1 Background

Chemical insecticides are extensively used to control mosquito growth, over use of these chemical insecticides has resulted into insecticide resistant in mosquitoes. The cases of insecticide resistant in mosquitoes have posed a major threat to the control of mosquitoes and mosquito-borne diseases in the tropical countries where these diseases are prevalent. In addition, resistance to insecticides is a major cause of rise in morbidity and mortality due to malaria and other vector-borne infections in Africa. Controls of such serious diseases are becoming increasingly difficult because of the high rate of reproduction and development of resistance to insecticides in mosquitoes (Severini et al., 1993). Chemical insecticides also caused several health hazards in humans and animals andare toxic to the environment. Bioaccumulation of these toxins in the environment results in ecosystem imbalance as the residue effects both target and non-target organisms (CDC, 2008). To overcome these problems, it is important to discover some new and potent insecticides from alternative sources such as plants and microorganisms (Sharma, 2001). In the quest for ecofriendly insecticides, various plants have been screened for their phytotoxic properties against insect pests and vectors. Some of these plants have been reported to possess toxic effect on mosquitoes and can be utilized as a potent source for mosquito control. Earlier, some plants such as Annona squamosa L., Gloriosa superba L., Millingtonia hortensis, Abuta grandifolia, Minthostachys setose, Azadirachta indica, and Hyptis suaveolens, have been reported to control mosquito population (Ciccia et al., 2000; Kaushik and Saini, 2008; Bagavan et al., 2009; Okigbo et al., 2010). Plant materials have received more attention since government of some countries have started restricting the use of many synthetic chemical insecticides because of their adverse effect on both human and their environment (Isman, 2000). However, plant base insecticides have been noted to have different effects on insects based on the type of botanical used, part of the plant used, solvent used for the extraction and types of phytochemical compounds present in the plant (Jeyabalan et al., 2003). Jenson et al. (2006) reported that crude plant extracts are highly efficient for the control of mosquitoes, rather than the purified compounds. The search for alternative pesticides and control measures that pose no risk or posing minimal risk to human health and the environment is of great interest from the preventive medicine point of view (World Health Organisation, 1999). Ocimum gratissimum L. (Lamiaceae) is one of those plants with insecticidal properties. The plant is known by different people of the world by different local names. It is commonly called Scent leave, ‘Nchu anwa’ or ‘Ahuji’ by the Igbo speaking populations of South Eastern Nigeria. ‘Effirin-nla’ by the Yoruba speaking people of South Western Nigeria and known as ‘Daidoya’ by the Hausa Speaking populations of Northern Nigeria (Odugbemi, 2006; Saliu et al., 2011). In India it is known by its several vernacular names, ‘Vriddhutulsi’ (Sanskrit), ‘Ram tulsi’ (Hindi), ‘Nimma tulasi’ (Kannada). The nutritional importance of Ocimum gratissimum centres on its importance as a seasoning because of its aromatic flavour that helps to improve the palatability of food (Okwu et al., 2006). O. gratissimum is an essential ingredient in the local cuisine popularly known as ‘pepper soup’ and relished by many in South Western Nigeria. It is an important herbal medicinal plant not only in Nigeria but also in the sub-Saharan Africa. In the southern part of the country, crude aqueous extract of O. gratissimum is commonly used in the treatment of epilepsy, high fever and diarrhoea (Effraim et al., 2003). In the savannah areas, decoctions of the leaves are used to treat mental illness (Akinmoladun, 2007). It is used by the Igbo community of south eastern Nigeria in the management of the baby’s cord, to keep the wound surfaces sterile. It is also used in the treatment of fungal infections, fever, cold and catarrh (Ijeh et al., 2005). Brazilian tropical forest inhabitants use a decoction of O. gratissimum in the treatment of digestive disorders (Madeira et al., 2003). They are also used for abdominal pains, sore eyes, and ear infections, for coughs, barrenness, and fever, convulsions, and tooth gargle, regulation of menstruation and as a cure for prolapsed of the rectum (Lexa et al., 2007). Hence, this research is aimed at exploring the efficacy of Ocimum gratissimum on adult Anopheles mosquitoes.

2 Materials and Methods

2.1 Extraction of plant powder and oil

Leaves of O. gratissimum were collected from Falowo Street, Off Danjuma in Akure South Local Government Area of Ondo State. The collected plant material was identified at the department of Crop Science and Pest Management of the Federal University of Technology Akure, Nigeria. The leaves of the plant were mildly washed in a bowl of clean water and subsequently air dried at ambient temperature (28±2^°C) for 30 days. The dried leaves were then pulverized into fine powder with the aid of an industrial electric pulverizing machine and the powder was kept separately in labelled plastic containers until use.

One hundred grams (100 g) of Ocimum gratissimum powder was weighed and soaked in 500 ml of Ethanol for 3 days. The oil was extracted from the powder after the third day with the aid of a muslin cloth by squeezing the mixture to ensure that the active ingredients in the powder get extracted alongside with the solvent. The extract was concentrated using the rotary evaporator and the oil extract was kept in an air tight clean plastic container until use.

2.2 Insect culture

Two grams (2 g) of dried granular yeast was made into paste and poured into a plastic bowl and placed in a shady area at the back of Biology Laboratory, School of Science, Federal University of Technology Akure, Ondo State, Nigeria. After 7 days mosquito larvae were collected and Anopheles mosquito larvae were identified and separated from the mixed culture using the morphological keys of Hopkins (1952). In the laboratory, the larvae were transferred into another plastic container where they were fed with yeast. Ten mosquito larvae of four replicates including the control were set up for the purpose of the experiment. Each replicate was introduced into the experimental cage so that the resulting adults will emerge in it. The emerged adult mosquitoes were used for the experiment.

2.3 Toxicity test

Different concentrations (1 g, 2 g, 3 g, 4 g and 5 g) of the plant powder were put in muslin cloths measuring 3 cm x 2 cm dimension and suspended with the aid of thread and cello tape at a distance of 6 cm from the top of the plastic containers (13 cm depth 12 cm diameter). Ten (0-24 hr old) adult mosquitoes were introduced into the plastic containers each containing the suspended bags of plant powder. Similarly, the effect of the oil extract was tested at 5-25 ml concentrations at 6 hour interval of exposure. The experiment was replicated four times, including the control and adult mortality was recorded at 6 hrs of intervals.

The fumigant effect of the plant oil on adult mosquitoes was tested by using the following concentrations 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, and 0.5 ml and pouring it on 2 cm×2 cm Whatman’s No.1 filter paper strips. These strips were allowed to air-dry for 1h thereafter placed in plastic containers. Ten (0-24 h old) adult mosquitoes were introduced into the containers and adult mortality was counted at 6 hrs of intervals.

The oil extract was further formulated into wax candle, the wax was heated for 10 mins at a temperature of 160^°C, after melting, different percentage of the oil extracts (10%, 20%, 30%, 40% and 50% concentrations) was dispensed into 90%, 80%, 70%, 60%, 50% of wax candle with the candle wick lengths of 4 cm, 6 cm, 8 cm, 10 cm, 12 cm, respectively and it was solidified for few minutes. The candle was burnt (forming a smoke generator) and exposed to the adult mosquitoes inside the mosquito net and the mortality was recorded at 6 hours intervals.

2.4 Statistical analysis

All data obtained were subjected to One-way Analysis of Variance (ANOVA) at 5% level of significance using SPSS 20.0 version. The means were separated using the New Duncan’s Multiple Range Test.

3 Results

3.1 Effect of Ocimum gratissimum powder on adult Anopheles gambiae

The results presented in Table 1 shows that there is significant difference in the effect of the powder of Ocimum gratissimum on the adult mosquitoes (p<0.05) at different intervals and weights. Cent percent mortality was recorded at 36 hours of exposure for all the concentrations of the powder used. Meanwhile, lowest mortality of 3.33% was recorded at 1 g at 12 hours of exposure. Concentrations at 2 g and 5 g of the powder showed 100% mortality at 30 hours of exposure followed by 1 g and 3 g which showed 93.33% and 80.0% respectively. Generally, it was observed that the adult mortality of mosquitoes increases as the concentration and time interval increase.

3.2 Fumigant effect of Ocimum gratissimum oil on adult Anopheles gambiae

Cent percent mortality was recorded at 30 hours of exposure at all the concentrations of O. gratissimum extract. No mortality was recorded at 5-25 ml of the extract at 6 hours of exposure (Table 2).

3.3 Fumigant effect of Ocimum gratissimum on adult Anopheles gambiae when formulated as Wax Candle

Table 3 shows that there is a significant difference in the mortality of adult Anopheles gambiae at different concentrations (P < 0.05). No mortality was observed at 0.1-0.2 g/mol at 6 hours of exposure and also for 12 hours exposure at 0.1 g/mol. Cent percent mortality was observed at 0.5 g/mol concentration for 30 hours. Furthermore, it was observed that no mosquito survived at all concentrations for 36 hours except the control where no mortality was recorded.

4 Discussion

The results showed that O. gratissimum extract has significant effect on the mortality of the adult Anopheles mosquitoes compared to the controls. O. gratissimum showed that the powder, oil and wax candle formulations showed 100% mortality at 2 g and 5 g of the powder, 5-25 ml of the oil and 0.5 g/mol of the wax candle at 30 hours of exposure. The high mortality of the adult mosquitoes recorded by the plant extract may be attributed to the active compounds present in this plant. The plant has been reported to contain flavonoids and Tannin (Gonzalez et al., 1991; Horie et al., 1993), terpenoids and alkaloids (Weidenfeld and Roder, 1991; Tyagi et al., 1995; Sur et al., 1997; Pari et al., 2000). All these compounds have high mortality on insect pests and vectors (Philipson and Wright, 1991). Furthermore, presence of flavonoid, Pectolinaringenin from C. philomids exhibited promising larvicidal activity against mosquito larvae (Muthu et al., 2012). Ocimum gratissimum also proved to be a good control for mosquitoes with the oil recording 100% mortality at 30 hours exposure at all the concentrations. The fumigant effect of the plant extract when formulated as oil and constituted as wax candle might be attributed to strong choky effect of the oil which blocked the spiracles of the mosquitoes which disrupt the respiratory activity of the insect vector. Similar effects of the oil have been reported by Ileke et al. (2013) on Callosobruchus maculatus. Likewise, Akinneye and Afolabi (2014) had earlier reported the fumigant effect of the powder of Cleisthopholis patents on adults An. gambiae while Akinkurolere et al. (2011) reported the fumigant effect of Xylopia aethiopica on An. gambiae. This suggests that botanicals with strong pungent odour are effective as larvicides and adulticides for controlling mosquito population.

5 Conclusion

In vector control programme, environmental safety is considered to be of paramount importance. This is because of the hazardous effect of insecticides to man and his environment. In order, to reduce the mammalian toxicity caused by over dependence on chemical insecticides, botanical insecticides have proven to be highly effective in reducing mosquito population and mosquito-borne diseases such as malaria, dengue fever, filariasis etc., and at the same time eliminating the environmental pollution caused by these chemical insecticides. O. grattissimum is an edible plant, relatively safe, inexpensive and readily available. In addition, the oil extract can easily be formulated into wax candle and made available in rural areas. This will further generate local employment, reduce on the use of expensive and imported chemical insecticidal products.

Aknowledgements

The author appreciates the contribution of Mr Ajayi of the Department of Crop Science and Protection for the identification of the plant used for this research. The contribution of Mrs E.T. Ojo of the department of Biology, Federal University of Technology Akure in the extraction of the plant’s oil is highly appreciated.

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