BackgroundThe resistance problem of dengue vectors to different classes of insecticides that are used for public health has raised concerns about vector control programmes. Hence, the discovery of alternative compounds that would enhance existing tools is important for overcoming the resistance problem of using insecticides in vectors and ensuring a chemical-free environment. The larvicidal effects of Azolla pinnata extracts by using two different extraction methods with methanol solvent against Aedes in early 4th instar larvae was conducted.MethodsThe fresh Azolla pinnata plant from Kuala Krai, Kelantan, Malaysia was used for crude extraction using Soxhlet and maceration methods. Then, the chemical composition of extracts and its structure were identified using GCMS-QP2010 Ultra (Shimadzu). Next, following the WHO procedures for larval bioassays, the extracts were used to evaluate the early 4th instar larvae of Aedes mosquito vectors.ResultsThe larvicidal activity of Azolla pinnata plant extracts evidently affected the early 4th instar larvae of Aedes aegypti mosquito vectors. The Soxhlet extraction method had the highest larvicidal effect against Ae. aegypti early 4th instar larvae, with LC50 and LC95 values of 1093 and 1343 mg/L, respectively. Meanwhile, the maceration extraction compounds were recorded with the LC50 and LC95 values of 1280 and 1520 mg/L, respectively. The larvae bioassay test for Ae. albopictus showed closely similar values in its Soxhlet extraction, with LC50 and LC95 values of 1035 and 1524 mg/L, compared with the maceration extraction LC50 and LC95 values of 1037 and 1579 mg/L, respectively. The non-target organism test on guppy fish, Poecilia reticulata, showed no mortalities and posed no toxic effects. The chemical composition of the Azolla pinnata plant extract has been found and characterized as having 18 active compounds for the Soxhlet method and 15 active compounds for the maceration method.ConclusionsOur findings showed that the crude extract of A. pinnata bioactive molecules are effective and have the potential to be developed as biolarvicides for Aedes mosquito vector control. This study recommends future research on the use of active ingredients isolated from A. pinnata extracts and their evaluation against larvicidal activity of Aedes in small-scale field trials for environmentally safe botanical insecticide invention.

Limited success for Aedes control program has impelled the necessities for new insecticide search. Hence, alternative plant compounds may be competent to overcome the pesticide resistance problem and to lead a chemical-free environment. Following go-green conceptions, larvicidal effects of the Azolla pinnata extracts using methanol and acetone solvent against Aedes albopictus late 3rd instar larvae were evaluated. The A. pinnata fresh plant from Kuala Krai, Kelantan, Malaysia (5° 31′ N 102° 12′ E) was used for crude extraction with Soxhlet apparatus using methanol and acetone solvents. Next, larvicidal test following WHO guidelines was tested against late 3rd instar to early 4th instar larvae of Ae. albopictus mosquitoes. Meanwhile, the chemical composition of extracts and their structures have been identified using GCMS-QP2010 Ultra (Shimadzu) fitted with Rtx-5MS capillary column (30 m × 0.25 mm inner diameter, ×0.25 μm film thickness; maximum temperature, 370°C), coupled to QP2010 Ultra (Shimadzu) MS. Results of methanol solvent showed the highest larvicidal activity against late 3rd instar to early 4th instar Ae. albopictus larvae with LC50 and LC95 values of 867 ppm and 1293 ppm at 24 hours, respectively, and 647 ppm and 972 ppm at 48 hours, respectively. Meanwhile, acetone solvent compounds were recorded with LC50 and LC95 values of 1072 ppm and 1302 ppm at 24 hours, respectively, and 904 ppm and 1126 ppm at 48 hours, respectively. Finally, the chemical composition of A. pinnata plant extracts has been characterized for 35 active compounds from methanol solvent and 37 active compounds with acetone solvent. In conclusion, A. pinnata plant bioactive molecules are efficient and could be developed as an eco-friendly, “go-green” approach for mosquitoes' larvicidal control programs. Thus, our study suggests that future research can be conducted on A. pinnata bioactive ingredients against Ae. albopictus larvae in small-scale field trials as botanical insecticide for environmentally friendly approach.

Dengue is vector-borne diseases with 390 million infections per year extending over 120 countries of the world. Aedes aegypti (L.) (Diptera: Culicidae) is a primary vector for dengue viral infections for humans. Current focus on application of natural product against mosquito vectors has been the main priority for research due to its eco-safety. The extensive use of chemical insecticides has led to severe health problems, environmental pollution, toxic hazards to human and nontarget species, and development of insecticide resistance on mosquitoes. Azolla pinnata is an aquatic fern and predominantly used as feed in poultry industry and as fertilizer in agricultural field for enhancing the fertility of rice paddy soil. The present study was conducted to explore the larvicidal efficacy of A. pinnata using fresh and powdered form against late third-stage larvae (6 days, 5 mm in larvae body length) of Ae. aegypti (L.) (Diptera: Culicidae). The larvicidal bioassays were performed using World Health Organization standard larval susceptibility test method for different concentration for powdered and fresh A. pinnata. Powdered A. pinnata concentration used during larvicidal bioassay ranges from 500ppm to 2000ppm; meanwhile, fresh A. pinnata ranges from 500ppm to 9,000,000 ppm. The highest mortality was at 1853 ppm for powdered A. pinnata compared with fresh A. pinnata at 2,521,535 ppm, while the LC50 for both powdered and fresh A. pinnata recorded at 1262 ppm and 1853 ppm, respectively. Finally, the analysis of variance (ANOVA) showed significant difference on Ae. aegypti larval mortality (F=30.439, df=1, p≤0.001) and concentration (F=20.002, df=1, p≤0.001) compared to powdered and fresh A. pinnata at 24-hour bioassay test. In conclusion, the powdered A. pinnata serves as a good larvicidal agent against Ae. aegypti (L.) (Diptera: Culicidae) and this study provided information on the lethal concentration that may have potential for a more eco-friendly Aedes mosquito control program.