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International Journal of Horticulture 2014, Vol.4, No.6, 24
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31
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4 Materials and Methods
4.1 Rearing of Insects
Newly emerged adult
C. maculatus
used for this study
were obtained from already existing culture in the
Postgraduate Research Laboratory of the Department of
Biology, Federal University of Technology, Akure,
Nigeria. They were subsequently reared inside 1 litre
Kilner jars, on un-infested cowpea seeds
Vigna
unguiculata
variety Ife brown obtained from
International Institute for Tropical Agriculture, Ibadan,
Nigeria. The culture was placed in an insect rearing cage
at ambient temperature of 28+2
o
C and 75+5% relative
humidity.
4.2 Identification and sexing of adult
Callosobruchus
maculatus
The identification and sexing of
C. maculatus
were
carried out in the Postgraduate Research Laboratory,
Department of Biology, Federal University of Techno-
logy, Akure, Ondo State using Binocular Microscope
based on observations of Halstead (1963), Appert (1987),
Odeyemi and Daramola (2000). Male have comparative
shorter abdomen and the dorsal side of the terminal
segment is sharply curved downward and inward. In
contrast the females have comparatively longer
abdomen and the dorsal side of the terminal segment is
only slightly bent downward (Ileke et al., 2013a). The
female also has two dark visible spots on their elytra
(Halstead, 1963; Odeyemi and Daramola, 2000).
4.3 Preparation of
Alstonia boonei
Leaf, stem bark and root of
Alstonia boonei
used for this
study were sourced fresh from Akola farm at
Igbara-Odo Ekiti, Ekiti State, Nigeria. These plant parts
were rinsed in clean water to remove sand and other
impurities, cut into smaller pieces before air-dried in the
laboratory. The cleaned dried plant parts were
pulverised into very fine powder using an electric
blender (Supermaster ®, Model SMB 2977, Japan). The
powders were further sieved to pass through 1mm
2
perforation. The powders were packed in plastic
containers with tight lids and stored in a refrigerator at
4
o
C prior to use.
4.4 Soxhlet Extraction of
Alstonia boonei
The solvents used for the extraction was Petroleum ether.
Thereafter, 200 g of the pulverised plant leaves, stem
bark and root were measured separately into a beaker
and packed in a thimble using muslin cloth and
extracted with 500 ml of Petroleum ether in a soxhlet
apparatus. In each case, the extraction was carried out
between 40~60
o
C. Excess solvent was recovered using
rotary evaporator vacuum. The resulting extract was air
dried in order to remove traces of solvent. From this
stock solution, different extract concentration of 1%, 2%,
3% and 4% were prepared separately as follows: 1%
concentration was made by diluting 0.1ml of oil in
9.9ml of solvent; 2% concentration was made by
diluting 0.2 ml of oil in 9.8ml of solvent; 3%
concentration was made by diluting 0.3ml of oil in
9.7ml of solvent. Similarly, 4% concentration was made
by diluting 0.4ml of oil in 9.6ml of solvent (Ashamo and
Akinnowonu, 2012; Ileke et al., 2013b).
4.5 The contact effect of
A. boonei
latex, oils and
extracts on
C. maculatus
1%, 2%, 3% and 4% concentrations of each oils of
A.
boonei
was mixed separately with 20g of un-infested
cowpea seeds in 250ml plastic containers. The oils and
seeds were thoroughly mixed using a glass rod and then
agitated for 5-10 min to ensure uniform coating. The
containers were left open for 30 min to allow solvent
traces to evaporate off. Two control experiments were
also set up without solvent and oil and solvent treated.
Ten pairs of teneral adult
C. maculatus
were
introduced into each of the containers and covered.
Four replicates of the treated and untreated controls
were laid out in Complete Randomized Design in insect
cage. Beetle mortality was observed after 4 days of
application. The beetles were confirmed dead when
there was no response to probing with sharp pin at the
abdomen. The total number of eggs laid per replicate
was recorded after 4 days of application. Percentage
adult mortality were corrected using Abbott (1925)
formular.
Where P
T
=corrected mortality (%)
P
O
=observed mortality (%)
P
C
=control mortality (%)
1
100
P- 100
P-P P
O
C O
T