Morin hydrate reduces fertility and survival, delays development, and weakens lipid reserves in Aedes aegypti

The Aedes aegypti mosquito is generally associated with arboviruses that cause yellow fever, dengue, zika and chikungunya. The most efficient way to control their populations is through application in breeding sites of highly toxic insecticides that can also impact non-target organisms and generate resistant populations. Therefore, the use of compounds is desirable. Morin hydrate has broad pharmacological applications based on its antioxidant potential, in addition to not having negative effects on mammals. Therefore, the objective of the present study was to investigate the effects of morin hydrate on A. aegypti survival, pupation rate, egg laying, triacylglycerol reserves, and expression of proteins related to lipid metabolism 24 h after exposure of larvae. For this, rearing media containing A. aegypti larvae with different concentrations of morin hydrate were formulated to evaluate the lethal concentration. Calculation of the expected lethal concentrations showed LC25 of 52.692 µM, LC40 of 111.121 µM, LC50 of 174.775 µM, LC75 of 575.083 µM, and LC90 of 1685.936 µM.  Twenty-four hours after treatment with morin hydrate, surviving larvae were transferred to morin-free water with food, and their pupation rate and fertility were evaluated. We observed that an increase in the concentration of morin hydrate induced a dose-dependent reduction in survival, doubled pupation time in survivors, and reduced the number of eggs laid by treated females during the larval stage by approximately 30% at concentrations exceeding 100µM. From this, the impact of 24 h on the triacylglycerol (TAG) stock was evaluated, in addition to evaluating the expression of proteins involved in lipid metabolism. Larvae 24h after treatment with 100 µM morin showed a reduction in TAG reserves of approximately 17%, while at 175 µM, there was a reduction of more than 33% in stocks and at 500µM there was a reduction of 61%. Furthermore, the lipolytic proteins TAGL1 and HSL were upregulated, while the lipogenic proteins FAS1, DGAT1 and GPAT1 were downregulated. Insulin-like receptors were also downregulated, unlike AKHr, which was also upregulated. These data demonstrate that morin hydrate reduces the survival and fertility of A. aegypti by affecting its lipid metabolism. Morin hydrate did not exhibit toxicity toward non-target organisms, demonstrating interesting potential for the control of mosquito populations.

Methods
Mosquito Rearing

The study utilized Aedes aegypti (Rockefeller strain) larvae from a laboratory colony maintained under controlled conditions (27±1°C, 80±10% relative humidity, 12h light/dark cycle). Larvae were fed commercial cat food, while adults received a 10% sucrose solution. Blood meals were provided to 4-day-old females for egg production. All procedures followed ethical guidelines approved by CEUA/UFRRJ (protocol #006/2022).

Chemical Exposure

Third-instar larvae were exposed to morin hydrate (10-2500 µM) dissolved in 0.1% ethanol for 24 hours under fasting conditions. A control group received only the 0.1% ethanol vehicle. Larval density was maintained at 1 individual per mL of solution throughout the experiments.

Biological Assessments

Mortality was evaluated after 24-hour exposure across four replicates (10 larvae/concentration). For chronic effects, surviving larvae were transferred to clean water and monitored for pupation and adult emergence over six days. Adult fecundity was assessed by counting eggs laid 72 hours post-blood meal.

Biochemical Profiling

Triacylglycerol levels were quantified using a commercial enzymatic assay, with results normalized to total protein content determined by the Lowry method. Ten larvae per concentration were homogenized for analysis, with five biological replicates per treatment.

Gene Expression Analysis

RNA was extracted from larvae (TRIzol method) and reverse-transcribed following DNase treatment. qPCR assays evaluated expression of lipid metabolism genes (Triacylglycerol Lipase 1, Brummer lipase, Hormone-sensitive lipase, Fatty acid synthase, Diacylglycerol acyltransferase 1, Glycerol-3-phosphate acyltransferase, Adipokinetic hormone receptor, and Insulin like receptor) using SYBR Green chemistry. Reference genes (Actin/α-Tubulin) ensured data normalization, with amplification efficiency validated for all primers.

Statistical Approach

Dose-response relationships were analyzed via Probit regression. Treatment effects were evaluated by ANOVA with post-hoc tests (Prism 8.0.2), considering p<0.05 significant. All data are presented as mean ± SEM from at least three independent experiments.