The potential use of *Azolla pinnata* as an alternative bio-insecticide

Dengue, transmitted primarily by Aedes aegypti and Aedes albopictus, remains a major public health problem in tropical and subtropical regions, with Malaysia reporting 80,000 cases and 113 deaths between January and August 2019. Existing physical controls (e.g., bed nets, protective clothing) offer only temporary relief, and chemical insecticides (e.g., temephos, pyrethroids) face growing resistance, as documented in Malaysian cities such as Kuala Lumpur and Penang. Resistance mechanisms include target-site alterations and increased metabolic detoxification. Continuous reliance on synthetic insecticides raises environmental concerns and selects for resistant vectors, underscoring the need for alternative, sustainable vector control tools. Plant-based products have shown promise as bio-insecticides and can act on multiple physiological and behavioral targets. Previous work on Azolla pinnata focused mainly on larvicidal effects and chemical composition using GC-based analyses, with soxhlet methanolic extraction showing strong larvicidal activity and minimal toxicity to non-target organisms. Building on this, the present study evaluates methanolic extracts of A. pinnata for adulticidal, ovicidal, and oviposition-deterrent effects against Aedes mosquitoes, and profiles its liquid-phase chemical constituents, aiming to inform development of plant-derived bio-insecticides aligned with sustainable control strategies.

The literature indicates widespread use of synthetic insecticides leading to resistance in Aedes populations, driven by target-site mutations and enhanced metabolic pathways. Plant-derived insecticides are highlighted as alternatives capable of multifaceted actions on mosquito physiology and behavior. Prior studies on Azolla pinnata reported larvicidal efficacy, identified insecticidal compounds via GC-MS (e.g., n-hexadecanoic acid, diethyl phthalate, neophytadiene, phytol derivatives), and found soxhlet methanolic extraction particularly effective. Reports also suggest repellent attributes of fresh A. pinnata in traditional use. However, adulticidal, ovicidal, and oviposition-deterrent activities of crude extracts and LC-MS-based profiling of low-volatility constituents were less explored, motivating the current comprehensive assessment.

Plant extraction: Azolla pinnata plants were collected from Kuala Krai, Kelantan (5°31′N, 102°12′E), dried, and blended. Soxhlet extraction was performed using 40 g of dried plant powder in a paper thimble (cotton wool to prevent sample escape) with 1 L methanol in a round-bottom flask, heated to methanol’s boiling point (~70 °C) for ~3 h until the siphon arm solvent became clear. Extracts were concentrated by vacuum evaporation and stored at −20 °C until use.

Aedes rearing: Eggs of Aedes aegypti and Aedes albopictus (from Vector Control Research Unit, Universiti Sains Malaysia) were hatched in dechlorinated water and reared at 25–30 °C, pH 6.95–7.03, relative humidity 80 ± 10%, and dissolved oxygen 5.5–6.1 mg/L. Larvae were reared to pupation; pupae were transferred to cages for adult emergence. Adults were provided with sucrose solution.

Adulticidal activity: Following WHO-guided paper impregnation methods, methanolic A. pinnata extract solutions were prepared at 30, 75, 125, 250, 500, 1000, 1500, 2000, 3000, 5000, and 6000 ppm. Filter papers (140 × 120 mm) were impregnated with test solutions and dried overnight; methanol-only papers served as controls. For each assay, 25 female mosquitoes (2–5 days old) were introduced into holding tubes for 1 h acclimation; then transferred to exposure tubes containing the impregnated paper for 1 h. Knockdown was recorded every 5 min over 60 min; mosquitoes were then held with 10% sucrose plus vitamin B complex for 24 h, after which mortality was recorded. A negative control used oil-impregnated paper with no mortality. Four replicates were conducted per species and concentration.

Ovicidal activity: Following Su and Mulla with modifications, 100 eggs of each species were immersed in trays containing methanolic A. pinnata extracts at 1500, 1000, 500, 250, or 125 ppm (control: distilled water). Five replicates per concentration were tested. Post-treatment, eggs were counted under a microscope and transferred to distilled water for 48 h to assess hatchability. Egg mortality (%) = (number of unhatched eggs/total eggs) × 100, based on unopened opercula.

Oviposition deterrence: Dual-choice assays were conducted in 30 × 30 × 30 cm cages with 15 gravid (5 days old) Ae. aegypti or Ae. albopictus females under room conditions, provided 10% glucose. Two 50 mL cups contained dechlorinated water (untreated) and two contained methanolic A. pinnata extract at 1500, 1000, or 500 ppm (separate cages per concentration). Whatman No. 1 filter paper strips were placed to provide oviposition substrate, with paper half-submerged. Cup positions were alternated diagonally between replicates. After 3 days, eggs on treated and untreated papers were counted under a stereomicroscope. Non-choice tests used a single treated cup per cage with 15 gravid females. Percent effective repellency (ER) was calculated as ER = ((NC − NT)/NC) × 100, where NC = eggs in control and NT = eggs in treatment.

Liquid chromatography–mass spectrometry (LC-MS): Analyses were performed on a Shimadzu LCMS system with an Agilent C18 column (2.0 × 150 mm) at 0.25 mL/min, 250 °C. The mobile phase was MeCN/H2O/HCOOH (3:92:5). Mass range covered polar compounds from 40–1300 amu. Compounds were identified based on retention times and mass spectra.

• Adulticidal activity: Mortality increased with concentration. Estimated lethal concentrations after 24 h were: Ae. aegypti LC50 = 2572.45 ppm; LC95 = 6100.74 ppm (regression: Y = −14.96 + 4.39X). Ae. albopictus LC50 = 2329.34 ppm; LC95 = 5315.86 ppm (regression: Y = −17.55 + 1.55X). Controls showed no significant mortality.
• Ovicidal activity: Egg mortality reached 100% for both species at ≥125 ppm (tested 125, 250, 500, 1000, 1500 ppm). At lower concentrations, 30 ppm caused 81% (Ae. aegypti) and 83% (Ae. albopictus) mortality; 75 ppm caused 90% and 91% mortality, respectively. Controls had full hatchability (0% mortality).
• Oviposition deterrence: In choice tests, mosquitoes laid eggs only in untreated cups at higher concentrations; effective repellency reached 100% at 125–1500 ppm for both species. At 30 ppm, effective repellency was 80% (Ae. aegypti) and 81% (Ae. albopictus); at 75 ppm, 89% and 90%, respectively. Non-choice tests showed 82% repellency at 30 ppm; 91% (Ae. aegypti) and 90% (Ae. albopictus) at 75 ppm; and 100% at ≥125 ppm. Ae. aegypti sometimes oviposited on sucrose-soaked cotton rather than treated oviposition substrates.
• Chemical profiling (LC-MS): Three likely active compounds were identified in A. pinnata extracts: 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol (RT 23.805 min; MW 576.4623; metabolite, antibacterial), pyridate (RT 3.771 min; MW 378.1163; pesticide), and nicotinamide N-oxide (RT 4.064 min; MW 138.0427; reported insecticidal-related activity). These support observed adulticidal, ovicidal, and repellent activities.

The study demonstrates that methanolic extracts of Azolla pinnata exhibit multi-modal bioactivity against Aedes vectors: moderate adulticidal effects with measurable LC50/LC95, strong ovicidal action achieving complete egg mortality at ≥125 ppm, and potent oviposition deterrence culminating in complete repellency at ≥125 ppm under both choice and non-choice conditions. Behavioral observations during adult bioassays (mosquitoes avoiding impregnated papers) and oviposition assays (preference for untreated water, or even sucrose-soaked cotton in non-choice tests) indicate that the extract functions as both a toxicant and a repellent. Identified LC-MS constituents—1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol, pyridate, and nicotinamide N-oxide—are consistent with bioactive roles and suggest a chemical basis for the combined toxic and deterrent effects. While adulticidal potency was moderate (slow-acting relative to conventional insecticides), the ability to use a single operational concentration near the adulticidal LC50 (~2572 ppm) across life-stage-targeted assays may simplify field applications. Overall, findings address the need for plant-derived, sustainable alternatives to synthetic insecticides and support further development of A. pinnata-based formulations for dengue vector control.

Azolla pinnata methanolic extracts showed promising bio-insecticidal properties against Aedes aegypti and Aedes albopictus, including moderate adulticidal activity, complete ovicidal activity at ≥125 ppm, and strong oviposition deterrence achieving full repellency at ≥125 ppm. LC-MS analysis identified likely active constituents underpinning these effects. The extract holds potential for development into plant-based bio-insecticides for integrated vector management. Future research should evaluate field applications, persistence and long-term efficacy, impacts on non-target organisms, and human health considerations, and optimize formulations (e.g., repellence creams or oviposition deterrents) leveraging identified compounds.

• Adulticidal activity was relatively slow with moderate LC50/LC95 values compared to conventional insecticides.
• Experiments were conducted under laboratory conditions; field efficacy and persistence were not assessed.
• Effects on non-target organisms and human health were not evaluated and require further study.
• LC-MS profiling indicated multiple constituents, and definitive attribution of activity to specific compounds remains challenging.