Chapter Three

3.0 Methodology
In terms of the insecticidal plant extracts, the recorded behavioral responses of the larvae to the

extract showed that besides causing prolonged prostration in the larvae, thereby preventing

foraging activity, the extract also displayed tarsal contact (antifeedant effect). Unlike most

synthetic insecticides, plant derivatives have been shown to possess multiple modes of

insecticidal attack, which could include cutting off food consumption by target insects,

deterrence of food selection, disturbance of physiological functioning (as happens with cardiac

glycosides and the steroidal hormone beta-ecdysone), inhibition of numerous processes in light

reception, message transmission, and motor outputs. The neurological effects might also be

accompanied, in many cases, by substantial mortality. The results of the mortality rate showed

that all the M. oleifera leaf extract concentrations tested caused significant concentration-

dependent mortality responses of the mosquito larvae.(Opoku-Bamfoh et al.2024)(Agbedahunsi

et al.2021)(Mondal et al.2022)

The experiment was performed in Plant science and Biotechnology laboratory located in Federal

University Gusau, Nigeria. The method of research used was the survey method using laboratory

instruments. The sample was caught by a standard dipper, and the net was not shaken. After

collection, the length of the larvae was measured, and the larvae were separately transferred into

single 0.25 ml capacity micro tubes labeled with code numbers denoting each treatment using a

Pasteur pipette. Then, 100 ul of the aqueous extracts of Moringa oleifera were added into the

plastic cups that were used to culture the larvae using a disposable syringe. The larvae were then

left undisturbed for either the recorded time or 12 hours, after which the adult mosquitoes were

monitored by placing two 250 ml plastic cups baited with sugar water on the shelf in the rearing
room where the treated larvae had been kept. One of the two cups containing sugar water was

offered as a control (untreated), and the other containing the same medium made up of the

aqueous extract that had been used to treat the larvae was offered to the adult mosquitoes.

3.1. Sampling and collection

In the Moringa oleifera plantation area (Yanwake), Moringa leaf extract in different

concentrations had different effects on adult houseflies and mosquitoes. Based on One-Way

ANOVA analysis for mosquitoes, there was a relative difference in adult Anopheles ciaraiae

survival. The results revealed that the effect (mortality rate) of different concentrations of

Moringa oleifera leaf extract on adult mosquitoes were significantly different. Based on Duncan

test, it was observed that adult Anopheles ciaraiae, when exposed to a lethal dosage of Moringa

oleifera leaf extract that resulted in 30 g/ml, showed the highest surviving days over 8 days when

compared to the control housefly using Lethal Concentration (LC) 50. Furthermore, LC100 in

adult Anopheles ciaraiae did not have a significant effect on the longevity of mosquito flies. The

first record of surviving days till death of the mosquito was annotated after 1 hour of exposure

with the 30 g/ml of LC 100. Therefore, if the same toxic effect of Moringa leaf extract persisted

in controlling Anopheline mosquitoes in Gusau, Zamfara, 30 g/ml of LC 100 would be the best

for use as a bio-insecticide with additional benefits of using the minimum possible concentration

as well as curtailing the cost of operation.(Idowu et al.2021)(Khan et al., 2021)(Ejeta et al.,

2021)

3) Results and Discussion

3.1. Evaluation of Mortality Rate in Adult Mosquito and Housefly subjected to Moringa leaf

extract in 'Yanwaki' area in Gusau, Zamfara State, Nigeria.

standard procedures. Collected adults of houseflies and mosquitoes were squished in a drop of

water on a slide. Then an unstained preparative mount was made. A cover slip was then applied

carefully from edge to edge to avoid formation of air bubbles. The gut contents were examined

under a light microscope and identified to genus level following available identification keys.

Identified pollen grains in the gut of adult houseflies and mosquitoes were identified using gross

evidence of pollen in the microscope. Each identified pollen grain was also collected by diluting

a drop of normal saline into the gut contents and placed onto a cover slip under the microscope,

then placed on a slide and identified morphologically. All the identified pollen were subjected to

1% I2KI. Results were first scored based on the number of identified pollen species at the family

level. In order to evaluate their food preference based on trophic group, the number of identified

pollen species belonging to the individual trophic group were total count summarized and

averaged.

Sampling and collection of mosquito and housefly adults was done from two areas according to

the availability of Moringa oleifera trees. The two areas are: Yanwake area in Gusau (Moringa

oleifera plantation) and Yanmangwarora Bypass (no Moringa oleifera plantation). The species of

mosquitoes and houseflies were collected using both wild and hand nets. The hand nets were

used in catching houseflies, while the wild nets were used in catching mosquitoes. The hand nets

were lightly swiped onto the housefly bodies, while the wild nets were swiped several times on

the resting spot of mosquito bodies. This is continued in other areas which have no Moringa trees

at all in order to isolate the control species of houseflies and mosquitoes.

The stock solution was disposed at room temperature under shade and kept for 24 hours to

confirm its maturity and to develop full strength, then filtered using millipore filter cloth to

obtain a pure solution with a dark brown precipitant. 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml,

70 ml, 90 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, and 400 ml from the stock solution in

different cap, plastic beakers were prepared. The stock solution was diluted to obtain different

concentrations of 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and 200%,

300%, and 400% respectively using 900 ml, 900 ml, 800 ml, 700 ml, 600 ml, 500 ml, and 400 ml

distilled water, and the formulations were carefully agitated to ensure even dispersion.

2.5 kilograms of observed fresh Moringa oleifera leaves were identified and obtained from "Yan

Mangwarora" bypass, Gusau, Zamfara, Nigeria. The leaves were left for one hour for the juice to

easily come out from the leaves, then weighed and blended thoroughly. The resulting juice was

decanted into a 10cm x 25cm (length) plastic container at intervals of 2 hours for one day and

allowed to settle for one day as well. A sheet of muslin (calico), a very thin lining, was spread

across the mouth of a 10cm x 25cm container and firmly attached. The settled juice-pure extract

suspended in the container due to sole gravitation and therefore dripped out slowly. The extract

was suspended for a period of time, worrying a little or no extract will be suspended. Already

prepared pressed juice will be automatically extracted. The resulting juice was poured out into a

5cm x 15cm (length) mild steel container and allowed to confirm that a greenish brown

precipitant forms with a clear solution produced. 10 liters of the clear solution was the stock

solution.