Mosquitoes are important vectors of diseases and nuisance pests. A mosquitos
principal food is nectar or similar sugar source. Mosquitoes can be an annoying,
serious problem in mans domain. They interfere with work and spoil hours
of leisure time. Their attacks on farm animals can cause loss of weight and
decreased milk production. Some mosquitoes are capable of transmitting diseases
such as malaria, yellow fever, dengue, etc. Malaria is transmitted by different
Anopheles species, depending on the region and the environment (Burfield
and Reekie, 2005). Mosquitoes are a major threat for over 2 billion people
in the tropics (Odalo et al., 2005).
To control mosquitoes, any type of control should involve careful consideration
of the biology of the mosquitoes and based on scientific surveillance. A response
to control nuisance mosquitoes may look very different from a response to control
disease-vectoring mosquitoes (Lee, 2000). In all cases,
larval mosquito control should be considered as the first option for abatement.
This involves location of larval habitats, followed by their modification or
treatment in such a way that the integrity of the habitat is preserved but the
mosquito larvae are reduced in numbers.
By controlling larval mosquitoes, the adults may never become problem. Larviciding
has the greatest control impact on mosquito populations because the larvae are
concentrated, immobile and accessible. Integration of disease treatment with
vector control (the latter, comprising of insect population control and personal
protection from mosquito bites) is considered the most effective means for diseases
control. Currently, repellents and Insecticide-Treated Bed (ITBs) nets represent
the most practical and economic methods of controlling vectors, the most common
mosquito repellent products available in the market contain DEET (N,N-diethyle-3-toluamide)
(Odalo et al., 2005). The search for effective
vaccines against malaria and other mosquito responsible diseases are still in
progress. Several studies have on focused natural products for controlling some
mosquitoes as insecticides and larvicides, but with varied results (Cavalcanti
et al., 2004).
Essential oil are natural volatile substances obtained from a variety of plants. Commercially, essential oil have many uses such as pharmaceuticals, flavor in many food products, odorants in fragrances and as insecticides. Also, particular emphasis has been placed on their antibacterial, antifungal and insecticidal activities.
Recently, various plants extracts, such as neem (Azadirachta indica, A.
jass), basil oil (Ocimum basilicum L., O. gratissimum L. and
O.americanum L.), citronella grass (Cymbopogon nardus Rendle),
galingale (Alpinia galangal L.), clove (Syzygium aromatium L.)
and thyme (Thymus vulgaris L.) have been studied as possible mosquito
repellents (Odalo et al., 2005).
Therefore, the purpose of this study is to determine the repellent activity of four accessions of basil (Ocimum basilicum L.) essential oil against Anopheles mosquito.
MATERIALS AND METHODS
Basil seeds used in these studies were obtained from 20 different parts of
Sudan; seeds of basil accessions were directly sown on 60 cm wide ridges at
the Demonstration Farm (Nishishiba), Fac. Agri. Sciences, University of Gezira,
Wad Madani, Sudan. Sowing was done on Feb. 20, 2005. Watering, weeding etc were
carried out as necessary. No chemicals (fertilizers or others) were applied.
Observations were made on growth and flowering of the plants weekly, the accessions
were given numbers, as previously reported by Abduelrahman
et al. (2009). Essential oil of 4 basil accessions obtained by steam
distillation, fresh leaves of plant material (150 g) was subjected to stem distillation.
The extraction was carried out at a rate not exceeding 3 mL min-1,
distillation was continued for 2-4 h. This procedure was repeated until at least
10 mL of oil had recovered. The volume of the obtained essential oil was dried
over anhydrous Na2SO4, stored in a dark bottle and kept
at 4°C until it analysis or were evaluated for mosquito repellency as described
Rearing of mosquitoes: The mosquitoes, Anopheles mosquitos larvae, were collected at various sites in Gezira state; the larvae were transferred separately in metal dishes (40 cm diameter) and their rearing was continued until pupation. The larvae were reared continuously for several generations and kept under 25-30°C, 70-80% relative humidity and photoperiod of 13:11 h (light/dark) on 4, August, 2006 to 15, September, 2006, in the insectary of Blue Nile Research and Training Institute, Wad Medani, Sudan. Larvae were fed on ground commercial biscuit. The adults were reared in humidified cages and supplied with 10% sugar solution and 10% multivitamin syrup supplied in plates. Female mosquitoes were periodically blood-fed on restrained rabbits to obtain protein used principally for egg production. Under these conditions, the full development from egg to adult lasted about 3-5 weeks. Batches of 3-5 day-old healthy female mosquitoes were used in the repellency bioassay.
Repellency tests: The repellency of the essential oils of 4 basil accessions
was evaluated using the human-bait technique (Schreck and
McGovern, 1989; WHO, 1996). The tests were carried
out at night as the test organism, anopheles, is a night biter. Evaluations
were carried out by placing the arms of volunteers inside 40x40x40 cm cages.
The temperature was maintained at 25-30°C and the relative humidity at 60-80%.
Three human volunteers were employed for one repellency tests. Both arms of
volunteers, which would later be placed one in each cage, were carefully covered
with thick paper, except for a cut area of 3x10 cm exposing the arm to mosquito
bites. This area (of 3x10 cm) was not treated with anything for control (one
arm) or pretreated by 0.1 mL of basil essential oil (the other arm). Each arm
of a volunteer was placed inside the cage containing 50 female mosquitoes for
3 min. Exposure was repeated every 30 min without renewing the essential oil
treatments. During the 3 min exposure the number of mosquitoes landing on each
3x10 cm test area was observed. Mosquitoes that land and bite are clearly distinguished
by the visible blood in their bodies. The number of these mosquitoes taken throughout
the test period 6 h (20:00 to 02:00 h) was used as the number of bites calculated
per hour. Each mosquito that has bitten once was removed from cage by the visible
blood in their bodies and replaced by a new one. This test repeated for all
4 accessions with same conditions.
Data analysis: The median protection time was used as a standard measure
of the repellency of the volatile oil against mosquito in the laboratory. Percent
protection from mosquito landing/biting or repellency was computed as compared
to control by the following equation (Tawatsin et al.,
where, C is the number of mosquitoes collected from control area and T is the number of mosquitoes collected from the treated areas of volunteers arms. Statistical analysis was conducted using a statistical analysis program, the Origin version 6.1.
RESULTS AND DISCUSSION
Insect repellency of the crude essential oils of four basil accessions:
Certain basil crude essential oils were claimed to have a repellent activity
towards mosquitoes (Tawatsin et al., 2001). To
test this claim the essential oils of four accessions of basil were selected
and tested for mosquito repellency on human volunteers using the method described
Table 1 shows the numbers of mosquitoes (Anopheles)
biting on the control and treated areas of the arm and Fig. 1
shows insect repellency of four volatile oils against Anopheles mosquito.
|| Number of mosquito bites on arms of volunteers
|*A. Accession; No. of bites are means of three replicates,
Control: Means of three replicates of untreated arms. The same volume (0.1
mL) of the essential oil from each accession tested was uniformly applied
(just ones) to a volunteers arm, which was exposed to the mosquitoes
and repellency evaluated
||GC-MS analysis (%) compositions of essential oils of 4 basil
accessions cultivated in Nishishiba
Insect repellency of four volatile oils against Anopheles
mosquito. The essential oil was applied only once onto hands of volunteers
and repellency followed for up to 6 h
The same volume (0.1 mL) of the essential oil from each accession tested was
uniformly applied (just once) to a volunteers arm, which was exposed to
the mosquitoes and repellency evaluated. All basil accessions showed complete
(100%) repellency toward mosquitoes till 1½ h of exposure of the volunteers
arm to the insects. Accessions No. 17 and 20 provided complete protection against
bites for 2 ½ and 2 h, respectively (Fig. 1). With
long exposure times observed repellency decreased markedly especially in accession
No. 10. This could be due to increased volatilization of the applied essential
oil with time, reducing efficacy.
Table 2 shows the GC-MS analysis (%) compositions of essential
oil of 4 basil accessions cultivated in Nishishiba. It is noteworthy that GC-MS
separations of the essential oil in the previously study (Abduelrahman
et al., 2009) for these accessions. We classify the basil accessions
analyzed according to the major essential oil constituents present. The 4 accessions
were classified into 3 classes where either one essential oil constituent dominated
in proportions exceeding 50% of the oil constituents or/otherwise, the first
two major constituents designed the group, the more dominant compound named
first. The major 3 classes (chemotypes) were: linalool chemotype (accession
No. 10), methyl cinnamate-linalool (accession No. 16) and geraniol-geranial
class (accession No. 17 and 20). An attempt was made to correlate between repellency
with essential oil chemical constituents. No clear-cut correlations were obtained.
One generalization that could be made, the two accession No. 17 and 20 which
provided complete protection against bites for 2- 2 ½ h, both have geraniol
as dominant component, other two accession No. 10 and 16 the dominant component
were linalool and M. cinnamate, respectively. So, may be the reason of
high protection due to present of geraniol, however, this claim may warrant
Chokechaijaroenporn et al. (1994) showed repellency
of the volatile oil of several basil species against Ae. Aegypti lasting between
15 and 105 min, depending on the basil type. On the other hand, in a study in
Guinea Bissau, West Africa, fresh Ocimum canum Sims (syn. Ocimum americanum)
could reduce biting by anopheline mosquitoes, about 63.6%. Kweka
et al. (2008) reported the repellency of plant species including
O. kilimandscharium and O. suave with high biting protection reached
(83 to 91%) and feeding inhibition (71.2 to 92.5%), respectively. It was observed
against three species of mosquitoes. Other essential oil e.g., oil of turmeric,
kaffir lime, citronella grass and citronella cream were shown to possess repellency
against mosquitoes (Assabgui et al., 1997; Palsson
and Jaenson, 1999; Rajkumar and Jebanesan, 2007,
2008; Oyewole et al., 2008;
Tawatsin et al., 2001). In this study, we tested
the repellent activity of four accessions of O. basilicum essential oil
against Anopheles mosquito. The results obtained from this study indicate
that all four essential oil of basil should promising repellent activity against
Anopheles mosquito. These results, somewhat, same with some earlier studies
in repellent activity of basil essential oil against mosquito; but these also
differ with some reported studies, the differences can be attributed to many
factors; perhaps the most obvious is the difference in concentration or amount
of essential oil, also some researchers obtained the oil by solvent extraction,
while we obtained by steam distillation. However, comparisons, such as the preceding,
are confounded by differences in the mosquito species tested, environmental
From knowledge of the essential oil components of the 4 accessions mosquito repellent activity could not be assigned to a particular compound. So, further investigations are needed to elucidate the four essential oil against a wide range of mosquito species and also to identify the active ingredient.
In conclusion, this study clearly demonstrated the potential of volatile oil derived from some basil species for use as topical repellents against nocturnal mosquitoes. Repellent activity is subject to improvement. These basil oil can, therefore, serve as a substitute for some synthetic insecticides which are expensive and have toxic residual effects to human.
We must acknowledge partial financial (grant No. MHESR, SR, 1, A, 13, 1-2005) support offered by the Basil Project at NOPRI, University of Gezira. This project is a kind research grant from the Ministry of Higher Education and Scientific Research, Khartoum. We also thank the Blue Nile Research and Training Institute, Wad Medani, Sudan for cooperation, made allowance and using their lab and insectary for this study.