Ephestia Experiment methodology

Here is one of my experiment that is still running, the metodhology was build up based on some preliminary experiment. the proposed titel will be around

Experiment Topic: The influence of Duration mating, Delaying mating and sex ratio on the fecundity and eggs hatchability of Ephestia cautella



Experiment Methodology

Experiments will be conducted in Entomolgy Laboratory, King Saud University. Incubator from Sterdium company (Pic.1) will be used and maintained at 25 + ˚C , 65 + 5 % RH and 15D:9L photo period during the experiments. HOBO logger is going to be used to monitor those temperature, RH and Light intensity (Pic.6)
Last instar larvae from colony that have wandering, will be collected using plastic bags attached on upside of the jars (Pic.2), subsequently those larvae will be transferred using fine brushes to small cups (height:2.5 cm, diameter: 7 cm) (Pic.3) , each cups will contain individual larvae and determined its sexes based on the presence of testes on dorsal side of abdomen. Those larvae are let to develop with small amount of diet till adult. Confirmation of the sex will be done on pupae based on the presence or absence of testes in dorsal side of the abdomen and on adult based on the edge shape of the abdomen.
The observation will be done daily to collect the emerging date data. Due to the different behavior of emerging time between male and female, and in order to have the same age of adult, many larvae with different age will be used. During the experiment, adult will be provided with 10% sugar solution (Shoukary, 1978).
On the date in which the pupae emerge to adult, they will be confined male and female to plastic jar (Pic.4). In gathering male and female, cages will be used to prevent adult escape (Pic.5). Male and female are confined according to some treatments, they are mating duration, delaying mating and sex ratio. Some parameters that are going to be observed for all treatment are:
1. The longevity period (male and female),
2. The number of eggs produced per day per individual female, and
3. Eggs hatchability per day per individual female.
Small cups (Pic.3) will be used for eggs container, each cup for one day collection and for one individual female. The larvae that already hatched will be counted and removed.
a. Mating duration
A pair of newly emerged adults of E. cautella (0-24 hours old) will be confined in a plastic jar (height: 6 cm; diameter: 7 cm) (Pic.4) for the periods: 0-24 hours; 0-72 hours; 0-120 hours; 0-168 Days; and Control (gathered until the end). The plastic jars are specially designed for mating, it is equipped with plastic mesh tied around with rubber band on the open side of the jar before the lid is attached. The position of the jars is flip down. The mesh is used to allow the eggs passing down to the lid while preventing the adult to go down to the lid, so the eggs will be save and easier to collect.
Number of replicates per treatment will be fifteen. Male of each replicate would be separated after those duration period and would be placed in a separate jar.
b. Delaying mating
There are three experiments within this topics. First experiment, A single male and female would be confined in a jar at age of 24, 48, 72, 120 hours for 24 hours mating, subsequently they will be separated into individual jars until their death.
Second experiment, A female at age of 0 hours, 48 hours , 96 hours and 144 hours days old each would be paired with a male at age of 24 h for 24 hours, subsequently the male is removed and provided directly with new 24 h old male, then again with the same pattern continuously until the females are death.
Third experiment, A female at age of 0 hours, 48 hours , 96 hours and 144 hours days old, each would be paired with a male at age of 24 h. This female is allowed to mate with male for 24 h only then separated individually for 24 h, after this separation the female are introduced with 24h old male, continuously until its death.
Number of replicates for each treatment will be fifteen. During delaying period female may lay sterile egg, they will be collected and counted as well.

c. Sex ratio
Female and male at 0-24 hours old will be confined according to several sex ratio (female:male) as follow; 1:1;1:2;1:3;2:1;3:1. The gathering of male and female will be up to it death. In each sex ratio design, Fifteen pairs will be used.


Prediction of the demand of adult during experiment are:
1. Mating duration
There are four treatment, each has fifteen pairs, so we need 60 male and 60 female of the same age.
2. Delaying mating
First experiment, there are four treatments, each has fifteen pairs, so we need 60 male and 60 female of the same age
Second experiment, there are four treatments, each has fifteen pairs, we need 60 female for once time and and 60 male daily till the female death
Third experiment there are four treatments, each has fifteen pairs, we need 60 female for once time and and 60 male every two days till the female death
3. Sex ratio
There are five treatment on this experiment, each has fifteen replication, so we need 120 of female and 120 of male at the same age
So total, if we conduct those experiment in one time we need 240 male and 360 female of the same age and 60 new emerge of male daily until female death

Prediction of experiment duration
Based on preliminary experiment we have some data:
1. On the situation in which male and female confined for their whole adult life span, the female have life span ranged from 4 to 15 days, while males have life span ranged from 2 to 9* days (data was token from 4 pairs)
2. If male and female were confined for certain mating duration ranged from 1 day to 5 days, the female life span would be 8 to 11 days, while male 9 days
3. If females were let to be alone, their life span would be 17 to 19 days (from three replications)