J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 362 http://jad.tums.ac.ir Published Online: December 31, 2019 Original Article Toxicity of Ocimum basilicum and Ocimum gratissimum Extracts against Main Malaria Vector, Anopheles gambiae (Diptera: Culicidae) in Nigeria Kayode David Ileke1; *Jacobs Mobolade Adesina2,3 1Department of Biology, School of Science, Federal University of Technology, Akure, Ondo State, Nigeria 2Department of Crop, Soil and Pest Management Technology, Rufus Giwa Polytechnic, Owo, Ondo State, Nigeria 3Insect Chemical Ecology Laboratory, Institute of Bioresources and Sustainable Development, Department of Biotechnology, Govt. of India, Takyelpat, Manipur, India (Received 20 June 2018; accepted 13 Oct 2019) Abstract 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 tox- icity 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. gratti- simum 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 concentra- tions 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 or- ganisms, 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. Keywords: Ocimum basilicum; Ocimum gratissimum; Insecticide; Anopheles gambiae; Clarias garipienus 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 infec- tious disease that causes the death of infants and this had led to public health concern through- out the tropical and subtropical regions (2). Al- teration 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 insecti- cide proves to be the only effective means of combating mosquito until its adverse effects on the environment and user health become pro- nounce (3). Malaria vectors have developed a resistant mechanism against chemical insecti- cides because of continuous application of syn- thetic products in our environment for their man- agement (1, 4, 5). The problems of these envi- ronmental and health hazards limit their suc- cess in vector control, which has led to the de- velopment 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 par- asitologists to control larvae, pupae, and adult mosquitoes (8, 10, 11). Ocimum species belong to the family Lami- *Corresponding author: Dr Jacobs Mobolade Adesina, E-mail: mobolade72@gmail.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 363 http://jad.tums.ac.ir Published Online: December 31, 2019 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 al- ternative to chemical mosquitocides, this study was undertaken to assess the toxicity of O. ba- silicum and O. gratissimum against pre-adult stages and adult malaria vector, Anopheles gam- biae and non-targeted aquatic organism, fin- gerlings of Clarias garipienus. Materials and Methods Mosquito rearing Anopheles gambiae mosquitoes were breed in the Hatchery Laboratory, Department of An- imal and Environmental Biology, Adekunle Ajasin University Akungba Akoko, Ondo State, Nigeria (7° 28' N, 5° 44' E) and maintained at ambient temperature of 28±2 °C with 12:12 light and dark photoperiod in 75±5% relative humidity. Plant Materials and Extractions Ocimum gratissimum and O. basilicum leaves were collected fresh from Supare Ako- ko, Ondo State (7° 26' 0" North, 5° 43' E). Plant leaves were authenticated by Plant Taxonomist in the Plant Science and Biotechnology De- partment, 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. ba- silicum leaves powders were soaked separate- ly in an extraction bottle containing absolute ethanol. Stirred occasionally with a glass rod and extraction terminated after 3 days. The re- sulting mixture was filtered and the solvent was evaporated using a rotary evaporator. Extracts were kept in a vial and preserved in the refrig- erator until further use. Larval, Pupal and Adult Mortality Bioassay Bioassay tests were carried out on Ocimum species extracts using five different concentra- tions (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) pre- pared according to the standard methods rec- ommended by WHO (13-15). Twenty larvae and pupae of An. gambiae were separately intro- duced 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 pa- pers 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. basili- cum 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 con- trol coils were also set up, one made without extracts and the second control made using syn- thetic chemical as positive control. One hun- dred 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. Per- centage 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 Envi- ronmental Biology, Adekunle Ajasin Univer- sity Akungba Akoko, Ondo State, Nigeria (7° http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 364 http://jad.tums.ac.ir Published Online: December 31, 2019 28' N, 5° 44' E). The fingerlings were accli- matized for 14d in a dechlorinated borehole wa- ter. Fingerlings were fed twice daily until 24h prior to their exposure to Ocimum species in accordance with the recommendation of Ad- ekunle Ajasin University, Akungba Akoko On- do 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 dai- ly 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, pu- pae 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 activ- ity of Ocimum grattisimum and O. basilicum extracts Ocimum grattisimum and O. basilicum sig- nificantly affects the mortality of An. gambiae larvae at all the concentrations tested (Table 1). The toxicity of O. grattisimum and O. ba- silicum leaves extracts were significantly (P< 0.05) different from the solvent treated and con- trol 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) dif- ferent 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% con- centration 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. basili- cum leaf extract were able to achieved 100% in- sect mortality at 0.5% concentration while O. grattisimum caused 95% adult mortality after 60 min of exposure period (Table 3). Ocimum grat- tisimum and O. basilicum leaves extracts effects were significantly (P< 0.05) different from sol- vent treated and the control at all tested con- centrations. The LC50 and LC90 of O. basilicum were lower than O. grattisimum in all stages of An. gambiae studied (Table 4). The lethal concen- tration 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. ba- silicum 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 suck- ing 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. gratti- simum extract which was not significantly dif- ferent from other treatments apart from untreat- ed 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. gratis- simum Extracts on Aquatic Habitat organism There was no mortality of fingerlings rec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 365 http://jad.tums.ac.ir Published Online: December 31, 2019 orded after 2d of water treatment (Table 6). However, 10.25% mortality were recorded on fingerlings water treated with 0.5% O. gratti- simum and O. basilicum extracts after 3d of treat- ment. Similarly, 10% and mortality of finger- lings were recorded in water treated with 0.4 % O. grattisimum and O. basilicum extracts after 4d of exposure period. At 0.5% concen- tration, 20% mortality were recorded in fin- gerlings water treated with O. grattisimum and O. basilicum extracts after 5 d of treatment. Table 1. Toxicity of Ocimum grattisimum and O. basilicum on Larvae of Anopheles gambiae Plant Extracts Concentration (%) 0.1 0.2 0.3 0.4 0.5 Ocimum grattisimum 55.00+3.75b 65.00+3.75b 85.00+3.75b 90.00+4.25b 100.00+0.00b Ocimum basilicum 60.00+4.25b 75.00+3.75b 92.50+4.20b 100.00+0.00b 100.00+0.00b Solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Each value is a mean ± standard error of four replicates. Means followed by the same letter along the column are not significantly different using Duncan’s new multiple range test. Table 2. Toxicity of Ocimum grattisimum and O. basilicum on Pupae of Anopheles gambiae Plant Extracts Concentration (%) 0.1 0.2 0.3 0.4 0.5 Ocimum grattisimum 45.00+3.75b 50.00+4.25b 70.00+4.25b 77.50+4.10b 90.00+4..25b Ocimum basilicum 50.00+4.25b 60.00+4.25b 75.00+3.75b 82.50+4.20b 100.00+0.00b Solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Each value is a mean ± standard error of four replicates. Means followed by the same letter along the column are not significantly different using Duncan’s new multiple range test. Table 3. Fumigant Toxicity of Ocimum grattisimum and O. basilicum on Anopheles gambiae Adults Plant Extracts Concentration (%) 0.1 0.2 0.3 0.4 0.5 Ocimum grattisimum 50.00+4.25b 60.00+4.25b 75.00+3.75b 85.00+4.25b 95.00+3.75c Ocimum basilicum 60.00+4.25b 75.00+3.75b 85.00+4.20b 95.00+0.00b 100.00+0.00c Solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b Untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Each value is a mean + standard error of four replicates. Means followed by the same letter along the column are not significantly different using New Duncan’s multiple Range test Table 4. LC50 and LC90 of Ocimum grattisimum and O. basilicum extracts An. gambiae Plants LC50 (LCL–UCL)% LC90 (LCL–UCL)% Larvae O. grattisimum 0.104 (0.007–0.168) 0.363 (0.232–0.494) O. basilicum 0.093 (0.013–0.143) 0.246 (0.165–0.770) Pupae O. grattisimum 0.145 (0.025–0.223) 0.728 (0.409–1.047) O. basilicum 0.121 (0.032–0.210) 0.485 (0.273–0.697) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 366 http://jad.tums.ac.ir Published Online: December 31, 2019 Adults O. grattisimum 0.118 (0.026–0.180) 0.519 (0.330–0.708) O. basilicum 0.088 (0.013–0.140) 0.305 (0.208–0.904) Keys: LC Lethal Concentration, LCL–Lower Concentration Limit, UCL–Upper Concentration Limit Table 5. Smoke toxicity of Ocimum grattisimum and O. basilicum extracts on Adults An. Gambiae Extracts Total number of Adult mosquitoes Fed Mosquitoes Unfed Mosquitoes % Protection Ocimum grattisimum 50 11.50±1.15a 38.50±2.24b 72.25±3.24b Ocimum basilicum 50 6.75±0.08a 43.25±2.44b 81.75±3.18b Control I (Synthetic Insecticide) 50 5.25±0.35a 44.75±2.37b 84.50±3.39b Control II (Untreated) 50 47.50+2.20b 2.50+0.04a 0.00 ± 0.00a Each value is a mean + standard error of four replicates. Means followed by the same letter along the column are not significantly different using New Duncan’s multiple Range test Table 6. Toxicity of Fingerlings treated with Ocimum grattisimum and O. basilicum extracts Extracts of plants Conc. Mortality%±S. E after 1day 2 days 3 days 4 days 5 days Ocimum grattisimum 0.1 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.2 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.3 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 0.4 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.04b 10.00+0.04b 0.5 0.00+0.00a 0.00+0.00a 10.00+0.04b 10.00+0.04b 20.00+1.14b Ocimum basilicum 0.1 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.2 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.3 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 0.4 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 10.00+0.25b 0.5 0.00+0.00a 0.00+0.00a 10.25+0.04b 10.00+0.02b 20.00+1.14b Untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a Each value is a mean ± standard error of four replicates. Means followed by the same letter along the column are not significantly different using Duncan’s new multiple range test Discussion The use of plant-derived products as mos- quitocides have proved to be an alternative ap- proach to the control of insect vectors since the use of synthetic insecticides have been discour- aged 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 ac- tive compounds presents in these plants such as alkaloids (8). The LC50 and LC90 of O. ba- silicum were lower than O. grattisimum in all stages of An. gambiae studied. The lethal con- centration of O. grattisimum and O. basilicum to achieve 50% mortality was lower in larvae stage (0.104% and 0.093% respectively) com- Table 4. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 362–368 KD Ileke and JM Adesina: Toxicity of … 367 http://jad.tums.ac.ir Published Online: December 31, 2019 pared to pupae (0.145% and 0.121% respec- tively) and adult (0.118% and 0.088% respec- tively) of An. gambiae. Similarly, the lethal con- centration of O. grattisimum and O. basilicum to achieve 90% mortality was lower in larvae stage (0.363% and 0.246% respectively) com- pared to pupae (0.728% and 0.485% respective- ly) and adult (0.519% and 0.305% respectively) of An. gambiae. Larvicidal, pupicidal and adul- ticidal activity of the Ocimum extracts has been collaborated ted by the findings of other stud- ies (1, 8, 19). The authors reported the effec- tiveness of O. sellio and O. basilicum essential oils on mosquitoes (19). Larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent ac- tivities towards three mosquito species were evaluated (1). Recently, Afolabi et al. (8) re- ported the adulticidal and repellent activity of different extracts of O. caninum and O. gra- tissimum against adult An. gambiae. Our pre- sent 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 organ- ism, 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. How- ever, the addition of O. americanum to diet of Sciaenops ocellapus did not significantly af- fect 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 man- agement. These findings have demonstrated to be one of the alternative approach to manage mosquito vectors than the use of synthetic chem- ical insecticides that causes adverse effect on humans, environment and on non-target aquatic organisms. Further studies should be conduct- ed to describe toxicological and histological ef- fects of Ocimum species on non-target organ- isms, fingerlings of C. garipienus. Acknowledgements The authors thank Dr Obembe of the de- partment of Plant Science and Technology, Ad- ekunle Ajasin University Akungba Akoko, Ni- geria for the identification of botanicals used and Mr Eniade of Hatchery Unit, Department of Animal and Environmental Biology, Ad- ekunle Ajasin University Akungba Akoko, Ni- geria for the provision of fingerlings of Clari- as garipienus. The authors declare that there is no con- flict of interest. References 1. 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