC50 values, and then calculated a co-toxicity coefficient, or CTC, for each mixture. Under the study’s classification, a CTC above 120 indicated synergy, 80 to 120 indicated additive action and a value below 80 indicated antagonism.

The tested mixtures were formulation A at 1:1, formulation B at 1:3 and formulation C at 1:5 by mass. Temephos alone had an LC50 of 0.017 mg/L, with a 95% confidence interval of 0.010 to 0.022 mg/L, whereas Bti alone had an LC50 of 8.059 mg/L, with a 95% confidence interval of 6.901 to 9.249 mg/L. These values describe potency in this particular laboratory assay and do not establish equivalent operational doses.

Mixed formulation A produced an LC50 of 0.024 mg/L, formulation B an LC50 of 0.048 mg/L and formulation C an LC50 of 0.067 mg/L. Their CTC values were 141.477, 140.299 and 150.009, respectively. The lower limits of all three confidence intervals remained above 120.

Formulation C was selected for the field trial because it had the greatest measured synergistic potency, rather than because it had the lowest LC50 when evaluated as a complete mixture. The field experiment was conducted in Yingjia Village from early July to late September 2024. During the trial period, temperatures ranged from 26 to 31 °C and rainfall ranged from 147.7 to 264.6 mm.

Staff from local disease-control centers selected 150 permanent water containers naturally infested with A. albopictus larvae. These included flowerpots, earthenware jars and cisterns with capacities of 4 to 25 liters. Fifty containers received formulation C every two weeks at twice the laboratory-derived 99% lethal concentration, or 2 × LC99.

Another 50 received the formulation once a month at four times that concentration, or 4 × LC99, and 50 remained untreated. The groups were positioned close to one another because mosquito movement and oviposition patterns could affect comparisons. Water levels were standardized against marked graduation lines before dosing, although evaporation and rainfall later caused some variation in active-ingredient concentrations.

Larval counts were made before treatment and on days 1, 3, 7, 15 and 30 after each application, with larvae returned to their containers after counting. The trial comprised three consecutive one-month cycles. Baseline mean larval densities did not differ significantly among the control, every-two-weeks treatment and monthly treatment groups: 6.94 larvae per container in the control, 6.29 in the every-two-weeks group and 7.12 in the monthly group, with F = 0.069 and p = 0.933.

Generalized linear mixed models with negative-binomial regression compared larval densities over time, including group, average temperature and rainfall during the preceding week as fixed effects and date as a random effect. Mean larval densities over the three cycles were zero in the every-two-weeks group; 0.28, 0.58 and 0.30 in the monthly group; and 8.52, 4.36 and 9.11 in controls. Both treated groups had significantly lower larval densities than controls at p < 0.001, but there was no significant difference between the two application schedules at p = 0.804.

The every-two-weeks regimen achieved a 100% observed reduction and a control-adjusted standard reduction of 100% in each of the three months. Under the monthly regimen, observed reductions were 96.07%, 91.85% and 95.79%, while standard reductions adjusted for background mosquito fluctuations were 96.80%, 87.03% and 96.79%. Control-container density changed by −22.78%, 37.19% and −31.21% relative to baseline, showing that larval populations continued to fluctuate seasonally even without treatment.

Standardized reductions therefore provided a conservative estimate when control density increased, a contrast that was especially relevant during the first and third months. The measured persistence of the mixture was one of the study’s notable features. In earlier laboratory work, Bti applied at 2 × LC99 produced mortality below 99.8% after one week and below 66.0% after two weeks.

A separate field study reported that 50% of sewers treated with temephos granules were reinfested with larvae after two weeks. In the present trial, the 1:5 mixture remained effective at sampling points extending to 30 days under the monthly regimen. The researchers also calculated that temephos at the LC99 level in formulation C represented 65.97% of the amount used in pure temephos.

This supports the narrower observation that the combination used less of the chemical component in that comparison. It does not by itself demonstrate reduced environmental contamination, lower application costs or a measurable resistance-management benefit. Several limitations prevent the field results from demonstrating eradication or long-term control.

Only three ratios, from 1:1 to 1:5, were tested, so mixtures containing more Bti were not evaluated. Resistance in the local mosquito population was not measured during the experiment, although earlier monitoring had detected a 12-fold temephos resistance ratio in Shaoxing relative to a susceptible strain. The apparent 100% reductions could also reflect temporary suppression of larval development, mortality at another mosquito life stage, oviposition avoidance or insufficient sampling rather than complete elimination of breeding.

The trial covered one village, three months and 150 containers, limiting generalization to other mosquito populations, seasons and environmental settings. The researchers noted that the mixture may retain activity against temephos-resistant populations, but that possibility requires direct testing, as do its long-term effects on insecticide resistance and the environment.

Subject of Research: Laboratory and field evaluation of a temephos–Bti mixture against Aedes albopictus larvae

Article Title: Synergistic larvicidal effects of temephos and Bacillus thuringiensis israelensis against Aedes albopictus: a laboratory and field study

Article References: Wu, Y., Liu, Q., Sun, J., Hong, Y., Fu, Y., Zhou, D., Ling, F., & Jiang, G. (2026). Synergistic larvicidal effects of temephos and Bacillus thuringiensis israelensis against Aedes albopictus: a laboratory and field study. Frontiers in Veterinary Science, 13, Article 1930489. https://doi.org/10.3389/fvets.2026.1930489

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1930489

Keywords: Aedes albopictus, Bacillus thuringiensis israelensis, temephos, larvicidal synergy, mosquito bioassay, field vector control, insecticide resistance, arbovirus vectors, dengue, negative-binomial regression, Synergistic, larvicidal