Chapter 3

Research Methodology

Mosquitoes are insects belonging to order Diptera, suborder Nematocera, and family

Culicidae. Some species of which are important vectors of diseases. This type of insect that comes

with different species in classification can transmit serious human diseases, causing millions of

deaths every year and the development of resistance to chemical insecticides resulting in

rebounding vectorial capacity. Plants may be alternative sources of mosquito control agents

(Kamaraj, C. 2011).

Dieffenbachia seguine (Dumb cane) is phytochemically investigated for toxicity,

antimicrobial and antioxidant activities (Oloyede, G. 2012). Some of sorts (alkaloids, saponins,

phenol, flavonoids, resins...) can be presumably found on its leaves through phytochemical

analysis and screening to which results a chloroform extract as a byproduct. The objective of this

study was to evaluate the larvicidal activity of the dumb cane leaf extract in overall capacity to its

effects against the larvae of Aedes Aegypti in which are the most commonly found mosquito

species on a tropical country like the Philippines and a prevalent vector of dengue fever.

The following will be the research designs discussing about the proper approach on

conducting the study, in able to gather specific and relevant results in which would be significant

to the data analysis procedure and conclusions for testing.

3.1 Mosquito Larvae Rearing

An outdoor culturing of Aedes Aegypti mosquito larvae will be maintained at conditions

of 28° - 36° C temperature. The larvae are gathered through the traditional mosquito larvae rearing

method. These larvae may be kept alive by putting dried yeast or fruit peelings on the water sample.

3.2 Larval Mortality and Bioassay

Several features and susceptibilities by the subjects can be measured wherein significant

for the study, such as the mosquito larvae’s mortality and bioassay. The approach would be sub-

divided into 5 groups (4 experimental/treated, 1 control), having the 4 treated group to be

distributed with different concentrations of dieffenbachia extract (2500; 5000; 7500; and 10000

mg/L). A number of 30 larvae per concentration will be present on each group and each container

must be capable of holding a minimum of 500 mL of tap water for the capability to sustain the

fourth larvae stage. Bioassay will be performed using these concentrations. For mortality studies,

the larvae will be introduced in samples containing various concentrations. The treatment would

be repeated five times and will be reported by the graph for the linear regression of data and for

further analysis. Two formulas will be used on calculating the percentage mortality, (1);and the

correctional formula when necessary, introduced by Abbot(1925) (2).

where n= the number of larvae, T=treated, C=control

3.4 Extraction
 Dieffenbachia seguine leaves will be set washed and air-dried for the traditional steam

distillation process. Further phytochemical screening can be done with the plant to determine the

active properties and characteristics of a plant for further studies. The collected extract from the

process will be calculated and used with fixed amount needed for each group.

3.4 Statistical Analysis

The analysis program ‘Probit’ (Finney, 1971), the lethal concentrations (µg/ml) for 50%,

and 90% of the mortality, LC50 and LC90, respectively, were at 24 hours after treatment. The 95%

confidence intervals, values, and degrees of freedom of the χ2 of fit tests, and regression equations,

will be recorded .Whenever the goodness of χ2 was found to be significant ( p <0.05 ), correction

factor was used in the calculation of confidence limits. Data from previous recorded ones were

subjected to analysis of variance (ANOVA).

Table 1. Larvicidal activity of Dieffenbachia seguine at various concentration applied for 24h to

fourth instars of Aedes Aegypti.