**2.3.2 Effect of temperature and salinity**

348 Aquaculture

respectively. Mean total number of eggs per sac was 21.7 ± 4.79 eggs. The total number of

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.

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).

under laboratory control condition (temperature 25°C; salinity 25 ppt).

eggs per sac ranged between 14.0 and 30.0 eggs per sac.

In general, there is no significant effect of different salinity on the production of eggs/sac of copepod for each temperature treatment (Table 2). Despite the large difference in salinity (5- 45ppt) , the number of eggs produced in each egg sac of a female was more or less the same when cultured under the same temperature. Decrement of temperature reduced the number of eggs/sac more than when they were exposed to the increment of temperature to 45°C. Copepods cultured in 25°C produced significantly high number of eggs if compared to 5°C and 45°C.


Table 2. The number of eggs/sac/female copepod *Pararobertsonia* sp. reared under different temperature and salinity

The number of offspring and their survival is shown in Figure 3. No nauplii survived to the next life-stage at 5°C. On the other hand, survival significantly increase (p<0.05) at 25°C. Copepods in 45°C treatment showed the same pattern of development as in low temperature (5°C). Increase of temperature up to 45°C still permit gravid females to produce high number of eggs/sac despite being exposed to different salinity although no survival to adult stages were observed.

Table 3 summarizes the data on maturation and generation time from the present study. Different temperatures were significantly affected (ANOVA, P<0.05) the maturation time of egg sacs while no significant difference (ANOVA, P>0.05) was observed among the salinities in every temperature treatments. The mean maturation time of egg sacs was shortest at high temperature (45°C) and longest at control temperature (25°C).

No data on generation time was recorded for 5°C due to the delay in the development of nauplii into the copepodite stage throughout the study period (20 days). At 45°C, the only data obtained was on generation time from nauplii to copepodite as no copepods survived to the adult stage. The mean generation time from copepodite to adult at 25°C was longest in low salinity (3.2 ± 0.84 days) and shortest in high salinity (2.0 ± 0.71 days). However, the mean generation time from nauplii to gravid female was longest in high salinity with 8.8 ±

Fig. 3. Mean number of offspring per egg sac per female harpacticoid *Pararobertsonia* sp. at low (5oC), control (25oC) and high (45oC) temperature and low (5ppt), control (25ppt) and high (45ppt) salinity. Key: T=Temperature; S=Salinity; L=Low; H=High; C=Control

1.10 days. The shortest generation time was in control condition 25°C and 25ppt (7.3 ± 0.96 days). The longest generation time from N1 to C1 was obtained from copepod reared in high salinity with 3.8 ± 0.45 days while the shortest of the generation time was observed in low salinity with 2.2 ± 0.45 days.

#### **2.4 Discussion**

#### **2.4.1 Reproductive biology and development of** *Pararobertsonia* **sp.**

Detail report on reproduction and development in copepods particularly harpacticoids were mostly from the temperate species (Hicks & Coull, 1983; Sun & Fleeger, 1995; William & Jones, 1999; Rhodes, 2003). *Pararobertsonia* sp. is a tropical harpacticoid that has been

5oC

25oC

45oC

Fig. 3. Mean number of offspring per egg sac per female harpacticoid *Pararobertsonia* sp. at low (5oC), control (25oC) and high (45oC) temperature and low (5ppt), control (25ppt) and

1.10 days. The shortest generation time was in control condition 25°C and 25ppt (7.3 ± 0.96 days). The longest generation time from N1 to C1 was obtained from copepod reared in high salinity with 3.8 ± 0.45 days while the shortest of the generation time was observed in

Detail report on reproduction and development in copepods particularly harpacticoids were mostly from the temperate species (Hicks & Coull, 1983; Sun & Fleeger, 1995; William & Jones, 1999; Rhodes, 2003). *Pararobertsonia* sp. is a tropical harpacticoid that has been

high (45ppt) salinity. Key: T=Temperature; S=Salinity; L=Low; H=High; C=Control

**2.4.1 Reproductive biology and development of** *Pararobertsonia* **sp.** 

low salinity with 2.2 ± 0.45 days.

**2.4 Discussion** 


Table 3. Maturation time of egg sacs (time between the appearance of egg and hatching), generation time from Nauplii I to Copepodite I, generation time from Copepodite I to Adult and generation time from Nauplii I to gravid female of *Pararobertsonia* sp. at different temperatures (5°C, 25°C and 45°C) and salinities (5 ppt, 25 ppt and 45 ppt). Means in the column with the same superscript are not significantly different (P>0.05).

successfully cultured in our laboratory environment for many generations since 2007, but the detail of the reproductive biology and development stage has never been reported before. The probability for a female *Pararobertsonia* sp. to produce multiple egg sacs from one fertilization event is very high, the same as for other harpacticoids. The gravid female can be gravid for several times in a short period without remating. Hicks & Coull (1983) reported that the number of sacs produced from a single copulation vary from four to 12 for ve species of *Tisbe* and three to 21 for other 21 species of harpacticoids. A female of *Tisbe biminiensis* produced up to nine egg sacs during its life (Pinto et al., 2001), while *Tisbe battagliai* fed on *Isochrysis galbana* Parke produced 5.3 ± 2.2 egg sacs when cultured under temperature 25°C and salinity 25 ppt (William & Jones, 1999).

Many researchers have shown that egg production in copepod is different for each individual. A study by Guérin et al. (2001) showed that the number of eggs per sac produced by *Tisbe holothuriae* has large fluctuation, ranging from the maximum of 133 to a minimum of only three eggs in an egg sac. Sun and Fleeger (1995) found that a harpacticoid *Amphiascoides atopus* (Diosaccidae) typically carries two egg sacs and the average brood size was 24 eggs per ovigerous female. The mean number of eggs per sac for *T. biminiensis* fed on *Nitzchia closterium* was 69.0 ± 24.6, *Tetraselmis gracilis* was 40.6 ± 16.1 and mixed of *N. closterium* and *T. gracilis* was 46.0 ± 17.1 eggs (Pinto et al., 2001). *Pararobertsonia* sp. in the present study produced the lower mean number of eggs per sac (21.7 ± 4.79) than above studies which could be related to the type of the given diet.

Maturation time as well as interval time between egg sacs for every individual of the same species is reported to be different and varies among each other (Tester & Turner, 1990). These differences may be due to inherent biological variability which is determined partly by genetic differences. In the present study the maturation time of egg sac of individual *Pararobertsonia* sp. showed wide variation (0 – 167 hours). Most egg hatching took placed within 36 to 47 hours, comparable to maturation time of most harpacticoid copepods which is within 48 hours (Dam & Lopes, 2003). Most females took the shortest time (within 11 hours) for the duration between hatching and the appearance of the next egg sac. By comparison, *T. biminiensis* took two days to produce a new egg sac (Pinto et al., 2001).

The lifespan of *Pararobertsonia* sp. cultured in temperature 25°C, salinity 25 ppt and fed on *Chaetoceros* sp. was about 31.2 ± 3.57 days, which is within the normal range. In comparison, lifespan of *T. biminiensis* cultured in temperature 28-30°C, salinity 34 ppt and fed on *N. closterium*, *T. gracilis* and mixed of both diet was about 29.0 ± 7.2, 32.9 ± 4.9 and 32.7 ± 4.6 days respectively (Pinto et al., 2001). William & Jones (1999) reported on lifespan of *T. battagliai* when fed on *Isochrysis galbana* Parke was 23 ± 4.9 at temperature 25°C and salinity 25 ppt. A number of factors including the difference in species, quantity and quality of the food source and environmental condition including temperature and salinity could affect the reproduction and lifespan of the species.

### **2.4.2 Effects of temperature on reproduction and development**

Temperature is often the most important environmental factor affecting the productivity of copepods in natural systems (Christou & Moraitou-Apostolopoulou, 1995; Siokou-Frangou, 1996). In general, the effects of temperature on marine copepods are well studied, but information on the effects of temperature for tropical copepod species are relatively limited. Many previous works have employed the effects of temperature on temperate harpacticoids species sush as *Tisbe* (Hicks & Coull, 1983; Miliou & Moraitou-Apostolopoulaou, 1991; Williams & Jones, 1994). Limited number of tropical species has been used as subject matter to study the effects of temperature in culture condition, which includes *Pararobertsonia* sp. (Zaleha & Farahiyah-Ilyana, 2010) and *Nitocra affinis* (Matias-Peralta et al., 2005).

Results from the present study showed how temperature affecting the offspring production, survival, maturation time of eggs and generation time in *Pararobertsonia* sp.The differences in temperature significantly affects the reproductive and development rate of *Pararobertsonia* sp. Low temperature delayed the development whereas high temperature increased the development rate but extreme temperatures (>45°C) could lead to mortality. Temperature stress may have negative effects on survival and reproduction. These findings were relatively similar to the study of Takahashi & Ohno (1996). The later study found that *Acartia tsuensis* (Copepoda: Calanoida) could develop normally from egg to adult within a temperature range of 17.5 to 30°C while optimum growth and minimum mortality were achieved at around 25°C. However, the development slowed at both lower and higher ends of temperature at 17.5°C and 30°C. Similar trends have also been observed in other copepod species, such as *Diaptomus pallidus* (Geiling & Campbell, 1978) and *Acrocalanus gibber* (McKinnon, 1996).

Increased and decreased temperature also affected egg production of *Pararobertsonia* sp. where the egg production at decrement or increment temperature was lower compared to control temperature. This is because *Pararobertsonia* sp. could not adapt the environmental stress that inhibits normal gonad development and consequently affected the average number of eggs produced per female. This study is consistent with previous study by

These differences may be due to inherent biological variability which is determined partly by genetic differences. In the present study the maturation time of egg sac of individual *Pararobertsonia* sp. showed wide variation (0 – 167 hours). Most egg hatching took placed within 36 to 47 hours, comparable to maturation time of most harpacticoid copepods which is within 48 hours (Dam & Lopes, 2003). Most females took the shortest time (within 11 hours) for the duration between hatching and the appearance of the next egg sac. By comparison, *T. biminiensis* took two days to produce a new egg sac (Pinto et al., 2001).

The lifespan of *Pararobertsonia* sp. cultured in temperature 25°C, salinity 25 ppt and fed on *Chaetoceros* sp. was about 31.2 ± 3.57 days, which is within the normal range. In comparison, lifespan of *T. biminiensis* cultured in temperature 28-30°C, salinity 34 ppt and fed on *N. closterium*, *T. gracilis* and mixed of both diet was about 29.0 ± 7.2, 32.9 ± 4.9 and 32.7 ± 4.6 days respectively (Pinto et al., 2001). William & Jones (1999) reported on lifespan of *T. battagliai* when fed on *Isochrysis galbana* Parke was 23 ± 4.9 at temperature 25°C and salinity 25 ppt. A number of factors including the difference in species, quantity and quality of the food source and environmental condition including temperature and salinity could affect

Temperature is often the most important environmental factor affecting the productivity of copepods in natural systems (Christou & Moraitou-Apostolopoulou, 1995; Siokou-Frangou, 1996). In general, the effects of temperature on marine copepods are well studied, but information on the effects of temperature for tropical copepod species are relatively limited. Many previous works have employed the effects of temperature on temperate harpacticoids species sush as *Tisbe* (Hicks & Coull, 1983; Miliou & Moraitou-Apostolopoulaou, 1991; Williams & Jones, 1994). Limited number of tropical species has been used as subject matter to study the effects of temperature in culture condition, which includes *Pararobertsonia* sp.

Results from the present study showed how temperature affecting the offspring production, survival, maturation time of eggs and generation time in *Pararobertsonia* sp.The differences in temperature significantly affects the reproductive and development rate of *Pararobertsonia* sp. Low temperature delayed the development whereas high temperature increased the development rate but extreme temperatures (>45°C) could lead to mortality. Temperature stress may have negative effects on survival and reproduction. These findings were relatively similar to the study of Takahashi & Ohno (1996). The later study found that *Acartia tsuensis* (Copepoda: Calanoida) could develop normally from egg to adult within a temperature range of 17.5 to 30°C while optimum growth and minimum mortality were achieved at around 25°C. However, the development slowed at both lower and higher ends of temperature at 17.5°C and 30°C. Similar trends have also been observed in other copepod species, such as *Diaptomus* 

Increased and decreased temperature also affected egg production of *Pararobertsonia* sp. where the egg production at decrement or increment temperature was lower compared to control temperature. This is because *Pararobertsonia* sp. could not adapt the environmental stress that inhibits normal gonad development and consequently affected the average number of eggs produced per female. This study is consistent with previous study by

(Zaleha & Farahiyah-Ilyana, 2010) and *Nitocra affinis* (Matias-Peralta et al., 2005).

*pallidus* (Geiling & Campbell, 1978) and *Acrocalanus gibber* (McKinnon, 1996).

the reproduction and lifespan of the species.

**2.4.2 Effects of temperature on reproduction and development** 

Miliou & Moraitou-Apostolopoulou (1991). The later study reported that a reduction in the number of egg sacs and the total number of offspring produced by the Greek strain of *Tisbe holothuriae* was observed when the temperature was lower or higher than the optimum (19°C). In addition, Ambler (1985) and Uriarte et al. (1998) revealed that egg production is normally lower at low temperatures and generally increase with increasing temperature up to a thermal threshold, after which decline begins. Such a trend has been reported for the egg production of *Acartia tonsa* (Ambler, 1985), *A. clausi* (Uye, 1985), *A. bifilosa* (Uriarte et al., 1998), *A. lilljeborgi* (Ara, 2001). A study on *Boeckella hamata* (Copepoda: Calanoida), showed that clutch size decreased with increased temperature (Hall & Burns, 2002).

Survival of *Pararobertsonia* sp. in this present study was solely affected by temperature rather than salinity. Some previous studies showed that temperature has a direct influence on the survival of copepod (Peterson, 2001). The negative effects of higher temperature on the survival of *Pararobertsonia* sp. are similar with other previous studies. Survival of copepods and cladocerans were better at low temperature than at high temperature (Moore et al., 1996). Chinnery & Williams (2004) found that survival, egg production and hatching rate of *A. bifilosa*, *A. clausi*, *A. discaudata*, and *A. tonsa* increased when temperature rise from 5 to 20°C. Although increasing temperature showed the positive effects, the development and survival reduces as temperature rises beyond a certain level (Chinnery & Williams, 2004). For example, temperatures greater than 30°C was unfavourable to survival, percent ovigerous females, and fecundity of *Pseudodiaptomus pelagicus* (Copepoda: Calanoida) (Rhyne et al., 2009).

Slight increment or decrement in temperature affected the maturation time of the egg sacs of *Pararobertsonia* sp. There are only few laboratory studies that have explored the relationship between temperature and development rate in the life history stages of harpacticoid copepods (Palmer & Coull, 1980). The present study revealed similar finding as previously described by Chandler et al., (2003). The later study reported that eggs of harpacticoid copepod, *Amphiasucus tenuiremis* were developed in two to three days at temperature 25°C and salinity 30 ppt. Temperature give significant effects on the maturation time of eggs sac of copepods (McLaren et al., 1969). Evidence from some laboratory studies proved that temperature has strong influence on reproductive and postembryonic development (Hicks & Coull, 1983; William & Jones,1999; Matias-Peralta et al., 2005). Rhyne et al. (2009) found that 26 to 30°C was the best range for nauplii production while 28 to 32°C was the best for fast maturation rate of nauplii. A study by Williams & Jones (1994) also noted that a benthic harpacticoid, *Tisbe battagliai* has their best temperature at 20°C and increasing of temperature towards 25°C decreased the production rate.

Harpacticoid copepods have short generation time as reviewed by Sun & Fleeger (1995), Chandler et al., (2003) and McKinnon et al. (2003). However, data on the duration of the larval stages of marine harpacticoid copepods and the influence of environmental factors (specifically temperature) and their potential interaction on postembryonic development are relatively limited (Williams & Jones, 1994). The generation time of *Pararobertsonia* sp. found from this present study clearly shown that temperature do affect the period between stages of life cycle. Increment in temperature decreased the development time while decrement in temperature increased the development time of *Pararobertsonia* sp. Hicks & Coull, (1983) found the similar finding where the development time of *Tisbe* sp. decreased with increasing temperature. *Tisbe* sp. required high temperature (up to 25°C) for faster development. In addition, mean development time of calanoid copepod, *Pseudocalanus newmani* decreased exponentially with increasing temperature and reached the shortest duration at 32°C (Rhyne et al., 2009). Similarly, Williams & Jones (1994) clarified that fastest development for harpacticoid copepod *T. battagliai* occurred in the warmer months of the year which regarding to the highest production of superior food. However, in the present study, food was given at constant concentration.

The development time of *Pararobertsonia* sp. at increment temperature was two times faster compared to control and decrement temperature. Low temperature can retard activity of organisms and consequently reduces the consumption of oxygen. Whereas, high temperature can increase oxygen consumption to one point where metabolic demands exceed energy reserved. A calanoid copepod, *Pseudocalanus newmani*, was reported to delay the development time from 20.9 to 42.3 days when temperatures decreased from 15 to 6°C (Lee et al., 2003). This trend was also observed in *A. clausi*, where development time delayed from 35.4 to 74.8 days when temperatures decreased from 10 to 5°C (Chinnery & Williams, 2004).

Some previous reports on the respond of tropical copepods to temperature stress presented similar finding with this present study. Milione & Zeng (2008) stressed the effects of temperature on both population growth and hatching rates. The later study suggested that for maximum population growth and egg hatching success of a tropical calanoid copepod, *A. sinjiensis* should be cultured at 30°C with a salinity of 30 psu. Likewise, Matias-Peralta *et al.* (2005) showed that the *N. affinis,* grow well and achieved highest maximum production (124.2 ± 2.6 offspring female-1) at temperature 35°C. In comparisons, Zaleha & Farahiyah-Ilyana (2010) reported that temperature of 25 ± 1°C and high salinity (25 ppt - 35 ppt) were the optimum condition for the maximum production (2.3 - 3.7 individual/ml) of a tropical *Pararobertsonia* sp. in the laboratory condition.

In this study, the survival and reproductive parameters of individual *Pararobertsonia* sp. in different temperature treatments showed wide variation. These differences caused by inherent biological variability and physiological response. Thermal stress caused energy to be allocated toward survival processes rather than reproduction. This may also be explained based on the study by Williams & Jones (1999) where they reported that nauplii production of *T. battagliai* ceased after 20 days at 25°C while lower temperature treatments continued to produce nauplii for 36 days. The adaptation of some individuals will be better than others in respond to environmental stress due to the attributes of some individuals to establish their population (Depledge, 1990; 1994). Every metabolic rate of zooplankton such as respiration and feeding rate is dependent on temperature (Heinle, 1969). In this present study, *Pararobertsonia* sp. grew well and achieved high reproductive activity at temperature 25°C, the same temperature for the stock which has been maintained for two years. In contrast, the copepods were exposed to daily change towards the required temperature in the other two experiments. This might be the reason for the different adaptability and productivity found in this study as they need to tolerate and respond to the environmental stress everyday and it could be affecting their physiological response.

#### **2.4.3 Effects of salinity on reproduction and development**

Although temperature is recognised as an important factor controlling reproduction in harpacticoid copepods, there are other factors which regulate reproductive activity including food resource availability, environmental stability and their effects on the

addition, mean development time of calanoid copepod, *Pseudocalanus newmani* decreased exponentially with increasing temperature and reached the shortest duration at 32°C (Rhyne et al., 2009). Similarly, Williams & Jones (1994) clarified that fastest development for harpacticoid copepod *T. battagliai* occurred in the warmer months of the year which regarding to the highest production of superior food. However, in the present study, food

The development time of *Pararobertsonia* sp. at increment temperature was two times faster compared to control and decrement temperature. Low temperature can retard activity of organisms and consequently reduces the consumption of oxygen. Whereas, high temperature can increase oxygen consumption to one point where metabolic demands exceed energy reserved. A calanoid copepod, *Pseudocalanus newmani*, was reported to delay the development time from 20.9 to 42.3 days when temperatures decreased from 15 to 6°C (Lee et al., 2003). This trend was also observed in *A. clausi*, where development time delayed from 35.4 to 74.8 days

Some previous reports on the respond of tropical copepods to temperature stress presented similar finding with this present study. Milione & Zeng (2008) stressed the effects of temperature on both population growth and hatching rates. The later study suggested that for maximum population growth and egg hatching success of a tropical calanoid copepod, *A. sinjiensis* should be cultured at 30°C with a salinity of 30 psu. Likewise, Matias-Peralta *et al.* (2005) showed that the *N. affinis,* grow well and achieved highest maximum production (124.2 ± 2.6 offspring female-1) at temperature 35°C. In comparisons, Zaleha & Farahiyah-Ilyana (2010) reported that temperature of 25 ± 1°C and high salinity (25 ppt - 35 ppt) were the optimum condition for the maximum production (2.3 - 3.7 individual/ml) of a tropical

In this study, the survival and reproductive parameters of individual *Pararobertsonia* sp. in different temperature treatments showed wide variation. These differences caused by inherent biological variability and physiological response. Thermal stress caused energy to be allocated toward survival processes rather than reproduction. This may also be explained based on the study by Williams & Jones (1999) where they reported that nauplii production of *T. battagliai* ceased after 20 days at 25°C while lower temperature treatments continued to produce nauplii for 36 days. The adaptation of some individuals will be better than others in respond to environmental stress due to the attributes of some individuals to establish their population (Depledge, 1990; 1994). Every metabolic rate of zooplankton such as respiration and feeding rate is dependent on temperature (Heinle, 1969). In this present study, *Pararobertsonia* sp. grew well and achieved high reproductive activity at temperature 25°C, the same temperature for the stock which has been maintained for two years. In contrast, the copepods were exposed to daily change towards the required temperature in the other two experiments. This might be the reason for the different adaptability and productivity found in this study as they need to tolerate and respond to the environmental stress everyday and

Although temperature is recognised as an important factor controlling reproduction in harpacticoid copepods, there are other factors which regulate reproductive activity including food resource availability, environmental stability and their effects on the

when temperatures decreased from 10 to 5°C (Chinnery & Williams, 2004).

was given at constant concentration.

*Pararobertsonia* sp. in the laboratory condition.

it could be affecting their physiological response.

**2.4.3 Effects of salinity on reproduction and development** 

evolution of particular life-history strategies. Some researchers revealed that salinity is one of the main environmental factors controlling species distribution, the rates of growth, developments in larvae stages and reproduction of harpacticoid copepod, especially on those with restricted capacity of osmoregulation (Miliou & Moraitou-Apostolopoulou, 1991; Miliou, 1996).

Generally, the results of this study apparently showed that *Pararobertsonia* sp. could tolerate in wide variation of salinities ranging from 5 to 45 ppt. Similar results were reported by Matias-Peralta et al. (2005). They clarified that *Nitocra affinis,* a tropical harpacticoid could tolerate in salinities from 10 to 35 ppt. In addition, study done by Sun & Fleeger (1995), they found that a harpacticoid *Amphiascoides atopus* was able to survive in a wide range of salinities (10 – 60 ppt). Different salinities showed to have different effects on offspring production and survival of *Pararobertsonia* sp. Conversely, there are no effects shown on the number of eggs per sac, maturation time and generation time. Devreker et al. (2009) reported that the combination of salinity and temperature have different effects on the physiology of an estuary calanoida copepod, *Eurytemora afnis*. High salinities are most stressful for *E. affinis* at high temperatures (Kimmel & Bradley, 2001). Survival of *E. affinis*  could strongly decreased when high salinities are combined with high temperatures (Gonzalez & Bradley, 1994).

High survival (more than 80%) of *Pararobertsonia* sp. was achieved under salinity 25 to 45 ppt. Extreme low salinity (5ppt) could give significant effect on their survival. Nevertheless, they can survive in the laboratory cultures when salinity dropped gradually into 0 ppt (personal observation). Supporting this finding, Gaudy et al. (1982) stated that decreases in salinity resulted to high mortalities of *Tisbe holothuriae*. Staton et al. (2002) found that there is a non-linear survival response of *Microathridion littorale* (estuarine harpacticoid copepod) to short term immersion of 24 hours in 3, 12 and 35 psu. Copepods that were transferred in the 12 psu showed the lowest survival rate.

Although *Pararobertsonia* sp. has been observed to survive better at higher salinity compared to lower salinity, the generation time from nauplii to gravid female was longer under high salinity. However, the generation time from copepodite to adult was shorter under high salinity. This difference could be related to the physiological difference existing between first naupliar and late copepodite stages. Similar result was reported by Hagiwara et al. (1995) for *Tisbe japonicus*. They found that development time of *T. japonicus* was fastest at higher salinities (16 -32 ppt) and growth rate tended to be slower in low salinity.

*Pararobertsonia* sp. were able to reproduce and survived from the first egg sac hatched as nauplii to gravid female under different salinities ranging from 5 to 45 ppt at temperature 25°C compared to higher and lower temperature. It is clearly shown that *Pararobertsonia* sp. could tolerate to salinity changes rather than temperature. As reported by Devreker *et al.,* (2009), the development of estuary calanoid copepod, *E. afnis* appeared to be more sensitive to temperature than salinity. Lee & Petersen, (2002) revealed that temperaturesalinity interaction effect on salinity and temperature tolerance. The tolerance to temperature and salinity stress is controlled by a group of regulatory genes as documented by Kimmel & Bradley (2001). The genotype controls the synthesis of proteins necessary for metabolic activity. Consequently, this difference in genotype modified the metabolic performance as a function of environmental conditions.

Under control temperature (25°C), *Pararobertsonia* sp. showed the ability to survive and even breed in a salinity ranged of 5 to 45 ppt. This might be due to its great adaptation and osmoregulatory ability. Kimmel & Bradley (2001) demonstrated that salinity variations induce synthesis or degradation of amino acids during osmoregulation. This generates an increase in the consumption of protein reserves as well as in energy requirements for enzymatic activity. Without energy renewal, this stress decreases copepod survivorship and causes death of the nauplius in the early stages. The present study suggests that there is no salinity stress at this range of salinity because the nauplii can develop normally to adult stage. However, harpacticoids did not survive when they were transferred directly into the salinity 5 ppt (personal observation). As reviewed by Staton et al. (2002), they noted that exposure of low salinity in more than 24 hours for *Microathridion littorale* (estuarine harpacticoid copepod) could lead to mortality.

*Pararobertsonia* sp. is regarded as an estuarine harpactiocid copepod which is considered to be exposed to large fluctuations of salinity due to tidal cycle daily. Therefore, this species could already adapted to salinity fluctuation more than the temperature changes, thus become less affected by the salinity changes in the experiment. Goolish & Burton (1989) confirmed that the variability in individual's physiological response to salinity changes was due to the salinity history of organism and species specific hereditary traits.

#### **3. Conclusion**

Harpacticoid copepods are potential candidate as live feed in aquaculture. They have most of the required characteristics to replace artemia and rotifers as starter food for newly hatched fish and shrimp larvae. Nevertheless, the mass production of copepods as live feed for aquaculture purposes is still at the experimental stage and success story is limited to only few copepod species. Understanding the basic biology of the species in culture condition will help in planning and handling the copepod culture for mass production. An example given in this chapter is the reproduction and development data of a tropical harpacticoid copepod, *Pararobertsonia* sp. This species could produce multiple egg sacs from a single copulation, with an average of 6.7 ± 2.47 egg sacs (ranging from 3.0 to 12.0 egg sacs) in 31.2 ± 3.57 days (average of lifespan). The production of eggs per sac was 21.7 ± 4.79, varies from 14 to 30 eggs. The maturation time of egg sac is variable with range from 0 to 167 hours (7 days). However, most of the eggs were matured within 36 to 47 hours (2 days). The interval time between egg sacs varies from 0 to 71 hours (3 days), but most of individual took 11 hours to produce the next eggs. In this present study it is clearly shown that reproductive biology of every individual of *Pararobertsonia* sp. are varies among each other. Temperature appears to give significant effect on reproduction and development of *Pararobertsonia* sp. compared to salinity. High temperature increased while low temperature delayed the development of *Pararobertsonia* sp. but extreme temperature could lead to the mortality. This is particularly true if the copepod is drastically exposed to the temperature of beyond the tolerant limit. On the other hand, this species has a wide range of salinity tolerance (5 to 45 ppt). However, direct exposure to that lowest or highest salinity could lead to the mortality as well.

#### **4. Acknowledgment**

The study was carried out as part of a research project 'Development of cyst in marine harpacticoid copepods' funded by National Oceanographic Directorate, Ministry of Science, Technology and Innovation, Malaysia.
