Mosquitoes are among the most important groups of arthropods with medical significance. They transmit several important parasitic and arboviral diseases, such as malaria, filariasis, dengue, yellow fever, and Rift Valley fever [1]. Malaria, caused by protozoans (Plasmodium spp.), can lead to high mortality and morbidity [2]. In 2019 there were 229 million reported malaria cases [2]. Malaria cases that occurred in 2020 were estimated to be 241 million, with 627,000 deaths reported from 85 countries. Around 95% of the malaria cases and 96% of malaria deaths were found in sub-Saharan Africa, with 80% of all malaria deaths in Africa estimated to be among children under the age of five [3].
Anopheles stephensi is a major malaria vector in South Asia and the Middle East, including the Arabian Peninsula [4], and is known to transmit both the major human malaria parasites Plasmodium falciparum and Plasmodium vivax [5]. In 2012, An. stephensi was first reported from the Horn of Africa as an invasive species in Djibouti. In 2016 and 2019, it was reported from additional countries, including Ethiopia, Somalia, and the Republic of Sudan [6,7,8]. If the species continues to spread across the continent, it is estimated that an additional 126 million people in Africa will be at risk of malaria [9]. In Ethiopia, this malaria vector was first reported from the Somali region in 2016 [10] and was later found to be widely present in urban areas of northeastern Ethiopia [11, 12]. The spread of this vector in different parts of the country has become a serious concern for malaria prevention and elimination strategies. The World Health Organization (WHO) has issued a warning about the invasion and spread of An. stephensi particularly in urging African national malaria control programmes and their partners to be vigilant in areas of risk and to improve and enhance their monitoring systems to identify and control this invasive mosquito species [13]. Recent evidence reveals that invasive An. stephensi from Ethiopia is a more competent vector for P. vivax than Anopheles arabiensis, the primary malaria vector in Ethiopia, in laboratory experiments [14].
Global malaria cases and deaths have been significantly reduced following the scaling up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) [15, 16]. However, widespread use of synthetic insecticides in controlling mosquito vectors has resulted in the persistence and accumulation of non-biodegradable chemicals in the ecosystem, development of resistance to insecticides in vectors, and toxic effects in non-target organisms [17,18,19]. As evidenced in recent studies from different parts of Ethiopia, An. arabiensis and An. stephensi have shown resistance to insecticides belonging to four of the chemical classes approved for IRS and ITNs, including DDT (organochlorine), malathion (organophosphate), bendiocarb and propoxur (carbamates), and alpha-cypermethrin, cyfluthrin, deltamethrin, etofenprox, lambda-cyhalothrin, and permethrin (pyrethroids) [20,21,22,23]. Reduced effectiveness of insecticides on these malaria vectors may aid in the invasion and establishment of An. stephensi.
The emergence of insecticide resistance necessitates an urgent need to develop new and improved mosquito control methods that are economical and effective and less toxic to non-target organisms and the environment. In this regard, botanicals, namely plant extracts and essential oils with insecticidal potential, are recognized as potent alternatives to replace the synthetic insecticides in mosquito control programmes due to their larvicidal, pupicidal, and adulticidal properties; these have also been shown to have oviposition inhibiting, repellent or insect growth regulatory effects, and may help us to find chemicals that are safe, biodegradable, and target specific [24,25,26,27].
Traditionally, plant-based products have constituted an important source of insecticides and other pharmaceutical drugs for many centuries; Calpurnia aurea, Momordica foetida, and Zehneria scabra are the foremost mentioned in Africa [28, 29]. In Ethiopia, these botanicals have been reported as having medicinal properties to prevent vector-borne diseases [30,31,32] and protect against insect pests [33, 34]. Moreover, these three medicinal plants are cheap and easily available. However, the bioactivities of these plant extract against invasive An. stephensi in Ethiopia has not been evaluated yet. This study evaluated the larvicidal and adulticidal activities of the aforementioned plant leaf extracts against An. stephensi under laboratory conditions.
Methods
Collection of plant samples
The fully developed fresh leaves of C. aurea and M. foetida were collected around Bahir Dar University campus located at 11°34′28.0″N, 37°23′53.4″E, at an altitude of 1801 m, while the leaves of Z. scabra were collected from Akaki District, Addis Ababa (8°49′40.5″N, 38°50′23.6″E, altitude 2117 m). The plant species were authenticated by a plant taxonomist from the National Herbarium in the Department of Plant Biology and Biodiversity Management, Addis Ababa University. The voucher specimens (MM-01 of M. foetida, MM-02 of C. aurea, and MM-03 of Z. scabra, respectively) were deposited at the National Herbarium, College of Natural and Computational Sciences of Addis Ababa University, Ethiopia.
Preparation of plant samples
The leaves were washed with water and air-dried separately under shade at room temperature for two weeks in the Insect Sciences Laboratory, Addis Ababa University. Finally, the dried leaves were manually ground by pestle and mortar through a sieve into a fine powder. The leaf powder was kept at room temperature in labeled air-tight plastic bags until used.
Extraction of the plant samples
The crude extraction processes were conducted in the Organic Chemistry Laboratory of the Department of Chemistry, Addis Ababa University. Seventy-two grams of each powdered material was soaked separately in 720 ml of hexane, methanol, and distilled water at a ratio of 1:10 (W/V) in Erlenmeyer flask. Afterward, the mixtures were sonicated by ultrasonic cleaner apparatus (USC-T, Malaysia) at 20 kHz frequency with 11 W power twice per day for 15 min for two days. The mixtures were left to settle for 10 min and then cooled at room temperature. The supernatant of the hexane and methanol crude extracts were filtered through Whatman No.3 (Whatman International Ltd., Maid stone, England) filter paper, while the aqueous extracts using suction were filtered through a Buchner funnel with Whatman filter paper No.1. The filtrates of hexane and methanol crude extracts were concentrated using a vacuum rotary evaporator (Rota-vapor-RE, Buchi Labortechnik AG, Flawil, Switzerland) under reduced pressure at 40oC, while the aqueous crude extracts were evaporated to dryness using a lyophilizer. The residue obtained from each plant extract was left to cool at room temperature to remove traces of solvent, and then finally, were collected separately in a Wheaton bottle glass containers and were preserved a refrigerated at 4 °C until used for experimentation.
Rearing of Anopheles stephensi
Larvicidal and adulticidal bioassays were conducted with colony larvae and adults of An. stephensi originated from the Awash Arba area, eastern Ethiopia. The mosquito larvae and adults were maintained at 28 ± 3oC temperature with 70 ± 10% relative humidity at the insectary of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. All equipment (cages, trays, incubators) containing mosquitoes have been deployed so that accidental contact and release was minimized.
The larvae (67 larvae/cm2) were kept in plastic trays (20 cm × 15 cm × 5 cm) containing de-chlorinated water (1.5 l) and were maintained under 12:12 h light and dark photoperiod cycles in the laboratory following the standard mosquito rearing procedure [35]. The larvae were fed with yeast (Saf-Instant yeast). The media were changed every three days. The pupae formed were collected by a glass beaker and transferred to 24.5 × 24.5 × 24.5 cm cages (Bug dorms) for adult emergence. Newly emerged adults were maintained in mosquito cages at 28 ± 3 °C temperature and 70 ± 10% relative humidity and fed sterile 10% sugar solution soaked in cotton pads within Petri dishes. The cotton was always kept moist with the solution and changed every day. In addition to sugar feeding, female mosquitoes were allowed to take blood meals from a restrained rabbit three times a week for egg development and oviposition. Moist filter papers in cups were placed inside rearing cages for oviposition by gravid female mosquitoes. The eggs were washed off with deionized water onto larval rearing trays and allowed to hatch into neonate larvae in the laboratory. Third to fourth instar larvae and bloodmeal starved 2 to 5 days old adult female An. stephensi were used continuously for larvicidal and adulticidal tests, respectively.
Preparation of stock solution
One gram of each crude plant extract was placed separately in 250 ml Wheaton glass bottles and dissolved in 100 ml of distilled water for methanol and aqueous extracts, while the hexane extracts were first dissolved in 4 ml of acetone and then added to 96 ml of distilled water to prepare 100 ml of a 1% stock solution. In the stock solution, one drop of emulsifier (Tween 80) was added to each extract at a concentration of 0.05%. From the 1% stock solution, concentrations of 25, 50, 100, 150, 200, 250, and 300 ppm for the larvae, and concentrations of 20, 40, 80, 160, and 320 ppm for the adult were prepared for exposure of the target mosquito.
Larvicidal bioassay
Larvicidal activities of each leaf crude extract were measured according to WHO standard procedures [35]. For larvicidal bioassay, hexane, methanol and aqueous crude leaf extracts of the test plants were screened at 25, 50, 100, 150, 200, 250, and 300 ppm of test concentrations. Twenty individuals of late third to early fourth instar larvae were kept in a 300 ml enamel cups containing 99 ml of distilled water and 1-ml of desired concentrations of crude solvent extracts of C. aurea, M. foetida and Z. scabra were added. In the same way, 99 ml of distilled water with 1 ml mixture of acetone (for hexane extracts) and Tween 80 were made up to 100 ml to serve as a negative control. In addition, temephos at the rate of 0.25 ppm within a similar volume of test cups was used as a positive control [36]. The experiment was conducted for 24 h at the optimum conditions of 28 ± 3 °C temperature and 70 ± 10% relative humidity under 12:12 light and dark photoperiod in the insectary. During the exposure period, no food was offered to the larvae. Each experiment was run three times on different days along randomly set up with appropriate negative control and standard check. Numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the averages of twelve replicates. The dead larvae included moribunds; those incapable of rising to the surface in each concentration of treatments, and the standards were combined separately and expressed as the average mortality to determine LC50 and LC90 values.
Adulticidal bioassay
Adulticidal activities of each crude solvent extract were performed using the CDC bottle bioassays [37, 38]. Most of the synthetic insecticides available for the control of adult mosquitoes, in particular An. stephensi were reported to be resisted by adults [12, 23], thus, this bioassay did not have any synthetic insecticide as a positive control. In this bioassay, aqueous, hexane and methanol crude leaf extracts of the three plants were screened at 25, 50, 100, 150, 200, 250, and 300 ppm concentrations. Two hundred fifty ml Wheaton bottles with screw lids were properly cleaned and dried, and then they were coated with 1ml solution of the desired concentrations of the tested extracts by swirling assuring complete coating of the bottle and its cap. Similarly, 1ml of acetone and emulsifier (Tween 80) solution was added to the control bottle handled as before.
Adult mosquitoes (20, aged 2–5 days) fed on 10% sucrose solution were released to each bottle, including the control bottle with the help of mouth aspirator. The opening of each bottle was closed with their lids after introduction of mosquito adults. The exposure time was set to 2 h. After that, the mosquitoes were transferred into 250 ml capacity plastic cups using aspirator, where they were provided with 10% sucrose solution and the mortalities were checked after 24 h. The experiment was done at 28 ± 3 °C temperature and 70 ± 10% relative humidity under 12:12 light and dark photoperiod in the insectary. The percentage of adult mortality was corrected using Abbott’s formula [39] when needed.
$$\% \text{Morality} = \frac{{\% \text{test}\,\text{morality} - \% \text{control}\, \text{morality}}}{{100 - \% \text{control}\, \text{morality}}} \times 100. $$
Data analysis
The mean percentage mortality was analysed using SPSS version 25.0 software (SPSS Inc, Chicago, IL, USA), and dose-dependent mortality also was performed by R software version 4.0.5. The percentage mortality of larvae and adult were checked for normality by 1-Sample Kolmogorov-Smirnov Z test (K-S). When the percent mortality did not conform to the normal distribution, the non-parametric equivalent tests of Kruskal-Wallis were followed. p-values were adjusted with the Bonferroni correction to adjust for the inflation of type I errors when several Mann–Whitney tests are performed [40]. The LC50 and LC90 values were calculated using a generalized linear probit model, considering a 95% confidence interval.
