MATERIALS AND METHODS

Experimental works on N. d. sinensis were conducted in the Endocrinology Laboratory, Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.

Tank preparation and maintenance: Different sizes of plastic aquarium tank were used for conditioning female broodstock (40x24x30 cm), conditioning male broodstock (35x17x25 cm), breeding (40x24x30 cm), separating ovigerous female (7x7x10 cm), conditioning larvae (26x16x16 cm) and life cycle observation (40x24x30 cm). Throughout this study, PVC pipe (Ø 16 mm) was used as shelter during molting. Aerated tap water was de-chlorinated and used as source of water. Aeration was provided throughout the study period.

Feeding and water quality: Broodstock and larvae were fed ad libitum with commercial shrimp feed containing 40% protein twice a day at 08:00 and 17:00 h. About 50% water change was carried out once a week. Few main water quality parameters measured in this study. The kits or apparatus used were; DO meter (dissolved oxygen and temperature) and API aquarium freshwater test kit (pH, ammonia, nitrite). Measurements were taken twice a week.

Broodstock conditioning: Matured males and females N. d. sinensis were bought from nearby aquarium shops. About 30 females and 30 males used were with average total length of 2.5±0.5 cm and placed into two separated conditioning tanks. Conditioning process takes about one month. Only females with saddle and healthy males were used.

Breeding: Ten pairs of matured male and female N. d. sinensis were placed in breeding tank. Observation was carried until female shrimp become ovigerous.

Larvae: Each of ovigerous females were weighed and moved to individual tank. Female was removed after the larvae hatched. Number of larvae produced by each female was counted and recorded. Larvae produced from the earlier experiment were placed and conditions in tanks for a week. After a week, only healthy larvae were used for the next experiment.

Life cycle: Thirty larvae (one week old) were placed in tank. Observation was carried out until all the larvae reached first maturity. Life cycle of N. d. sinensis was presented into illustration form. Total length (TL) of N. d. sinensis was measured from rostral tip to posteromedian margin of the telson, excluding posterior setae following Yang and Ko (2003). Larvae and eggs were observed and measured using ocular micrometer placed in the microscope eyepiece of Leica DM750, compound microscope with scale bar. Photographs were taken using Canon S95 digital camera. Ranges of water quality parameters observed throughout this study were recorded.

RESULTS

N. d. sinensis reached first maturity at around 75 days old with TL 2.3±0.2 cm. Upon reaching maturity, the presence of orange colored ovaries can be seen through the cephalothorax region of the females (Fig. 1). Appendix masculina on the second pleopod (Fig. 2) were observed in males. Mating process usually take place 1-3 day(s) after the introduction of male and female into the same tank. Female molted prior to mating.

Both molting and mating occurred rapidly in less than 10 sec. During mating process male and female of N. d. sinensis will be facing each other and sperm were deposited into the genital opening of female N. d. sinensis using its appendix masculina. The eggs were fertilized when it passed through the sperm on the way to the brood pouch. The pouch was formed by pleopods and overhanging of pleura of the female. A day after mating, female was observed to carry fertilized eggs. The eggs were incubated in the brood pouch until hatch.

Fertilized eggs were oval in shape, vary from greenish to yellowish in color. The egg size was comparatively large, with an average length 1.19 mm. Thin ribbon-like filament binds the eggs in grape-like bunches and attached it to the female's pleopod. The membrane can hardly be distinguished since it is thin, transparent and lying close to the surface.

Fig. 1:	Matured female N. d. sinensis with visible orange colored ovaries (arrow) in the cephalothorax region

At 27°C, embryonic development of N. d. sinensis lasted for 15 days. The eggs became lighter in color and translucent just before hatching. Newly hatched larvae look like the miniature version of the adult (Fig. 3) with an average TL of 2.3±0.5 mm. Figure 4 showed the number of larvae produce by females.

Fig. 2:	Appendix masculina (arrow) on the second pleopod of matured male N. d. sinensis

Fig. 3:	Newly hatched larvae of N. d. sinensis

Fig. 4:	Linear regression of fecundity (number of larvae produce) based on weight of female N. d. sinensis

It ranged from 21-51 larvae per hatching, increased linearly (R² = 0.9587) with the size of the female. Larvae reached juvenile stage after 60 days. At this stage, male and female were undifferentiated. Juveniles take 15 days to reach adulthood and first maturity. Complete life cycle of N. d. sinensis is presented in Fig. 5.

During the study period, it was found that larvae and adult shrimp cultured in freshwater were susceptible to clitellate annelids, Holtodrilus sp. (Fig. 6) resulted in high mortality.

Fig. 5:	Life cycle of N. d. sinensis

Fig. 6(a-c):	Holtodrilus sp. were distributed all over the body surface of N. d. sinensis cultured in freshwater, (a) Holtodrilus sp. attached to the naupliar appendages of larvae, (b) Holtodrilus sp. elongated (arrow) ready to move and (c) Tooth (arrow) on the jaws of Holtodrilus sp.

Table 1:	Ranges of water quality parameters measured during the study period

Holtodrilus sp. with body length 0.5-0.6 mm were found distributed all over the body surface of the host, with a higher concentration around the appendages. Culture of this species in slight saline condition between 5-10 ppt can effectively treat this annelid. Water quality parameters like dissolved oxygen (DO) was more than 4 mg L^-1, temperature 27-28°C, pH 6.5-7.5 and ammonia and nitrite less than 0.1 mg L^-1, respectively (Table 1).

DISCUSSION

In this study, N. d. sinensis was found to be able to reached first maturity as early as 75 days old, which is similar to those reported by Demas (2007). Female molted prior to mating, possibly induced by the released of certain chemical substance into the surrounding water. Somehow, before or during molting, the release of this substance allows the male to detect gravid female in the water column. This signaled the male that female is ready to spawn.

Newly hatched larvae of N. d. sinensis look like the miniature version of adult similar to that observed by Yang and Ko (2003). Morphological differences were only observed when N. d. sinensis reached maturity with the appearance of endopod and appendix masculine in males, which is similar to the report by Englund and Cai (1999). N. d. sinensis lacked of planktonic larval stage, therefore, it is a completely suppressed type based on the type categorized by Lai and Shy (2009). Numbers of egg produced per female of freshwater shrimp is depending on the size of the female itself. Smaller females produced fewer eggs compared to larger females (Ketse, 2006). In the present study, the size (weight) of female N. d. sinensis had a linear and positive relationship (R² = 0.959) with number of larvae produced. Similar observation was reported on Caridina gracilirostris (Heerbrandt and Lin, 2006).

Female N. d. sinensis was observed to incubate and fan their eggs in the brood pouch. According to Adiyodi and Adiyodi (1994) this kind of parental care will results in higher survival rate of larvae produced. Similar to those reported by Schram (1986), N. d. sinensis eggs were incubated in brood pouch until hatching.

Infestation by clitellate annelids on N. d. sinensis is quite common. Similar observation on others freshwater shrimp was reported by Liang (1963) and Liu (1984). This study suggested it is better to culture N. d. sinensis larvae in slightly saline water (5-10 ppt) since clitellate annelid, Holtodrilus sp. will not be able to tolerate saline water. Study by Niwa et al. (2005) stated that clitellate annelids, Holtodrilus truncatus infestation on freshwater shrimp can be treated with saline water.

CONCLUSION

Neocaridina denticulata sinensis can successfully breed in laboratory conditions using a simple experimental setup. This shrimp is able to reach first sexual maturity earliest at 75 days old. It reproduces throughout the year. It is recommended to culture newly hatched larvae in slight saline water (5-10 ppt) to avoid infestation and mortality due to clitellate annelid. The findings of this study can be used as a guideline for culturist interested in the mass production of this ornamental shrimp for the aquarium industry.

ACKNOWLEDGMENT

Author would like to thank the Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia for providing the materials and facilities for this research.