Larvicidal activity of Artemsia capillaris and Setaria palmifolia and Macaranga tanarius extracts against Aedes aegypti (Culicidae)

Larvicides are an effective method of vector management to control mosquito-borne diseases such as dengue. Growing awareness of environmental concerns with improperly used synthetic insecticides is driving interest in plant-derived biopesticides, perceived as being safer and to which insects are less likely to evolve resistance. This study examined hexane and ethyl acetate extracts of two plants, Artemisia capillaris and Setaria palmifolia, previously identified as having repellent abilities against a biting midge, for any larvicidal ability against the yellow fever mosquito, Aedes aegypti. Only the hexane extract of Artemisia capillaris had an effect, causing almost immediate knockdown of larvae faster than a Bacillus thuringiensis positive control. At doses above 300 ppm, all or nearly all larvae would die within 24 hours, while at lower doses a percentage would recover even if they appeared moribund. The LC50 and LC90 were calculated as 187.60 and 526.24 ppm at 24 hours and 128.31 and 332.26 ppm at 48 hours, respectively. Future research can focus on developing effective formulations for this larvicide, determining the physiological mechanism behind the knockdown effect, or testing its effects on other insects and other mosquito life stages.

Methods
The essential oils were those from the original study. Briefly summarizing those methods, Artemisia capillaris and Setaria palmifolia leaves were collected on a single day in Taipingshan National Forest Recreational Area, ground while fresh, and 50 grams extracted in a Soxhlet extractor with hexane at 70 °C for 24 hours and again with ethyl acetate at 78 °C for 24 hours. Essential oils were separated from the solvents under a vacuum using a rotary evaporator. Two separate sets of extractions were done for each species, and the resulting oils combined for long-term storage in amber vials in the dark at room temperature. For this study, these four extracts (one hexane and one ethyl acetate extract per species) were dried in their vials under room temperature in a fume hood. The dry residues were dissolved to saturation (100% stock solution) with approximately 2 mL of dimethyl sulfoxide (DMSO).

The mosquitoes were an Aedes aegypti strain sourced in Tainan City, Taiwan, provided by the Vector-borne Infectious Diseases Lab, National Taiwan University. Larvae were kept in RO water inside an incubator maintained at 28 °C, 80% relative humidity, and a 16:8h (light/dark) light cycle, and fed ad libitum with crushed goldfish feed.

Following international standard protocols, the larvicidal bioassays were conducted in 250 ml beakers with 100mL RO water after adding 25 four-day-old Aedes aegypti larvae, five grains of fish feed, and either 0.1mL DMSO (negative control), 0.1mL of DMSO-plant extract solution, or 0.2mL of 50% v/v DMSO aqueous solution suspended with VectoBac WDG, a commercially available Bacillus thuringiensis israelensis (Bti) larvicide (positive control). Beakers were incubated under the same conditions as larva rearing. The initial test concentrations were 500 and 1000 ppm. The concentrations to determine the LC50 and LC90 were 50, 100, 150, 200, and 250 ppm, decided after a series of hit-and-trial larvicidal assays. The status of the larvae was recorded every hour for the first six hours, then each beaker was sealed with plastic wrap perforated for gas exchange and the statuses recorded every 24 hours. Observations were done for 10 days (240 hours) for initial tests, four days for the toxicity determining tests, or until adults emerged or all larvae had died. Larvae were recorded as alive, dead, or inactive [twitching periodically but lacking controlled locomotion and sinking to the bottom]. Each bioassay had three replicates.

The effects of dose and time were analyzed using R v 4.4.1 with two-way repeated measures ANOVA, and Tukey’s HSD test was used for post hoc analyses on the dose effect on the observation of each day. The recorded mortality rates of each of the concentrations on the 24th and 48th hours after the bioassay began were used to calculate LC50 and LC90 using R 4.4.1 with logit dose-response as in previous studies.