**2.3 Results**

346 Aquaculture

Data recorded were analysed using statistical analysis of variance (ANOVA) at 0.05 level of probability. Kolmogorov–Smirnov tests were used to test for the normality respectively before the comparisons. The differences on the effects of temperature and salinity were compared using Univariate ANOVA. One-way ANOVA was used to test the differences on the effects of pH. Tukey's multiple comparison procedure was used to compare the significant differences between treatment means. All statistical analyses were performed

Seawater was filtered through GFC membrane filter after passed through UV treatment before use. A 500 ml and 1000 ml beakers were used as culture vessel. The culture was maintained at temperature between 26°C to 27°C and salinity between 22 to 26 ppt. Culture was fed daily with 1 ml of baker's yeast (0.02 g/L) (Nanton and Castell, 1998) and with mixed algal (*Isochrysis* sp*.*, *Nannochloropsis* sp*.*, *Chaetoceros* sp.) at the density of 1 x 106 cell/ml. No aeration was provided for the 500 ml and 1000 ml stock cultures. The partial replacement of seawater was done every three days and full replacements at every month by passing the cultures through a 45 µm mesh net. The trapped copepods were then

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

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

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

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

using SPSS program, version 11.5.

**2.1.1 Establishing a stock culture** 

transferred into new culture vessel.

microscope for each individual.

copulation.

were determined.

**2.1.2 Determination of eggs' number** 

### **2.3.1 Lifespan, egg production and development in** *Pararobertsonia* **sp.**

Lifespan of 30 individual females of *Pararobertsonia* sp. was determined in this experiment by measuring the length of time from first day of hatching as nauplii until they died under laboratory control condition at temperature 25°C and salinity 25 ppt. The average lifespan of a female *Pararobertsonia* sp. was 31.2 ± 3.57 days with a minimum and maximum of 26.0 and 40.0 days respectively.

The interval time between the production of egg sac is measured as time between hatching and the appearance of the next egg sac from one fertilization event. The maturation time of egg sac was determined as the time between the appearance of eggs and its hatching time. A female of *Pararobertsonia* sp. appears to produce several egg sacs from one fertilization event. The total number of egg sac per female had large fluctuation and significantly different among the 30 individual copepods observed. Total number of egg sacs per female was 6.7 ± 2.54 egg sacs in average, where the minimum and maximum was 3.0 and 12.0 egg sacs respectively. Mean total number of eggs per sac was 21.7 ± 4.79 eggs. The total number of eggs per sac ranged between 14.0 and 30.0 eggs per sac.

The time of formation between first egg sacs and the next was recorded within 0 to 11 hours. The egg sac could be seen in the oviduct within minimum hours range from 0 to 11 hours and maximum hours range from 60 to 71 hours after the previous egg release. The time between hatching and the appearance of the next egg sacs was significantly frequent within 0 to 11 hours with 65.88 % and less frequent within 60 to 71 hours with 1.18 % (Figure 1).

Fig. 1. The percentage (%) of interval time between egg sacs of a female *Pararobertsonia* sp. under laboratory control condition (temperature 25°C; salinity 25 ppt).

A female could have different maturation time for each interval of their production of egg sac. Figure 2 shows the percentage (%) of maturation time for each individual female to carry their eggs until its hatching. Time (hours) recorded for eggs to be released and hatched

Fig. 2. The percentage (%) of eggs and their maturation time for a female *Pararobertsonia* sp. under laboratory controlled condition (temperature 25°C; salinity 25 ppt).

was within 0 to 11 hours until 156 to 167 hours. However, most of the eggs (39.50 %) were matured and hatched within 36 to 47 hours while only 1 % took 0 to 11 hours, 96 to 107 hours and 156 to 168 hours to hatch. It shows that the maturation time of eggs was shorter at early and longer for the late egg sacs produced.
