Toxicity of Ocimum basilicum and Ocimum gratissimum Extracts against Main Malaria Vector, Anopheles gambiae (Diptera: Culicidae) in Nigeria

Background: Anopheles gambiae (Diptera: Culicidae) transmit malaria parasite that causes malaria fever in humans, causing millions of deaths every year among infants in tropical countries. This study was undertaken to assess the toxicity of Ocimum basilicum and Ocimum gratissimum against pre-adult stages and adult malaria vector, An. gambiae and non-targeted aquatic organism, fingerlings of Clarias garipienus. Methods: Ethalonic extracts of O. basilicum and O. gratissimum were prepared according to the method described by WHO. The larvae and pupae of An. gambiae were exposed to plant extracts for 24h and their mortality was recorded. Toxicity of Ocimum species on non-targeted organism, fingerlings of C. garipienus was also investigated. Results: Ocimum basilicum showed remarkably potency against pre-adult stages and adults An. gambiae causing 100% mortality at 0.4% concentration within 24h of treatment. The LC50 and LC90 of O. basilicum were lower than O. grattisimum in all stages of An. gambiae studied. Ocimum basilicum and O. gratissimum extracts significantly reduced the number of bites by the vector given a range of 72.25% to 81.75% protection. Ocimum species at the tested concentrations did not significantly reduce the number of fingerlings introduced. Conclusion: Ocimum species at the tested concentrations did not significantly reduce the numbers of non-targeted organisms, fingerlings introduced. Therefore, O. basilicum and O. gratissimum could be used to reduce malaria prevalence in the endemic areas of Nigeria as it poses no threat to aquatic organisms.


Introduction
Arthropod insect vectors are blamable for spreading serious human diseases like malaria, encephalitis, yellow fever, dengue and filariasis (1). In Africa, malaria is a vector-borne infectious disease that causes the death of infants and this had led to public health concern throughout the tropical and subtropical regions (2). Alteration in the natural environment have also contributed to the widespread of the disease and change in behavior of the vector toward chemical insecticides.
In the beginning, synthetic chemical insecticide proves to be the only effective means of combating mosquito until its adverse effects on the environment and user health become pronounce (3). Malaria vectors have developed a resistant mechanism against chemical insecticides because of continuous application of synthetic products in our environment for their management (1,4,5). The problems of these environmental and health hazards limit their success in vector control, which has led to the development of eco-friendly, biodegradable and readily available plant-based mosquitocides with low-cost implication (3,6,7). Botanicals are considered as one of the harmless sources for controlling insect vectors and stored products pests (8,9). Botanical based insecticides have been reported by many entomologists and parasitologists to control larvae, pupae, and adult mosquitoes (8,10,11).
Ocimum species belong to the family Lami-aceae. Recent research has investigated the health benefits associated with Ocimum essential oils. Studies revealed the anti-viral, anti-microbial, antioxidant, and anti-cancer properties of the plants (12). Hence, in view of an increasing interest in developing plant-based insecticides as an alternative to chemical mosquitocides, this study was undertaken to assess the toxicity of O. basilicum and O. gratissimum against pre-adult stages and adult malaria vector, Anopheles gambiae and non-targeted aquatic organism, fingerlings of Clarias garipienus.

Plant Materials and Extractions
Ocimum gratissimum and O. basilicum leaves were collected fresh from Supare Akoko, Ondo State (7° 26' 0" North, 5° 43' E). Plant leaves were authenticated by Plant Taxonomist in the Plant Science and Biotechnology Department, Adekunle Ajasin University, Akungba Akoko, Ondo State (7° 28' N, 5° 44' E). The leaves were rinsed in distilled water to remove any form of impurities, air-dried in laboratory and ground into powder.
About 150g of O. gratissimum and O. basilicum leaves powders were soaked separately in an extraction bottle containing absolute ethanol. Stirred occasionally with a glass rod and extraction terminated after 3 days. The resulting mixture was filtered and the solvent was evaporated using a rotary evaporator. Extracts were kept in a vial and preserved in the refrigerator until further use.

Larval, Pupal and Adult Mortality Bioassay
Bioassay tests were carried out on Ocimum species extracts using five different concentrations (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) prepared according to the standard methods recommended by WHO (13)(14)(15). Twenty larvae and pupae of An. gambiae were separately introduced into the treated water, solvent treated and untreated water was set as control. Each treatment was replicated four times. Mortality was observed over 24h of treatment.
Twenty An. gambiae adults were introduced into a test-tube that contain suspended filter papers soaked with 0.1%, 0.2%, 0.3%, 0.4% and 0.5% Ocimum species extracts separately in four replicates for adult bioassay according (2,3). Mortality of adult insect was accessed after 2h of post-exposure. Percentage larvae, pupae and adult mortality were corrected (16).

Mosquito Coil Toxicity Bioassay
Chemical mosquito coils were mimic to form plant-based coils derived from O. basilicum and O. gratissimum separately. This is done by thoroughly mixed 10ml of 50% concentrated plant materials with 5g coconut shell, charcoal powder and distilled water to form semi-solid material that solidify with time under shade (3). Coil toxicity bioassay was evaluated using a glass chamber size 100× 70× 30cm. Two control coils were also set up, one made without extracts and the second control made using synthetic chemical as positive control. One hundred 2-3d old adult An. gambiae, fed with 10% sucrose solution was released into the chamber for 60min. The procedure was repeated four times on separate days. Mortality of adult insect was accessed after 60min of post-exposure. Percentage adult mortality was corrected using the methods described by Abbott (16).

Non-targeted Aquatic organism Bioassay
Fingerlings of C. garipienus of not more than 4-5wk old were collected from the Hatchery Laboratory, Department of Animal and Environmental Biology, Adekunle Ajasin University Akungba Akoko, Ondo State, Nigeria (7° 364 http://jad.tums.ac.ir Published Online: December 31, 2019 28' N, 5° 44' E). The fingerlings were acclimatized for 14d in a dechlorinated borehole water. Fingerlings were fed twice daily until 24h prior to their exposure to Ocimum species in accordance with the recommendation of Adekunle Ajasin University, Akungba Akoko Ondo State, Nigeria Ethical Committee. Twenty fingerlings were exposed to each concentration tested, fed with fish pellets (1mm) and daily monitored for 5d. Mortality was recorded daily for four days. They were considered dead when no visible movement was observed when agitated and probe with a sharp object.

Analysis of Data
The percentage of mortality of larvae, pupae and adults were calculated and corrected relative to the associated controls using Abbott's formula. Lethal concentrations (LC50 and LC90) and their 95% confidence limits were determined using Probit analysis (17).

Result Larvicidal, Pupicidal and Adulticidal activity of Ocimum grattisimum and O. basilicum extracts
Ocimum grattisimum and O. basilicum significantly affects the mortality of An. gambiae larvae at all the concentrations tested ( Table  1). The toxicity of O. grattisimum and O. basilicum leaves extracts were significantly (P< 0.05) different from the solvent treated and control experiment. Ocimum basilicum extract was the most toxic causing 100% mortality at 0.4% concentration within 24h of post-exposure and its effect was not significantly (P< 0.05) different from O. grattisimum extract. Ocimum grattisimum and O. basilicum extracts were able to cause 100% mortality at 0.5% concentration within 24h of post exposure and its effect was significantly (P< 0.05) different from solvent treated and control.
Similarly, O. basilicum caused 100% pupae mortality within 24h of treatment at 0.5% concentration and its effect was significantly not different from O. grattisimum extract who caused 90% pupae mortality ( Table 2).
The results of fumigant toxicity of O. basilicum leaf extract were able to achieved 100% insect mortality at 0.5% concentration while O. grattisimum caused 95% adult mortality after 60 min of exposure period (Table 3). Ocimum grattisimum and O. basilicum leaves extracts effects were significantly (P< 0.05) different from solvent treated and the control at all tested concentrations.
The LC50 and LC90 of O. basilicum were lower than O. grattisimum in all stages of An. gambiae studied ( Table 4). The lethal concentration of O. grattisimum and O. basilicum to achieve 50% mortality was lower in larvae stage (0.104% and 0.093% respectively) compared to pupae (0.145% and 0.121% respectively) and adult (0.118% and 0.088% respectively) of An. gambiae. Similarly, the lethal concentration of O. grattisimum and O. basilicum to achieve 9 % mortality was lower in larvae stage (0.363% and 0.246% respectively) compared to pupae (0.728 % and 0.485% respectively) and adult (0.519 % and 0.305% respectively) of An. gambiae.

Mosquito Coil Toxicity
Smoke toxicity of O. grattisimum and O. basilicum extracts on An. gambiae adults were presented in Table 5. Ocimum grattisimum and O. basilicum extracts and the positive control (Baygon insecticide) were not able to give 100% protection against adult A. gambiae from sucking blood. Positive control (Baygon insecticide) had the lowest number of fed mosquito (10.5). This is followed by O. basilicum extract which had 6.75 and 11.5 fed mosquitoes in O. grattisimum extract which was not significantly different from other treatments apart from untreated control that recorded 47.5 fed An. gambiae. Ocimum grattisimum, O. basilicum extracts and positive control were able to achieve 72.25%, 81.75% and 84.5% Protection which was not significantly from each other.

Toxicity of Ocimum basilicum and O. gratissimum Extracts on Aquatic Habitat organism
There was no mortality of fingerlings rec-

Discussion
The use of plant-derived products as mosquitocides have proved to be an alternative approach to the control of insect vectors since the use of synthetic insecticides have been discouraged due to their food and environmental health concerns, toxicity to untargeted organisms and insect vector resurgence rates have made their exploitation undesirable (18).
According to the results of our study, the extracts of the Ocimum species caused high mortality of An. gambiae larvae, pupae and adults. The high lethal effects on pre-adults and adults An. gambiae may be ascribed to the active compounds presents in these plants such as alkaloids (8). The LC50 and LC90 of O. basilicum were lower than O. grattisimum in all stages of An. gambiae studied. The lethal concentration of O. grattisimum and O. basilicum to achieve 50% mortality was lower in larvae stage (0.104% and 0.093% respectively) com-pared to pupae (0.145% and 0.121% respectively) and adult (0.118% and 0.088% respectively) of An. gambiae. Similarly, the lethal concentration of O. grattisimum and O. basilicum to achieve 90% mortality was lower in larvae stage (0.363% and 0.246% respectively) compared to pupae (0.728% and 0.485% respectively) and adult (0.519% and 0.305% respectively) of An. gambiae. Larvicidal, pupicidal and adulticidal activity of the Ocimum extracts has been collaborated ted by the findings of other studies (1,8,19). The authors reported the effectiveness of O. sellio and O. basilicum essential oils on mosquitoes (19). Larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species were evaluated (1). Recently, Afolabi et al. (8) reported the adulticidal and repellent activity of different extracts of O. caninum and O. gratissimum against adult An. gambiae. Our present study, O. basilicum and O. gratissimum extracts significantly reduced the number of bites by the vector given a ranged of 72.25% to 81.75% protection.
Preliminary study on non-targeted organism, fingerlings of C. garipienus showed that Ocimum species at the tested concentrations did not cause lethal effects on the numbers of fingerlings introduced. No previous study on the effects of O. basilicum and O. gratissimum extracts on non-targeted organism, fingerlings of C. garipienus was found in literature. However, the addition of O. americanum to diet of Sciaenops ocellapus did not significantly affect the reproductive parameter and whole body composition after 7wk (20).

Conclusion
Extracts of O. grattisimum and O. basilicum are promising in disease-vector mosquito's management. These findings have demonstrated to be one of the alternative approach to manage mosquito vectors than the use of synthetic chemical insecticides that causes adverse effect on humans, environment and on non-target aquatic organisms. Further studies should be conducted to describe toxicological and histological effects of Ocimum species on non-target organisms, fingerlings of C. garipienus.