Insecticidal Effect of Ethnobotanical Plant Extracts Against Anopheles Arabiensis (Diptera: Culicidae) Under Laboratory Condition

Background: The emergence and spread of resistant strains of malaria vectors to chemical insecticides are becoming major problem for malaria vector management. Natural plant products play a vital role to resolve the current challenge of malaria control. Objective: The current study was conducted to evaluate insecticidal effect of ethnobotanical plant extracts against the primary malaria vector, Anopheles arabiensis in Northwestern Ethiopia. Methods: Primarily, ethnobotanical plants used for Anopheles mosquito control was surveyed in Dangur district, Northwestern Ethiopia. Insecticide susceptible strains of Anopheles arabiensis mosquito were reared in insectary of tropical and infectious diseases research center, Assosa university. The larvicidal and adulticidal potentials of frequently used plant extracts against susceptible strains of laboratory colony were evaluated. Result: A total of fteen plants were identied as ethnobotanical plants helping the local people for mosquito control. Azadirachta indica, Ocimum lamiifolium, Ocimum americanum, Moringa oliera leaf, and Moringa oliera seed species of local plants were found to be frequently used to kill and/or repel mosquitoes in the study district. All the plant extracts were found to have potential larvicidal activity against 4 th instar larvae of An. arabiensis and only ethanol and methanol extract of A. indica and O. lamiifolium were found to have potential adulticidal effect against adult of An. arabiensis. The highest larvicidal activity was observed in ethanol extract of A. indica with 95% larval mortality and lowest LC50 of 40.73 ppm and LC90 of 186.66 ppm. The highest adulticidal activity was observed in methanol extract of A. indica with 75% adult mortality at 300 ppm and lowest LC50 of 106.65ppm and LC90 of 1293ppm. The lowest larvicidal and adulticidal activity was observed in methanol extracts of O. lamiifolium with 63.35% larval mortality and leaf extract of M. oliera with 50% adult mortality at 300 ppm, respectively. Conclusion: ethanol extract of A. a remarkable larvicidal effect against An. and thus it


Introduction
Anopheles arabiensis is the principal malaria vector in Ethiopia having a wide distribution while An. pharoensis, An. funestus, An. nili, and An. stephensi have secondary role in malaria transmission in the country [1,2,3]. Vector control is a crucial prevention tool to mitigate the diseases. Long-lasting insecticidal nets (LLINs) and Indoor Residual Spray (IRS) are among the most effective malaria vector management strategies recommended in Africa. However, the wide spread of insecticide resistant strains of Anopheles mosquito is challenging the chemical insecticides-based malaria control strategies. Insecticide susceptibility tests carried out in Ethiopia have shown different levels of resistance by the principal vector to insecticides in use for IRS and/or to treat nets [4,5]. For these reasons, looking for alternative option is becoming major interest of scientists and policy makers working in the area.
Traditionally or culturally, different communities use different plants in various forms to protect themselves against mosquitoes and other insect bites [6,7]. Naturally occurring compounds and their derivatives are of increasing interest for the development of new insecticidal compounds against malaria vectors. Plants possess a wide range of bioactive phytochemicals that are selective, biodegradable, and have minor or no adverse effects on non-target organisms and the environment [8]. Reports indicated that the essential oils and extract of local plants have a promising larvicidal, adulticidal, and repellent activities against malaria vectors [9,10].
Gathering information about ethnobotanical plants used in particular society and evaluating their e cacy is important. Therefore, the main objective of the present study was to investigate the insecticidal effect of ethnobotanical plant extracts against An. arabiensis under laboratory condition.

Descriptions of the Study Area
Ethnobotanical collection was conducted in in three purposively selected kebeles namely; Manbuk 01, Kitil, and Adipopo of Dangur district, Northwestern Ethiopia. Dandur district is situated 687 km away from the capital city of the country, Addis Ababa, in the Northwest Ethiopia (Fig. 1).
The district has an estimated total population of 68,653. Geographically, the district lies at latitude and longitude of 11°30′N and 35°50′E, respectively with elevation ranging from 672-2731meter above sea level. Based on the information from metrological data in 2021, the area has mean annual rainfall ranging from 700 to 1400 mm per year and mean annual temperature ranging from 26-35℃ per year.

Ethnobotanical Data Collection Techniques
Semi-structured interviews were prepared for inhabitants/key informants of the three kebeles (Manbuk 01, Kitil, and Adipopo) and it was used as a guide following Martin [11]. A total of 20 key informants were identi ed based on the recommendations of local authorities and knowledgeable elders who were suggested by respective kebele elders. Key informants were interviewed to identify plants they use to repel and/or kill insects in general and mosquitoes in particular.

Voucher Specimen Collection and Identi cation
Voucher specimens of the reported plants as repellant against mosquito vectors were collected and identi ed with taxonomic keys [12].

Preparation of Crude Plant Extracts
Plant extraction was conducted in the microbiology laboratory, Assosa University. The leaves and seeds of the plant were washed using clean water thoroughly and air-dried under shade at room temperature for 15 days.
Then, the dried materials were ground separately to powder using a grinding mill. The powdered material was macerated with ethanol and methanol using Erlenmeyer asks and placed on an orbital shaker at 60 rpm at room temperature for 72 hours [13]. Then the plant extract was ltered through cotton and subsequently with Whatman lter paper (12.5 cm size). Filtrates were concentrated using a rotary evaporator to remove solvents from the extract. The crude extracts were then collected in small volume beakers and kept in the freezer until used mosquito e ciency tests.

Mosquito Rearing
Larvae of Anopheles arabiensis were obtained from Tropical and Infectious Diseases Research Center insectary laboratory, Assosa University. Mosquitoes were reared using the standard procedures. They were maintained at yeast a 3:1. When Pupae were formed no food was supplied and transferred to the cup that contains water by disposable pipettes and put in screened cages for adult emergence. The adults were fed on 10% glucose solution soaked in cotton pads, in addition to an animal (rabbit with shaved back and belly) blood meal given to the females twice per week. A petri dish lined with a moist cotton piece and covered with lter paper was put inside each cage for eggs lying [14]. By doing so, 4th instar larvae and 2-5 day of adults were continuously available for different bioassay tests.

Larvicidal Activity
Preparation of test and control solutions 200mg of the dried crude ethanol and methanol extract of each plant were placed in a standard measuring ask and distilled water to prepare 20mL of 1% stock solution. 0.1mL of Tween 80 was used as an emulsi er. From the 1% stock solution, concentrations of 50, 100, 150, 200, 250, and 300 ppm were prepared by adding the appropriate volume of dilution. 0.1mL of Tween 80 was made up to 100 mL distilled water to serve as the negative control solution [14].

Larvae bio-assay with crude leaf extract
In the rst phase of bio-assay, the mosquito activities of frequently used plants ethanol and methanol of crude extracts of the leaves and seed were screened at 300 ppm concentration. Batches of 20 4th instar larvae of An. arabiensis were transferred using a dropper to 200 mL glass beakers each containing 100 mL of water and one batch as a negative control for each concentration. Each test was run three times. The test containers were held at 25 ± 2℃ and 80 ± 10% relative humidity with a photoperiod of 12 hours light followed by 12 hours dark. Larvae mortality was recorded after 24 hours of exposure in each concentration of test solutions [14].
Determination of LC50 and LC90 of the crude leaf and seed extract of test plants Based on the preliminary screening, ethanol and methanol of the leaf and seed of plant extract were selected and subjected for dose-response bioassay at the concentrations of 50-300 ppm. The average mortality after 24 hours was recorded and used to determine LC50 and LC90 values [14].

Adulticidal Activity
Adult bio-assay with crude leaf extract The triplicate series contained twenty females of An. arabiensis in each tube. In the rst phase of bio-assay, the mosquito activities of ethanol and methanol crude plant extracts were screened at 300 ppm concentration. This concentration was impregnated into lter papers (12 × 15 cm). Only distilled water is used as a negative control.
The impregnated papers were air-dried for 5 minutes and then inserted into an exposure tube in the WHO testing kit. Twenty 2-5 days of old blood-starved female mosquitoes were introduced into the holding tube and held for 1 hour to acclimatize. The mosquitoes were then transferred by gentle blowing in the exposure tube. After 1 hour in the exposure tube, mosquitoes were then transferred back to the holding tube to recover. A pad of cotton soaked with 10% glucose solution was placed on the mesh screen to feed recovering experimental and control mosquitoes. At the end of the 24 hours recovery period, Mosquito mortality was recorded and the percentage mortality was calculated [15].
Then Percentage of mosquito mortality was calculated by using the formula: Determination of LC50 and LC90 of the crude leaf extracts of test plants Based on the preliminary screening ethanol and methanol leaf of plant extract were selected and subjected for dose-response bioassays at the concentrations of 50-300 ppm. The average mortality after 24 hours was recorded and used to determine LC50 and LC90 values [14].

Data Analysis
Data from all replications were pooled and mean percent mortalities of larva and adult mosquitoes that are treated with crude leaf extract of the plants were determined by analysis of variance (ANOVA) using SPSS version 20. Fishers Least Signi cant Difference (LSD) was used to investigate statistical signi cance between the different test plants against mosquito mortality. The difference between means was considered statistically signi cant at P < 0.05. LC50% and LC90% and other statistics at 95% ducial limit of lower con dence limit and upper con dence limit and Chi-square values were determined using dosage mortality probit regression analysis of SPSS program version 20 to determine their larvicidal and adulticidal e cacies [14].

Ethnobotanical Plant Species that used to repel Mosquito in the Study Area
Fifteen local plant species used to repel mosquitoes were reported from the study area and identi ed (Table 1).

Larvicidal Activity of Crude Leaf and Seed Extracts of the Test Plants
The highest mortality was recorded in ethanol leaf extract of A. indica (95%). However, the methanol leaf extract of O. lamiifolium recorded the lowest activity with mortality of 63.35% (Table 2).  *LC50-Lethal concentration that kills 50% of the exposed larvae, LC90-Lethal concentration that kills 90% of the exposed larvae, UCL = Upper con dence limit, LCL = Lower con dence limit, X 2 -chi-square, df-degree of freedom

Adulticidal Activity of Crude Leaf and Seed Extracts of the Test Plants
The highest mortality was recorded in methanol leaf extract of A. indica (75%) ( Table 4). However, the ethanol and methanol leaf extract of M. oli era lowest mortality with 50% (Table 4). No adult mortality was observed in the negative control during the experiment.  *LC50-Lethal concentration that kills 50% of the exposed adult, LC90-Lethal concentration that kills 90% of the exposed adult, UCL = Upper con dence limit, LCL = Lower con dence limit, X 2 -chi-square, df-degree of freedom

Phytochemical Screening of Methanol and Ethanol Crude Leaf and Seed Extracts of Test Plants
Phytochemical screening was conducted for all methanol and ethanol crude plant extract to test the presence of alkaloids, avonoids, terpenoids, tannin, saponin, and phenols and the results were presented in Table 6. High larval mortality was caused by ethanol leaf extract of A. indica with mortality 95%. This nding is similar to the report of Okumu et al. [22]. where hexane leaf extract of A. indica was found to cause 100 % mortality against larvae of An. gambiae at 1000 ppm although the concentrations are different. This could be due to the presence of excess bioactive secondary metabolites.
However, this nding disagrees with nding of Vilayatakar et al. [23] that reported the aqueous extract of M. oleifera leaves against larvae of Anopheles which has shown 60% mortality at 500ppm. Similarly, acetone extract of O. lamiifolium oil was found to have high larvicidal activity against An. arabiensis [24]. Various factors might be responsible for the larvicidal activity, but the difference in larvicidal activities in the current nding could be due to locality of the plants, and different solvents.
Methanol leaf extract of A. indica cause adult mortality with mortality 75% which could be due to the presence of bioactive secondary metabolites. This nding is in line with the earlier nding of Kamaraj et al. [25]. who reported adulticidal e cacy of methanol against Cx. gelidus Theobald.  Map of the study area