Culturing Rotifers

Introduction

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In 1989, I received an M.Sc. degree in Zoology (Hydrobiology) from Biological Department of Nankai University, Tianjin, China.

I have been studying aquatic organisms on a shrimp farm for over ten years. The article on rotifer production is the summarization of my several years work. There are some new ideas about the production procedures and principles in this article. In order to share my experience with other people and promote the development of rotifer production technology in aquaculture all over the world, I published the article.

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"The article was published in Aquaculture Magazine 1996 22(3):16-22."

Production and Application of Rotifers in Aquaculture

Jin Guan Li 13-3-603, Wangdingdi, Tianjin 300191, China

Abstract

This paper describes the ecological principles and procedures in the production of the rotifer Brachionus plicatilis in outdoor earthern ponds in north China.

In high eutrophication waters, copepods, small shrimp and fish, the predators of rotifers, can not survive longer and are ultimately eliminated from the waters. But rotifers have higher ability to tolerate eutrophication than the predators. The rotifers can survive, grow and reproduce very well in high eutrophication waters.

So we artificially create high eutrophication waters to mass produce the rotifer Brachionus plicatilis. Commercial availability of rotifer resting eggs could be the solution by eliminating the need to maintain stock cultures and reduce the chances for contaminations with ciliates and pathogenic bacteria. We have produced dry rotifer resting eggs ( cysts ). One gram contains 2,000,000 resting eggs with about 80% of hatching rate at 28 C and 20 ppt in 36 hours.

Introduction

Rotifers are valuable live food for larval fish and crustacean culture. Several characteristics of rotifers, including their nutritional quality, body size and relatively slow motility have contributed to their usefulness as good prey for active larvae ( Snell et al., 1984 ).

The rotifer Brachionus plicatilis has been most widely used as essential food source in raising marine fish, shrimp and crab larvae due to its tolerance to the marine environment ( Lubzens, 1987; Dhert et al., 1994 ).

There are a large array of culture methods to produce the rotifer B. plicatilis. These can be sorted into three basic methods: (1) batch culture, (2) semicontinuous cultures and (3) feedback culture systems.

The widely applied batch culture procedure appears to be simple, using microalgae and/or bakers' yeast or other formulated products as food source. The development of the larval rearing industry is primarily due to the advancement of mass culture technology of the marine rotifer B. plicatilis ( Fulks et al., 1991 ). But rotifer mass cultures are still sometimes unstable due to the change of bacterial flora or contamination of other species ( Hino, 1993 ).

The rotifer B. plicatilis is an euryhaline species. In north China along the Bohai Bay, great populations of rotifers appear in natural brackish waters near village and/or processing plants in April, when Chinese shrimp and crab larvicultures have begun. The wild rotifers are harvested by fishermen and sold to local hatcheries at price about 1 US dollar per kilogram, which includes about 60,000,000 live rotifers.

With the rapid development of Chinese white shrimp Penaeus chinensis and the freshwater crab Eriocheir sinensis larvicultures, the supply of wild rotifers can not reach the demand. In 1988, Chinese experts invented rotifer mass production technology. Since 1988, we have been producing the rotifer B. plicatilis in outdoor earthern ponds planedly.

Principles

We observed that: in nature, great populations of the rotifer B. plicatilis only appeared in waters with high eutrophication near village and/or processing plants of aquatic and/or poultry products. In these waters there is no copepods, the main predator of rotifers. If waters includes copepods, the rotifer population is very small or there is no rotifers in the waters.

Our observations can be easily explained by ecological principles. Domestic sewage and waste water is about 99.9 percent water and 0.02-0.04 percent solids of which proteins and carbohydrates each compose 40-50 percent and fats 5-10 percent.

In other words, sewage includes mostly biodegradeable pollutants such as human fecal matter, animal wastes and certain dissolved organic compounds ( carbohydrates, urea, etc. ) and inorganic salts such as nitrates and phosphates of detergents and sodium, potassium, calcium and chloride ions. Under natural processes most of the biodegradeable pollutants of sewage are rapidly decomposed and when they accumulate in large quantities, they create eutrophication.

The great amount of basic nutrients such as ammonia, nitrogen, nitrate, nitrites and phosphates stimulate algae growth and lead to green algae blooms. Under these conditions, copepods and other predators ( small shrimp and fish ) can not survive longer and are ultimately eliminated from the waters. But rotifers have higher ability to tolerate eutrophication than copepods, small shrimp and fish. The rotifers can survive, grow and reproduce very well in high eutrophication waters. So great populations of rotifer B. plicatilis apear in the brackish eutrophication waters.

In nature waters with low N and P content, copepods are predators of rotifers. Once appearance of copepods, the density of rotifers can not be large. In opening outdoor earthern ponds, it is impossible to avoid entering of copepods.

We used ecological principles into rotifer production procedures. Artificially create high eutrophication waters by applying fermented bean cake and chicken manure, the grow of copepods is inhabited and then the copepods are eliminated from the system. But this circumstance is very suitable for mass production of rotifers.

Rotifer production

1. Construction of earthern ponds

Rectangular ponds are recommended with suitable size of about 1,000 sq. m. of surface area and about 1 m of water depth. 5-10 ponds are needed according to demands for rotifers. The rotifer B. plicatilis is an euryhaline species, but the optimal salinity for rotifer growth is 10-20 ppt. So the ponds should be constructed near freshwater source.

2. Applying manure and algae culture

In north China during early spring as soon as the ice melts, sea water and freshwater are pumped directly into the earthern ponds. Salinity remains about 15 ppt. Disinfecting the ponds is not needed. About 500 kg of fermented bean cake and chicken manure ( 1:4 ratio ) are applied into each ponds. The dominant marine algal species are good food sources for rotifer growth. The algae can provide rotifers with sufficient protein and especially essential fatty acids. Practical applications have proved that the produced rotifers are suitable for larvicultures of marine shrimp, crab and fishes. The bacterial populations in these ponds can provide rotifers with proteins and especially essential vitamins such as VB12 for rotifers growth ( Yu et al., 1988 ).

3. Inoculation of live rotifers or hatching of resting eggs

In end of March at about 15 C, wild rotifer populations have appeared, which are harvested to inoculate the rotifer ponds at initial density of 1-5 rotifers per l. In later years, wild rotifers are not needed, because there are a great number of rotifer cysts on the bottom of the rotifer ponds. They can easily hatch at suitable conditions. In regions where there is no rotifers, commercial rotifer cysts are recommended to start rotifer mass cultures.

Rotifer culture practice requires large volumes of phytoplankton. The volume ratio between rotifer and algal ponds is 1:3 ( Hagiwara, 1989 ). So 3/4 of ponds are used only as algal culture ponds. The algal ponds must not be used to culture rotifers, because if all the ponds are inoculated with rotifers, the rotifers can rapidly consume all the algae and the algal culture will not forever recover.

2-3 Rotifer ponds are selected to hatch the resting eggs. DO and light significantly affect the hatching of rotifer resting eggs ( Hagiwara et al., 1989). The rotifer resting eggs are located on the bottom of the rotifer ponds, where DO is very low or zero and light is not sufficient. So the algae water in the rotifer ponds should be pumped into other ponds, and then the empty ponds are exposed to sun and air in order to oxidize the reductive pond bottom. 2-3 Days later brackish water ( 10-20 ppt; sea water + freshwater ) is pumped into the empty rotifer ponds upto a level of 20-30 cm, so that the resting eggs on the bottom of the ponds receive sufficient sunlight.

Meanwhile a number of resting eggs float from the bottom to the water surface ( Hagiwara et al., 1985 ). At 15 C after about 72 hours, most of the eggs have hatched. The rotifer resting eggs on the bottom of algae ponds with about 1 m of water depth do not hatch due to the deficiency in DO and light.

4. Daily management and harvest of rotifers

After hatching of the resting eggs, the algae water should be pumped into the rotifer ponds. Fresh sea water or brackish water and fermented bean cake should be added to the algae ponds to promote growth of the algae population. After 10 days later in early April, the rotifers are ready for harvest at a density of about 100,000 ind./l at 15 C.

The production yields are about 10 billion rotifers per day per pond. By pumping continuously algae water into the rotifer ponds and changing part of the water in these ponds, the production period can last for a long time ( a month, or longer ). The harvested rotifers can be transferred into other algae ponds to start another rotifer production cycle. Introduction of boiled soya-bean milk as a food source for the rotifers can enable a reduction of the culture ponds required for green algae.

Snell et al ( 1987 ) described 2 important indicators for assessing the physiological condition of rotifer mass cultures. The first is swimming activity which is measured by observing rotifer swimming over a grid with 1 mm square. Lowered swimming activity indicates the physiological stress in the rotifer mass culture.

In this case, the water should be partly changed. Unionized ammonia has been recognized as an important factor restricting rotifer reproduction and causing mass culture instability ( Yu et al.,1986 ), but the rotifer B. plicatilis can tolerate higher ammonia concentration than copepods. In practical production of rotifers in case of appearance of copepods in the rotifer ponds, we can effectively kill the copepods by adding 500-1,000 grams of the commonly used fertilizer urea.

The second indicator for assessing the status of rotifer mass cultures is egg ratio, which is the number of eggs carried by females divided by the number of females. Egg ratios are most useful for predicating future reproductive output of rotifer populations. Egg ratios typical of exponentially growing B. plicatilis populations at 25 C ranged from 0.5-1.2 ( Snell et al., 1987 ). Rotifer populations reproducing at a replacement level ( stationary phase ) had egg ratios between 0.13-0.5. Once egg ratio fell below 0.13, populations declined.

We find that appearance of male rotifers is the 3rd indicator for assessing the status of rotifer mass cultures. The monogonont rotifer B. plicatilis reproduce both asexually and sexually. Under optimal culture conditions, the rotifer reproduces by parthenogenesis.

Rotifer bisexual reproduction is affected by both internal and external factors ( Hagiwara et al., 1991 ). The ability of rotifers to undergo mictic multiplication and to generate the resting eggs is thought to develop in an evolutionary process to cope with severe environmental changes ( Fukusho, 1989 ). Under severe conditions, the rotifers undergo bisexual reproduction. So the appearance of male rotifers indicate the culture medium need to change.

Application of rotifers in aquaculture

The harvested rotifers are rinsed with fresh sea water three times before use. Only live rotifers are feeded to larval fish and crustacean. In north China along Bohai Bay, hatcheries produce larvae of red sea bream, black sea bream, Chinese white shrimp and fresh water crab.

Daily rotifer consumption by red sea bream and black sea bream have been extensively studied ( Fukusho, 1989 ). The relationship between the total length of larva ( L mm ) and the daily rotifer consumption ( F ) can be expressed by the following equation: 3.934 F = 0.303 L , where 3.92 mm < L < 10.05 mm ( Kitajima et al., 1976).

The density of rotifers in the culture water will be very important when larvae and juveniles are reared. 3-10 Rotifers per ml is necessary for red sea bream, while 1-3 rotifers per ml is sufficient for black sea bream ( Fushimi, 1983 ). Estimates suggest that one red sea bream larva requires 12,000 to 15,000 rotifers over 25 days untill it reaches 10 mm in length ( Kafuku et al., 1983).

Practical application of rotifers in larviculture of Chinese white shrimp P. chinensis have demonstrated that rotifers have more advantages than Artemia nauplii. Higher survival rate of shrimp larvae can be obtained by using rotifers as main food from Z2 to M2 stages of shrimp larvae.

In recent years, larviculture of Chinese fresh water crab E. sinensis have developed rapidly. Successful mass production of the rotifer B. plicatilis has inhanced development in larviculture of the crab.

Production of rotifer cysts

The need for continuous maintenance of live stock cultures of Brachionus either for laboratory investigations or aquacultural purposes requires considerable routine effort and involves the risk of bacteriae and ciliates contamination.

Commercial availability of rotifer eggs could be the solution by eliminating the need to maintain stock cultures and reduce the chances for contamination with ciliates and pathogenic bacteria. Furthermore, the rotifer cysts could also be disinfected prior to hatching out in a new culture inoculum.

Under optimal culture conditions, B. plicatilis reproduce by parthenogenesis, each female produce several eggs at a time which upon hatching reach the reproductive stage in a few days only. But in undesired conditions, they reproduce by bisexual and produce resting eggs ( cysts ) which deposit on the bottom of ponds. Rotifer cysts remain dormant and hatch after stimulation by specific external conditions.

The extent of resting egg production is determined by both internal and external factors. The most important internal factors are the age of the parental female and her genotype. The external factors include temperature, photoperiod, population density and grouping, and both qualitative and quantitative aspects of diet ( Pourriot et al., 1983 ). In nature, the most important factor affecting cysts production of rotifers is the supply of diets. In spring, rotifer cysts hatch and rotifer populations rapidly grow. When the diets are consumed out, the rotifers begin to produce cysts.

On a hatchery we produce rotifer cysts by adjusting only the diet supply. First we culture rotifers with algae and yeast at 25 C and 20 ppt under natural sunlight. When rotifers reach a density of 10,000 ind./l, the diets supply are gradually reduced to zero. 10 Days later, we harvest the cysts by draining off the culture water and the resting eggs are collected by sieving through a net. Then the cysts are purified and processed to a dry form. 1 gram of processed rotifer cysts include 2,000,000 eggs with about 80% of hatching rate at 28 C and 20 ppt in 36 hours.

References

Dhert P., P. Sorgeloos, 1994. Live feeds in aquaculture. Proceedings of Aquatech'94, in press.

Fukusho, K., 1989. Biology and mass production of the rotifer, Brachionus plicatilis. Int. J. Aq. Fish. Technol., 1:232-240.

Fulks, W., K. L. Main, 1991. Rotifer and Microalgae Culture Systems. The Oceanic Inst., Honolulu, USA. 364pp.

Fushimi, T., 1983. III-5 ingestion by fish larvae and juveniles. The rotifer, Brachionus plicatilis-Biology and mass culture ( Japan. Soc. Sci. Fish. ed.), Koseishya-Koseikaku, 69-93, Tokyo.

Hagiwara, A., 1989. Recent studies on the rotifer Brachionus plicatilis as a live food for the larval rearing of marine fish. La mer 27:116-121.

Hagiwara, A., A. Hino, 1989. Effect of incubation and preservation on resting egg hatching and mixis in the derived clones of the rotifer Brachionus plicatilis. Hydrobiologia 186/187:415-421.

Hagiwara, A., A. Hino, R. Hirano, 1985. Studies on the appearance of floating fertilized eggs in the rotifer Brachionus plicatilis. The Aquaculture 32(4): 207-212.

Hagiwara, A., C. S. Lee, 1991. Resting egg formation of the L- and S-type rotifer Brachionus plicatilis under different water temperature. Nippon Suisan Gakkaishi 57(9):1645-1650.

Hino, A., 1993. Present culture systems of the rotifer Brachionus plicatilis and the function of micro-organisms. In: C. S. Lee, M. S. Su and I. C. Liao (eds). Finfish Hatchery in Asia: Proc. Finfish Hatchery in Asia'91. TML Conf. Proc. 3:51-59, Tungkang Marine Lab., Taiwan Fisheries Research Inst., Tungkang, Pintung, Taiwan.

Kafuku, T., H. Ikenoue, 1983. Modern methods of aquaculture in Japan. Developments in aquaculture and fisheries sciences, 11. Kodansha Ltd., Tokyo and Elsevier, Amsterdam. 216 pp.

Kitajima, C., K. Fukusho, H. Iwamoto, H. Yamamoto, 1976. Amount of rotifers Brachionus plicatilis, consumed by red sea bream larvae, Pagrus major. Bull. Nagasaki Pref. Inst. Fish., 1:105-112.

Lubzens, L., 1987. Raising rotifers for use in aquaculture. Hydrobiologia 147:245-255.

Pourriot, R., T. W. Snell, 1983. Resting eggs in rotifers. Hydrobiologia 104:213-224.

Snell, T. W., K. Carrillo, 1984. Body size variation among strains of the rotifer Brachionus plicatilis. Aquaculture 37:359-367.

Snell, T. W., M. J. Childress, E. M. Boyer, 1987. Assessing the status of rotifer mass culture. J. World Aquacult. Soc. 18:270-277.

Yu, J., A. Hino, R. Hirano, K. Hirayama, 1988. Vitamin B12-producing bacteria as a nutritive complement for a culture of the rotifer Brachionus plicatilis. Nippon Suisan Gakkaishi 54:1873-1880.

Yu, J., K. Hirayama, 1986. The effect of un-ionized ammonia on the population growth of the rotifer in mass culture. Bull. Jap. Soc. Sci. Fish. 52: 1509-1513.

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