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Larvicidal and Pupicidal Activities of
P. glandulosus
and
C. rigidus rigidus
Leaf Essential Oils against Three Mosquito Species
11
blocked roof gutters especially in the Far North
Region of Cameroon. This would offer an eco-friendly
and less expensive way which may replace DDVP, the
conventional chemical, to
reduce the problem of these
mosquitoes known as “enemy number one of mankind
woldwide”, especially where the examined plants are
largly available and used in
Cameroon. However,
carrying out studies on mode of action in mosquito
physiology, persistance, synergistic effect of the two
plant essential oils and field trial in the nearest future
still need to be done.
3 Materials and Methods
3.1 Collection of plant materials
The fresh leaves of
P. glandulosus
Hook f (Lamiaceae)
and
C. rigidus
R. Br. (Myrtaceae) were collected in
October 2011 (6:00 am-11:00 am GMT) in Ngaoundere
(latitude 7° 22' North and longitude 13° 34' East,
altitude of 1100 masl), located in the Adamawa region
(plateau), Cameroon. The plants were less than
one-year old and only the green leaves were harvested.
These plants were identified for confirmation at the
National Herbarium of Cameroon, where voucher
specimens were deposited with the following voucher
number: 18564/SRF/CAM and 41168HCN for
C.
rigidus
and
P. glandulosus
, respectively. Leaves
were dried at room temperature of (25±3)
℃
and
(81±2)%
RH, and then ground in powder using
electric grinder until the powder passed through a 0.4
mm mesh sieve. The powder was stored in opaque
containers inside a refrigerator at -4
℃
and
transported by road in February 2012 to Faculty of
Pharmaceutical Siences, Agulu, Nnamdi Azikiwe
University, Awka, Anambra state, Nigeria by road
where the experiments were carried out and then
stored in a freezer at -4
℃
until needed.
3.2 Test organisms
The larvae of
Ae. aegypti
and
Cx. quinquefasciatus
were collected from WHO/National Arbovirus and
Vector Research Centre Enugu, Enugu state, Nigeria.
The larvae of
An. gambiae
were collected from Awka
market, Anambra State, Nigeria inside the gutter and
identified to WHO/National Arbovirus and Vector
Research Centre Enugu, Enugu state, Nigeria. All the
different genera larvae were then brought into the
insectarium in the Faculty of Pharmaceutical Sciences,
Agulu, Nnamdi Azikiwe University, Awka, Anambra
State, Nigeria where they were reared. The larvae of
Ae. aegypti
and
Cx. quinquefasciatus
were fed with
chicken feed (grower) mixed with fish feed in 3:1
ratio. Those of
An. gambiae
fed on ground chicken
feed (grower), yeast and fish feed in 3:1:2 ratios. On
every alternate day, the water from the culture bowl
was changed carefully until pupation. The feeding was
continued till the larvae transformed into the pupal
stage. The larvae and early pupae were maintained at
(28 ± 4)
℃
, (80±5)% RH and under 12 Light: 12 Dark
photoperiod cycles.
3.3 Extraction of essential oils
The method adopted by Diksha et al. 2012 and Aihua
et al. 2009 was used for extraction and isolation of the
essential oils in the Department of Pharmaceutical and
Medicinal Chemistry. The fresh dried powdered leaves
were completely immersed in distilled water, then
hydro-distilled in a full glass Clevenger-type
apparatus (India) to give reddish-yellow oil for
P.
glandulosus
and very importantly white-clear oil for
C.
rigidus
. During heating, generated steam ruptured the
cell walls of the leaves and released the oil present in
leaves. This process was continued for four hours for
maximum oil recovery. The oil was allowed to stand
for sufficient time, to be clear, and then it was
collected carefully after draining out condensed water.
The oil extracted from sample was found containing
fraction of water, which was removed by adding small
amount of anhydrous sodium sulphate. The essential
oil samples were rolled with aluminium foil and
stored in the dark at -4
℃
. The amount of oil obtained
from plant materials was calculated as: Oil (% w/w) =
Observed mass of oil (g)/Weight of sample (g) × 100
(Aihua et al., 2009).
3.4 Larvicidal and pupicidal bioassays
Larval and pupal bioassays were conducted according
to the WHO standard procedure (WHO, 2005) to
determine the toxicity of the plant essential oils
against
Ae. aegypti, Cx. quinquefasciatus
and
An.
gambiae
4
th
instar larvae and early pupae. Stock
solutions of the essential oils were made using Tween
80 as emulsifier to facilitate the dissolving of material
in water. The stock solutions were further diluted up to
10 ml by adding tap water. From these stocks, various