Organisms of E. serrulatus were isolated from water samples collected from a pond in Damyang, Korea in 2002. To secure a pure culture of copepod population, sequential generations of E. serrulatus were started from a single egg-bearing female and was grown to a stock culture.

Chilomonas paramecium, a free-living protozoan organism that was isolated from a water sample of a rice field at Damyang, Korea, was used as live food for E. serrulatus. A pure culture of C. paramecium clone proliferated from a single organism was obtained by two rounds of cloning by limiting dilution (Freshney, 1987). For the routine laboratory maintenance of C. paramecium and E. serrulatus, the culture medium (GM medium) was prepared by boiling 1 liter of bottled mineral water (SoonSoo100, CocaCola Co., Korea) for 10 minutes with 1 g of soybean, Glycine max. The GM medium was stored at 5℃ until used. Fresh medium was prepared every month. Before each culture was started, the GM medium was supplemented with 1 pre-boiled wheat grain per 10 ml of medium as a nutrient source, as suggested by Kumazawa (2000). Routine laboratory culture of E. serrulatus was maintained in disposable petri dishes (87 x 15 mm, Green Cross Medical, Inc. Korea).

To investigate the growth efficiency of C. paramecium under different infusion media, C. paramecium was cultured in a 24-well flat bottom tissue culture plate (Corning, New York) started at a density of 4 x 10³/ml in 2 ml of media prepared with various grains. These infusion media were constituted with 1 liter of bottled mineral water boiled for 10 minutes with 1g grains of either Triticum aestivum (wheat, TA medium), Glycine max (soybean, GM medium), Sesamum indicum (sesamine, SIM medium), Hordeum vulgare (barley, HV medium), Coixlachryma-jobi (Job's tears, CJ medium), or Setaria italica (foxtail millet, SIA medium). The infusion media were stored at 5℃ until used. All cultures were kept at room temperature (20-23℃) and were exposed to 400 lux illumination of fluorescent lighting for 12 hours everyday. The number of C. paramecium was counted using a Neubauer counting chamber after staining the organisms with 0.4% New methylene blue. Each data point represents the average of the triplicated samples.

The microculture slide for rearing copepods was prepared using a microscope slide glass (75 x 25 mm, Marienfeld, Germany) on which a cover glass (22 x 40 mm, Marienfeld, Germany) was mounted at a height of 0.8 mm using a couple of polypropylene bars (0.8 x 1.0 x 22 mm) fixed with a silicone-based sealant (Fig. 1A). The microculture cell space between the slide glass and cover glass held approximately 700 µl of medium. Before a copepod culture was initiated, each microculture cell was filled with the GM medium that contained C. paramecium at a density of 4 x 10³/ml. A single egg-bearing female E. serrulatus was pipetted into the cell space after which the microculture slide was placed in a disposable Petri dish (87 x 15 mm, Green Cross Medical, Inc. Korea, Fig. 1B). Three layers of wet paper towel with deionized distilled water were placed in the bottom of the petri dish to provide humidity and to minimize the evaporation of culture medium. A couple of wood stick was placed between the wet paper towel and the microculture slide. Petri dishes with microculture slides were kept at room temperature (20-23℃). When multiple slides are used simultaneously, a stainless steel pan with an aluminum slide tray placed on top of three layers of wet paper towel was used. Culture slides were exposed to 400 lux illumination of fluorescent lighting for 12 hours everyday.

Growth and reproduction of E. serrulatus were determined by monitoring the time taken for hatching and the extrusion of egg sacs by in situ observation of copepods individually cultured in microculture slides under the microscope. After all eggs in the first egg sacs of an adult female hatched into nauplii, the female adult copepod was removed from each slide. The duration of developmental stages from egg to nauplius 1, copepodite 1, and adult male and female was monitored and the body length from the rostrum to the end of furcal rami, together with the body width at cephalothorax, was measured. Once nauplii became adults and mating done, they were removed from the cell except for one egg-bearing adult female per slide which was monitored daily thereafter for the number of broods produced and the duration between brood formations. Any nauplius hatched from the subsequent broods produced by the adult female in each microculture cell was removed to avoid the overpopulation which could affect the life span of the adult female. The brood number which was the total number of broods a female produced during its life time was recorded for each animal. The culture was continued until the female copepod in each slide died. Approximately two thirds of media in each microculture cell was replaced daily with fresh GM medium containing C. paramecium at a density of 4 x 10³/ml as live food.

The proliferation rate of C. paramecium cultured in six different infusion media is shown in Fig. 2. Among the tested media, the GM medium provided the highest proliferation rate of C. paramecium at 12th day of culture at which an average of 5,297/ml of C. paramecium was recorded. The SIA medium resulted in a peak number of C. paramecium at the 8th day after culture, while the TA medium showed the lowest growth rate.

Growth parameters of E. serrulatus from nauplius 1 to adult stages cultured in microcultue slide in terms of size and duration is shown in Table 1. The average period of eggs hatched from each female copepod to become nauplius 1 in the microculture slide took an average of 1.5 days, while an average of 7.7 days was taken from nauplius 1 to become the copepodite 1 stage. The average period of nauplii 1 hatched from eggs to become egg-bearing adult females took 29.3 days. The average length and width of an nauplius 1 were 80.1 and 120.0 µm which were increased to 283.8 and 845.2 µm in the adult female stage (Fig. 3). The number of broods produced by an adult female copepod ranged from 1 to 8 (mean, 4.5), and the average life time of copepod was 44.7 days (Table 2). Continuous passage of copepods was successfully maintained in the microculture environment as long as sufficient medium and food were provided.