DOI: 10.5061/dryad.gxd2547wt
title: Larvicidal activity of Artemsia capillaris and Setaria palmifolia and Macaranga tanarius extracts against Aedes aegypti (Culicidae)

description: 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.
description: The essential oils were those from the original study. Briefly
 summarizing those methods, <em>Artemisia capillaris</em> and
 <em>Setaria palmifolia</em> 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
 <em>Aedes aegypti</em> 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 <em>ad libitum</em> 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 <em>Aedes aegypti </em>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 <em>Bacillus
 thuringiensis israelensis</em> (<em>Bti</em>) 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 LC<sub>50</sub> and
 LC<sub>90</sub> 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 <em>post hoc</em> analyses on the dose effect on
 the observation of each day. The recorded mortality rates of each of the
 concentrations on the 24<sup>th</sup> and
 48<sup>th</sup> hours after the bioassay began were used to
 calculate LC<sub>50</sub> and LC<sub>90</sub>
 using R 4.4.1 with logit dose-response as in previous studies.
description: # Larvicidal Activity of *Artemsia capillaris* and *Setaria palmifolia*
 and *Macaranga tanarius* Extracts Against *Aedes aegypti* (Culicidae)
 [https://doi.org/10.5061/dryad.gxd2547wt](https://doi.org/10.5061/dryad.gxd2547wt) ## Description of the data and file structure This repository is associated with Huang, Tsai, and Shelomi (2025). Larvicidal Activity of *Artemsia capillaris *and* Setaria palmifolia *Extracts Against *Aedes aegypti* (Culicidae). Currently under peer review. This study examined hexane and ethyl acetate extracts of two plants, *Artemisia capillaris* and *Setaria palmifolia*, and ethyl acetate extract only of *Macaranga tanarius*, for any larvicidal ability against the yellow fever mosquito, *Aedes aegypti*. Only the hexane extract of *Artemisia capillaris* had an effect, causing almost immediate inactivity in 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 inactivity effect, or testing its effects on other insects and other mosquito life stages. This repository contains the raw data for the experiments as Excel files. In the files, we referred to the initial tests (500 ppm & 1000 ppm, to see if an extract works) as "stage 1," hit-and-trial tests as "stage 2," and toxicity determination tests as "stage 3." There might be a few vague or likely mistaken parts from the raw data that were not interpreted in the same way during the making of data for analyses and the raw data sheets organized *post hoc*. The issue may typically happen in data from stage 1, where recording methods were not well-established. The data marked with orange in the Raw Data Sheets denote those that were not used in data analyses. The data that were actually used in the analyses for the publication are from: 20230918 (stage 1), 20231024 (stage 1), 20240126 (stage 2, added *post hoc*), 20240202 (stage 3), 20240713 (stage 3), 20240719 (stage 3), and the (2) part of 20240807 (stage 1 redone). All queries about this data should be directed to Lin-Jie Huang. ### Files and variables #### File: Artemisia Hexane Extract Stage 2&3 Mortality (with Days-Mortality Graphs).xlsx **Description:** This file has the mortality data for larvae exposed to different dosages (0, 50, 100, 150, 200, 250, and 300 ppm) of hexane extract of Artemisia capillaris at 0, 24, 48, 72, and 92 hours. Each replicate is a separate worksheet. The data from the first two worksheets is combined and averaged into the third. Variables: * Mean and Standard Deviation for Mortality rate (%) * Hours after exposure * Dose Concentration #### File: Artemisia Hexane Extract Stage 2&3 Mortality (with old Concentration-Mortality Graphs).xlsx **Description:** This file has several worksheets, each representing one replicate on the date that is the name of the worksheet.The tables from A1-K6 have the mean and standard deviation mortality rate as a percentage at different ppm doses of hexane extract of Artemisia capillaris at 0, 24, 48, 72, and 92 hours, if one excludes knockdown (defined in the paper as "inactive" larvae that were "twitching periodically but lacking controlled locomotion and sinking to the bottom"). The tables from N1-X6 are the same, but the knocked-down/inactive larvae are counted as dead. The tables from A10-J29 have the number of deaths, which was used to calculate the means and SDs for the tables A1-K6. The tables from N10-W29 have the number of deaths, which was used to calculate the means and SDs for the tables N1-X6. The table from A33-J37 applies Abbott's Formula Correction to the data from A1-K6. The tables from A39 to L55 are the raw data for total number of dead and alive larvae in each replicate (three per dose) at each time. Two worksheets titled "Averages" are the mean values for the tables preceding them. This is the data used to calculate LC50 and LC90 with online tools, and the resulting graph is provided, but was not used in the final paper. The data from A1-N31 and P1-X31 is the sum of the number of dead larvae at each dose (rows) at each time (the different tables) for each experiment (column), plus average (columns M and W) and standard deviation (columns N and X). The same exact data but transposed is re-pasted in the rest of the worksheet, but can be ignored as it was not used in the final paper. Variables: * Mortality Rate * Number living larvae * Number dead larvae * Hours after exposure * Dose concnetration #### File: Graphs - Emergence.xlsx **Description:** This file contains excel worksheets for the mean adult emergence rates from pupae of larvae for the five examined extracts: hexane (Hex) and ethyl acetate (EtAc) extracts of *Artemsia capillaris* and *Setaria palmifolia* and *Macaranga tanarius*. The data is tables A3-G14. This data was the inital test used to check for larvicdal activity. Variables: * Mean adult emergence rate (columns B-D) and standard deviation (columns E-G) * Days after exposure (rows) * Dosage concentration (columns) #### File: Graphs - Mortality.xlsx **Description:** This file contains excel worksheets for the mean larval mortality rate after exposure to the five examined extracts: hexane (Hex) and ethyl acetate (EtAc) extracts of *Artemsia capillaris* and *Setaria palmifolia* and *Macaranga tanarius*. The data is tables A3-G14. This data was the inital test used to check for larvicdal activity. Variables: * Mean larval mortality rate rate (columns B-D) and standard deviation (columns E-G) * Days after exposure (rows) * Dosage concentration (columns) #### File: Raw Data Sheets.xlsx **Description:** An excel file containing the raw, unprocessed data used to fill other files. Each worksheet represents the experiment of a different date. Lin-Jie Huang's notes and observations are included. Variables: * Number of larvae alive, inactive, semi-inactive, molting, dead, with other notes. * Time of observation * Treatment (negative control, *Bti* positive control, different dosages of text extract) #### File: Stage\_1\_Emergence.xlsx **Description:** This file contains excel worksheets for the number of emerged adults (A3-G36) and the mean and standard deviation adult emergence rates (I3-O34 and Q3-W14 are the same exact data) from pupae of larvae for the five examined extracts: hexane (Hex) and ethyl acetate (EtAc) extracts of *Artemsia capillaris* and *Setaria palmifolia* and *Macaranga tanarius*. The same data segregated by sex is provided from rows 39-89. Variables: * Number of emerged adults * Mean adult emergence rate * Standard deviationfor emergence rate * Days after exposure (rows) * Dosage concentration (columns) * Sex #### File: Stage\_1\_Mortality\_(Corrected).xlsx **Description:** This file contains excel worksheets for the number of dead larvae (A3-G36) and the mean and standard deviation larval mortality rates (I3-O34 and Q3-W14 are the same exact data) for larvae exposed to the five examined extracts: hexane (Hex) and ethyl acetate (EtAc) extracts of *Artemsia capillaris* and *Setaria palmifolia* and *Macaranga tanarius*. Variables: * Number of dead larvae * Mean larval mortality rate * Standard deviation for larval mortality * Days after exposure (rows) * Dosage concentration (columns)