**2. Reproductive biology and effect of environmental factors**

The first step to understand how to culture harpacticoid copepod is to know the reproductive biology of this animal when kept under culture condition where temperature and salinity would easily change. Copepods such as *Calanoides acutus*, *Rhincalanus gigas*, *Calanus propinguus*, *Paraeuchaeta Antarctica* showed a higher metabolic rate in the summer than in the winter (Kawall et al., 2001). It seemed that increasing in temperature will increase the respiration rate and it has positive correlation with the relative biomass of copepods (Le Borgne, 1982). For harpacticoid copepods, a number of researchers have documented that temperature is the major factor controlling the reproductive activity in the harpacticoid copepods. For example, the optimum temperature for the maximum production of the *Tisbe battagliai* in the laboratory condition was 20°C (Williams & Jones, 1999).

The optimal conditions for the temperate is differ from the tropical species due to its natural environment. Local adaptation in term of reproductive traits could happen in order for the copepod to maximise available energy with changing temperature. This was shown by the population of harpacticoid *Scottolana canadensis* from different lattitude (Lonsdale & Levinton, 1986; 1989). William & Jones (1999) noted that under optimal conditions in the laboratory (high quantity of algal food and temperature 20°C), *Tisbe battagliai* developed rapidly from hatching to the adult stage, have a rapid generation time and produce in quick succession, numerous broods containing large numbers of offspring. Females that were reared at 15°C lived twice as long as compared to 25°C. Increasing in temperature (>25°C) will decrease the number of offspring per day.

Harpacticoid copepods has been found to be a good candidate in aquaculture industry since they have a high reproductive potential, short generation time, high population growth, flexible in diet and tolerate a wide range of environmental factors such as temperature and salinity (Sun & Fleeger, 1995; Støttrup & Norsker, 1997). They provide a broad spectrum of prey sizes suitable for fish larvae (Gee, 1989). There are reports that indicate the availability of enzyme in harpacticoid copepods which enable the organisms to convert any type of their organic food into lipids stored in their body (Nanton & Castel 1998; Drillet et al. 2011).

The prominent problem in using copepods as live feed in aquaculture industry is to get high yield due to several reasons which mainly related to the culture technique. Pure culture is difficult to maintain in ponds or in hatchery on a continuous basis. Although continuous cultivation of marine copepods has been achieved, but it is only for a small number of species. Several species of harpacticoid copepods that have been cultivated either for commercial use or laboratory level are *Tisbe holoturidae, Tisbe beminiensis, Nitokra lacustris* and *Tigriopus japonicus*. As reviewed by Rippingale and Payne (2001), harpacticoid copepods especially *Tisbe* spp (Støttrup & Norsker, 1997) and *Tigriopus* spp (Carli et al., 1995) were the easiest to culture. Nevertheless, the technology to economically culture marine copepods to get highly enough yield is still being developed to date (Marcus & Murray, 2001; Rhodes,

Lacking in information about the basic biology, reproduction and response of copepods in rearing environment could be one of the reason why mass culture technique is yet to be successful for copepod production. Harpacticoid copepods in particular need special attention as they are predominantly benthic in nature. This chapter will discuss on

The first step to understand how to culture harpacticoid copepod is to know the reproductive biology of this animal when kept under culture condition where temperature and salinity would easily change. Copepods such as *Calanoides acutus*, *Rhincalanus gigas*, *Calanus propinguus*, *Paraeuchaeta Antarctica* showed a higher metabolic rate in the summer than in the winter (Kawall et al., 2001). It seemed that increasing in temperature will increase the respiration rate and it has positive correlation with the relative biomass of copepods (Le Borgne, 1982). For harpacticoid copepods, a number of researchers have documented that temperature is the major factor controlling the reproductive activity in the harpacticoid copepods. For example, the optimum temperature for the maximum production of the *Tisbe* 

The optimal conditions for the temperate is differ from the tropical species due to its natural environment. Local adaptation in term of reproductive traits could happen in order for the copepod to maximise available energy with changing temperature. This was shown by the population of harpacticoid *Scottolana canadensis* from different lattitude (Lonsdale & Levinton, 1986; 1989). William & Jones (1999) noted that under optimal conditions in the laboratory (high quantity of algal food and temperature 20°C), *Tisbe battagliai* developed rapidly from hatching to the adult stage, have a rapid generation time and produce in quick succession, numerous broods containing large numbers of offspring. Females that were reared at 15°C lived twice as long as compared to 25°C. Increasing in temperature (>25°C)

important aspects in rearing harpacticoid copepods as live feeds in aquaculture.

**2. Reproductive biology and effect of environmental factors** 

*battagliai* in the laboratory condition was 20°C (Williams & Jones, 1999).

will decrease the number of offspring per day.

2003; Drillet et al., 2006).

Salinity affects the functional and structural responses of invertebrates through many aspect such as changes in total osmo-concentration, relative proportion of solutes, coefficient of absorption and saturation of dissolved gases (Kinne, 1964). Changes in total osmoconcentration could considerably modify rates of metabolism and activity. This is because of the influence of salinity variations on several biochemical and physiological mechanisms which the function is essential for survival in marine organisms (Fava & Martini, 1988). A study on harpacticoid copepod, *Tigriopus californicus* showed that high salinity slowed down their growth rate and delayed the maturation time. A decrease in reproductive rate at increasing salinity caused moderate decrease in total number of clutches (Dybdahl, 1995). Gaudy et al. (1982) reported that the egg sac production and offspring survival of *Tisbe holothuriae* increased at higher salinities (28-30 ppt). However, the net reproductive rate of *T. holothuriae* declined at salinities above and below 38 ppt (Miliou & Moraitou-Apostolopoulou, 1991). The production of fewer offspring per egg sac and an increment in mortality had been observed.

The salinity tolerance was different at each life cycle stage, and in different sex (Damgaard & Davenport, 1994). In most species, the narrowest tolerance rate is during very early stages, which later increased and finally decreased again in the adult stage. The narrowest range of tolerance is often associated to the period of embryonic development. Nauplii are commonly less sensitive, and during early growth could even tolerate wider salinity range and more pronounced to salinity fluctuations than adult individuals (Milione & Zeng*,* 2008).

#### **2.1 Investigation on reproductive biology of a harpacticoid copepod,** *Pararobertsonia* **sp.**

A harpacticoid copepod, *Pararobertsonia* sp. was first collected during an ecological survey in an estuary in Terengganu, Malaysia (5° 02.260' N, 103° 17.821' E). The estuary is located at the river mouth of Merchang River, meeting the South China Sea. It is inhabited by large coverage of small seagrass, *Halodule pinifolia* and oysters farming activities by local people. During field sampling, a 62µm net was towed horizontally on the surface of exposed seagrass patches to catch the copepods. The trapped samples were placed into container filled with seawater and aeration was provided. The collected samples were transported back to Institute of Oceanography Laboratory, Universiti Malaysia Terengganu (UMT).

In laboratory, isolation process was carried out to separate *Pararobertsonia* sp. from other benthic copepods. Harpacticoids carrying egg sacs were isolated from samples under a Leica ZOOM 2000 dissecting microscope using an Irwin Loop and placed individually into a petri dish 60 X 15 mm containing 5 ml of filtered-autoclaved seawater (Støttrup & Norsker, 1997) and 1 ml of baker's yeast (0.02 g/L) (Nanton & Castell, 1998). The experiment was maintained closest to the normal habitat condition at temperature 26°C to 27°C and salinity 24 to 26 ppt. Each plate was initiated with single gravid copepod to ascertain monospecific culture. The culture was maintained separately until the identification was carried out for species confirmation.

After being isolated and identified, there are several steps need to be completed before culture of harpacticoid can be successfully maintained in laboratory for any investigation. These include activities such as breeding and up-scaling under laboratory condition. Water as culture media need to be treated and conditioned to meet the copepod's environment requirement.

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 using SPSS program, version 11.5.
