**2.1.2 Determination of eggs' number**

Total of 150 to 200 individual copepodids regardless of sex were isolated from the stock culture and observation was made several times everyday for any gravid female. After few days, 30 gravid females were collected and transferred into a vial and fixed with a few drops of 5% buffered formalin. The number of eggs was counted under Leica DME compound microscope for each individual.

### **2.1.3 Determination of lifespan, egg production and development**

Prior to the experiments, 30 mating pairs were isolated and placed individually in a small petri dish (60 X 15 mm) containing 5 ml new culture medium. The cultures were maintained at temperature 26-27°C and salinity of 22-26 ppt. *Chaetoceros* sp. was offered at the density 1 x 106cell/ml. Each dish was examined daily for the first appearance of the egg sac before removing the males. This was done to ensure the number of sacs per female from its first copulation.

Each dish was examined daily at six hours intervals each day to ascertain the time of egg release and the appearance of the next egg sac. Once a female released all eggs, it will be transferred to a new dish with fresh culture medium for further observation. This was repeated until all of the females died. The daily routine consisted of checking each individual for egg development and duration under a Leica ZOOM 2000 dissecting microscope. Food was added every other day in the same concentration of 1 x 106 cell/ml. The total number of egg sacs per female, maturation time of egg sacs (time between the appearance of the egg sac and its hatching), interval time between egg sacs (time between hatching and the appearance of the next egg sac for one fertilization) and lifespan of female were determined.

#### **2.2 Effect of temperature and salinity on reproductive biology of a harpacticoid copepod,** *Pararobertsonia* **sp.**

Gravid females of *Pararobertsonia* sp. from the first copulation were selected for this study as to ensure that the next generation (nauplii) were exposed to the specified temperature and salinity regime designed in the experiment. Table 1 summarizes the combination of treatments designed for the harpacticoids.


Table 1. Combination of different temperature and salinity treatment for the harpacticoid copepod culture (Key: T=Temperature; S=Salinity; L=Low; H=High; C=Control)

Nine different treatments were prepared (TL SL, TL SC, TL SH, TC SL, TC SC, TC SH, TH SL, TH SC and TH SH) to determine the effects of different temperature and salinity on the reproduction and development of *Pararobertsonia* sp. The experiment was conducted in three different temperatures; 5°C, 25°C and 45°C. In each temperature setup, copepods were treated with three salinity levels; 5 ppt, 25 ppt, and 45 ppt. All copepods were gradually exposed to the lower (5°C) and higher (45°C) temperature from the control value (25°C) before the experiment started to avoid shock effect which would instantly kill the animal. The same treatment was also applied for salinity exposure.
