Lab. No. 6. Production and Application of Rotifers in Aquaculture

Transcription

1 Lab. No. 6 Production and Application of Rotifers in Aquaculture Rotifers are valuable live food for larval fish and crustacean whose small mouth cannot accept larger preys. The rotifer Brachionus plicatilis is, up to now, the only live feed that can be used in their very early larval stages. Several characteristics of rotifers have contributed to their usefulness as good prey for active larvae of marine fish, shrimp and crab, these include: nutritional quality: can have an excellent nutritional profile (if fed the right feeds) small body size: are easily consumed by almost all marine larvae relatively slow motility. its tolerance to the marine environment. easiness to culture it in large scale rapidly and inexpensively In fact, the development of the larval rearing industry is primarily due to the advancement of mass culture technology of the marine rotifer Brachionus plicatilis. Biology of rotifers Taxonomy PHYLUM: Nemathelminthes or Aschelminthes CLASS: Rotatoria ORDER: Monogononta FAMILY: Brachionidae Morphology Rotifers are among the smallest filterfeeding metazoans. Composed of a fixed number of about 1,000 cells, their growth is obtained by plasma increase, not by cellular division. They filter small particles by means of a ciliated annular organ, the corona, located in the anterior part of the body. The corona is also used for its whirling locomotion, hence the name of the Class Rotatoria. Whereas many species spend their life span attached to a substrate by means of their retractile foot, Brachionus plicatilis that is the main species cultured for finfish larval rearing world-wide, is a planktonic, unattached rotifer. Fig Brachionus plicatilis: female and male The rotifer s body is differentiated into three distinct parts: head, trunk and foot. The head carries the ring organ or corona, which is easily identifiable by its ring of cilia. The retractile corona assures locomotion and a whirling water movement which facilitates the ingestion of small food particles (mainly algae and detritus). 25

2 The trunk contains the digestive tract, consisting in a mastax that grinds the ingested particles, the oesophagus, the stomach with gastric glands and the intestine. The excretory system consists of paired protonephridia with terminal cells (cyrtocytes), the duct and the bladder. The genital organ is unpaired (Monogononta) or paired in the Seisonidea and Bdelloidea. The joint external opening for bladder and oviduct is called the cloaca. The foot is ending in one or four toes bearing pedal glands that secrete an adhesive substance in crawling and sessile rotifers. Two different morphotypes of B. plicatilis exist: the small (S) type and the large (L) type. They differ in their lorica length: 130 to 340 µm (average 239 µm) for the L-type and 100 to 210 µm (average 160 µm) for the S-type. There are also differences in weight, shape of occipital spines and optimal growth temperatures (L-type rotifers have a wider temperature range while S-type rotifers have a higher temperature resistance). S-type rotifers are suitable as first food for fish larvae with a mouth opening smaller than 100 µm at first feeding, such as gilthead seabream, groupers, and rabbitfish. Fig. Brachionus rotundiformis and Brachionus plicatilis Life history The life span of rotifers is measured in days and depends on culture temperature, but in a controlled environment and at 25 C, it has been estimated to range around 7 days. At this temperature, larvae become adults after 0.5 to 1.5 days and then females start to lay eggs approximately every four hours. A female will typically produce up to 20 eggs during her lifetime and can carry up to 7 eggs simultaneously. The reproductive activity of Brachionus is influenced by temperature as illustrated below. Effect of temperature on reproductive activity of Brachionus plicatilis The reproduction of B. plicatilis can be either sexual (called mictic reproduction) or asexual (amictic or parthenogenetic reproduction). Only the latter is adopted for rotifer 26

3 mass culture due to its faster rate and also due to the absence of males, which are useless as fish feed not having a functional digestive tract. In the amictic reproduction the offspring are clones genetically identical to their mothers, i.e. all newly born rotifers are diploid females. Such multiplication can go on for months in a population kept in proper rearing conditions. Depending on environmental conditions, each female may produce about 20 amictic eggs. Males are only produced after a sudden change in the environment, when females produces haploid (n chromosomes) eggs. Males and females breed, and the result is mictic resting eggs, analogous to Artemia cysts, which will hatch amictic females again. Stock culture of rotifers Fig Parthenogenetical and sexual cycle of Brachionus plicatilis Preparation of the culture medium The same procedures and precautions described in the algal production labs apply to rotifer culture. The culture water (seawater diluted with tap water to a salinity of 25 ppt) is aerated, prefiltrated over a 1 µm filter bag and disinfected overnight with 5 mg.l -1 27

4 NaOCl. The next day the excess of NaOCl is neutralized with Na 2 S 2 O 3 and the water is filtered over a 0.45 µm filter. The only enrichment added to the rotifer culture medium, be it either a log-phase algal culture or treated seawater plus artificial diet, is represented by the addition of vitamin B12 (cyanocobalamin) as a fertility booster for the rotifers. Its dosage is usually 1 ml of B12 stock solution per litre. The vitamin is added together with the inoculum. To prepare the stock solution put 0.1 g of vitamin B12 into a sterilised 1-liter graduated Pyrex bottle and fill with sterilized DW to the mark. When fully dissolved, store in the refrigerator. Warning: never sterilise vitamin solutions. For stock cultures, the rotifers can be obtained from the wild, or from research institutes or commercial hatcheries. However, before being used in the production cycle the inoculum should first be disinfected. The most drastic disinfection consists of killing the free-swimming rotifers but not the eggs with a cocktail of antibiotics (e.g. erythromycin 10 mg.l -1, chloramphenicol 10 mg.l -1, sodium oxolinate 10 mg.l -1, penicillin 100 mg.l -1, streptomycin 20 mg.l -1 ) or a disinfectant. The eggs are then separated from the dead bodies on a 50 µm sieve and incubated for hatching and the offspring used for starting the stock cultures. However, if the rotifers do not contain many eggs (as can be the case after a long shipment) the risk of loosing the complete initial stock is too big and in these instances the rotifer should be disinfected at sublethal doses; the water of the rotifers being completely renewed and the rotifers treated with either antibiotics or disinfectants. The treatment is repeated after 24 h in order to be sure that any pathogens which might have survived the passage of the intestinal tract of the rotifers are killed as well. The concentration of the disinfection products differs according to their toxicity and the initial condition of the rotifers. Orientating concentrations for this type of disinfection are 7.5 mg.l -1 furazolidone, 10 mg.l -1 oxytetracycline, 30 mg.l -1 sarafloxacin, or 30 mg.l -1 lincospectin. Fig Stock cultures of rotifers kept in 50 ml centrifuge tubes. 28

5 At the Laboratory the stock cultures for rotifers are kept in sterilized 50 ml conical centrifuge tubes (Fig. 6.3) at 28 C ± 1 C. The tubes are exposed to the light of two fluorescent light tubes at a distance of 20 cm (light intensity of 3000 lux on the tubes). Inoculation of the tubes is carried out with an initial density of 2 rotifers.ml-1. The food consists of marine algae cultured according to the procedure previously described in the preceding labs. The algae are centrifuged and concentrated to cells.ml -1. The algal concentrate is stored at 4 o C in a refrigerator for a maximum period of 7 days, coinciding with one rotifer rearing cycle. Every day the algal concentrate is homogenized by shaking and 200 µl is given to each of the tubes. If fresh algae are given instead of the algal concentrate 4 ml of a good culture is added daily. After one week the rotifer density should have increased from 2 to 200 individuals/ml. The rotifers are rinsed, a small part is used for maintenance of the stock, and the remaining rotifers can be used for upscaling. Furthermore, after some months of regular culture the stock cultures will be disinfected as described earlier in order to keep healthy and clean stock material. However, the continuous maintenance of live stock cultures of Brachionus does not eliminate the risk of bacterial contamination. Mass culture of rotifers Mass culture parameters and conditions What follows in the column marked as preferable range, gives a reasonable example of mass culture conditions to be maintained in order to develop rotifer cultures properly. Aeration is a critical element in rotifer culture with yeast and/or artificial diets. A proper balance must be maintained between: adequate dissolved oxygen level, i.e., at least 4 ppm; sufficient turbulence to keep rotifers and food in suspension; a culture medium without excessive turbulence, which causes stress and resuspends bottom sediments (flocks). Population dynamics Rotifer population dynamics under mass rearing conditions follow different phases, mimicking those of microalgae: 29

6 the lag-phase, when, just after the inoculum, rotifers begin to consume the phytoplankton of their culture medium and the number of both egg-bearing individuals as well as the quantity of amictic eggs increases; the log-phase (or exponential phase), where rotifers reproduce very fast and population growth is exponential; the transitional phase (or declining growth), where growth rate slows down and egg-bearing rotifers become rarer; the decline phase, where almost only old rotifers without eggs are found and their number decreases rapidly as death rate exceeds growth rate. To be used as inoculum, the rotifer population must still be in the middle of its logphase with at least 20% fertility rate (measured as percentage of eggs over total rotifers. Populations in their last declining phase, characterised by limited motility, scarce repletion and absence of egg-bearing animals, should always be discarded. Upscaling rotifer cultures For upscaling processes, a clean culture of egg-rich rotifers from a 0.5-l flask is usually inoculated directly into a 5 to 10-l flask, bypassing the 2-l flask stage. The inoculum should provide an initial density of 10 to 20 rotifers per ml. A larger inoculum results in a faster pace of population growth. Rotifers should always be inoculated in algae cultures which have not yet reached their peak growth (that is their log-phase). Environmental conditions should be the same ones at which algae are kept. However, aeration can be reduced to diminish the amount of foam and bottom sediments produced by the metabolic activity of rotifers. Vitamin B12 is routinely added to all new vessels, at 1 ml of stock solution per litre of algal culture. Mature rotifer cultures from small vessels (5 liters or larger) are poured into bags with an algal population that has not yet entered its full log phase. Five to ten % inoculum is used at this stage, i.e. one 5-liter vessel is used to inoculate one 100-l bag, following the rule of the new culture starting at rotifers/ml. Using a sterile volumetric cylinder, add 1 ml of the Vitamin B12 per litre of culture; mark the date on the bag surface, together with the origin of the inoculum and the serial number to facilitate its handling and record keeping. Feeding Rotifers are filter feeders, accepting small particles up to 30 µm in size including bacteria, algae, yeast and protozoa. There are basically two main feeding methods for rearing rotifers: 1) a combination of algae and baker s yeast and 2) a totally artificial diet. For its reliability and higher output, the latter is progressively replacing the first method. The mass production on algae and yeast is performed in a batch or semi-continuous culture system. Mass production on algae Undoubtedly, marine microalgae are the best diet for rotifers and very high yields can be obtained if sufficient algae are available and an appropriate management is followed. However, the culture of microalgae as a sole diet for rotifer feeding is costly due to the 30

7 labour intensive character of microalgae production. In most places, however, pure algae are only given for starting up rotifer cultures or to enrich rotifers. Usually, large amounts of cultured microalgae, such as the marine alga Nannochloropsis, are usually inoculated in the cultivation tanks together with a starter population containing 50 to 150 rotifers.ml -1. Mass culture on yeast Baker's yeast Saccaromyces cerevisiae has a small particle size (5-7 µm) and a high protein content. It is a common staple in the rotifer mass production process. It is a labour and cost sparing food the complete replace of natural rotifer diet by baker's yeast however were characterized by varying success and the occurrence of sudden collapses of the cultures. Most probably the reason for these crashes was explained by the poor nutritional value of yeast for rotifers, it is believed that the bacteria associated with the yeast represent the true food. Protocol: 1. fill the tank with sterilized sea water diluted with tap water to obtain a 25 ppt salinity; check the chlorine content of tap water and, if present, neutralize it with an excess of sodium thiosulphate. Take care to leave enough space for the algal cultures to be supplied as food (about 30% of the tank volume). 2. place the air diffusers and switch on the aeration; 3. place the traps for ciliates and impurities; 4. select the most suitable rotifer bags to be used for inoculum, checking for contaminants and using only clean batches; 5. filter the selected batch or batches 6. inoculate the tank to achieve an initial density of rotifers/ml. This is considered day 0; 7. add algal culture as 20% of tank volume to provide rotifers with their initial food; as usual, the algae should be in their log-phase and from non contaminated cultures, even if of different species. 8. the next day (day 1) fill the remaining 10% volume with algal culture; 9. on the tank file, record all information on the culture growth, food distributed and environmental parameters monitored (see below for details); 10. from day 1 on, feed with bakers yeast at the following rates, according to the recorded rotifer density. Rotifer density (No./ml) Daily feeding rate (g yeast/million rotifers) less than to100 2 more than The daily amount of fresh yeast is divided into 4 equal rations fed at 2 and 8 am, 2 and 10 pm. Feed immediately and discharge any leftover. Prepare fresh for every meal. 31

8 Enrichment rotifers are what they eat A major constraint of the yeast-fed rotifers method is the absolute necessity to improve the otherwise very poor nutritional quality of rotifers before their distribution to fish larvae. This means that, rotifer cultivated on usually needs to be supplemented with essential fatty acids and vitamins to suit the larval requirements of the predator organisms. To improve rotifer culture and upgrade the nutritional value for fish larvae during mass culture and before their harvest they are fed with special feeds and integrators. In the past (although still practiced in some countries) their nutritional value was upgraded by an enriching process before their harvest through feeding them with microalgae rich in PUFA and vitamins. At present, the enrichment is provided by specially formulated artificial diets like Selcoâ products. This oil emulsion gives excellent results in terms of high levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and vitamin C, which was not possible with the only use of algae. Moreover, labour, time, investment and running costs are spared. Enrichment by Algae The high content of the essential fatty acid eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in some microalgae such as Nannochloropsis occulata, Isochrysis galbana and Chlorella have made them excellent live food diets for boosting the fatty acid content of the rotifers. Rotifers submerged in these algae (approximately algae.ml -1 ) are incorporating the essential fatty acids in a few hours time. However, the culture of microalgae as a sole diet for rotifer feeding is costly due to the labour intensive character of microalgae production. Most of the time the rotifers are boosted in oil emulsions (see below) and fed to the predators which are kept in green water. This green water, consisting of ± algal cells.ml -1 (Tetra-selmis, Nannochloropsis, or Isochrysis) is applied to maintain an appropriate HUFA (but also other components) content in the live prey before they are eventually ingested by the predator. Procedure: use selected algae; Isochrysis, Chlorella sp., or Nannochloropsis spp. enrich for at least 8 h; maximum rotifer density: 500/ml; microalgae density: Isochrysis 5 million cells/ml; Chlorella sp. or Nannochloropsis spp. 12 million cells/ml; resulting average total PUFA content of enriched rotifers: ± 7 mg/g dry weight Fig. 6.4 Enriched rotifer 32

9 It is important to know that, the content of nutrients decreases rapidly in rotifers that are not immediately consumed by fish larvae. In starving rotifers the total PUFA loss reaches 60% after 6h at 18 C. Even in green water, i.e. with microalgae, this loss remains important (about 40% after 6 h). To prevent this degradation in nutritional quality, enriched rotifers not immediately fed to fish should be stored in containers at low temperature between 5 and 10 C. Oil emulsions One of the cheapest ways to enrich rotifers is by using oil emulsions. Although homemade emulsions can be prepared with egg lecithin and fish oils. Commercial emulsions are generally more stable and have a selected HUFA composition. Home-made emulsions The first emulsions were made from (n-3) HUFA rich fish oils and emulsified with egg yolk and seawater. Recently, more purified oils containing specifically high levels of the essential fatty acids have been used. Since the stability and storage possibility of these products is relatively low they are usually made on the spot and used immediately. Mass culture and enrichment with Culture Selco as food A different technique based on a compound feed has been developed a few years ago by the Belgian Company INVE SA. The product, named Culture Selco (CS) is a dry and complete rotifer diet that does not require algae and is also effective as enrichment medium. Particle size (5 to 7 µm) and physical characteristics ensure an optimal uptake by rotifers. The feed composition includes proteins (>35%), lipids (>15%, of which 23% are PUFA), carbohydrates (30%), carotenoids and other micronutrients as minerals and vitamins A, D3, F and C. The average daily production of rotifers fed on CS ranges consistently from 45 to 60% of the initial rotifer density. In addition rotifers are enriched with high levels of the essential (n-3) PUFA and vitamins. This diet has made rotifer mass culturing reliable and predictable, and has consistently reduced the need for algal cultures and their associated labour and facilities requirements. New rotifer cultures can be easily started from old ones, thanks to their enhanced fertility. Harvesting/concentration of rotifers Small-scale harvesting of rotifers is usually performed by siphoning the content of the culture tank into filter bags with a mesh size of µm. If this is not performed in submerged filters the rotifers may be damaged and result in mortality. It is therefore recommended to harvest the rotifers under water; concentrator rinsers (Fig. 5.6). Aeration during the concentration of rotifers will not harm the animals, but should not be too strong so as to avoid clogging of the rotifers, this can be very critical, specially after enrichment. For large-scale harvesting, a double submerged filter is used. The inner filter has a mesh size of µm to retain larger particles, flocks of agglomerated food particles and ciliates which would rapidly clog the finer filter. The outer filter has a 50 µm filter mesh. Its capacity should be large enough to keep safely the whole rotifer population for the time needed to complete harvest and rinsing. 33

10 Fig Side and upper view of a concentrator rinser containing a filter with a mesh size of 50 µm and equipped with an aeration collar at the bottom. Both filters are placed inside a large wheeled container full of water to avoid pressure build-up from the outgoing water that would smear rotifers against the net. A gentle air bubbling along the inner side of the filter helps to keep the filter free from clogging. At harvest, rotifers are rinsed with fresh sea water before being fed to fish or utilized as inoculum for new tanks. Application of rotifers in aquaculture Only live and enriched rotifers are fed to larval fish and crustacean. The density of rotifers in the culture water will be different according to the type and age of larvae. Production and use of resting eggs For the mass rearing of rotifers as larval food the amictic way of should be favored. However, when the interest is in production of resting eggs for use as a storable off-theshelf product mixis needs to be induced. These resting eggs, also called cysts, are relatively large (their volume is almost 60% of that of a normal adult female, Fig. 6.6), are ideal for storage and transport and can be used as inocula for mass cultures. 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. 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. 34

11 Figure. Microscopic view of resting eggs (length µm; a. at same magnification as two amictic females; b. at high magnification On a hatchery rotifer cysts can be produced by adjusting only the diet supply. First rotifers are cultured 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, the cysts are harvested by draining off the culture water and the resting eggs are collected by sieving through a net. Resting eggs can be also harvested by replacinge the water by brine so that resting eggs will float and can be collected from the water surface. Then, the cysts are purified and processed to a dry form. Dry resting eggs can be stored for more than one year.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. When placed in seawater, rotifer cysts hatch in about 24 hours at 25 C under light conditions. Newly-hatched rotifers undergo asexual reproduction. There are several advantages of using rotifer cysts to initiate mass cultures. The use of stock cultures is not required which considerably reduces labor cost and algal production costs. Moreover, the upscaling from stock culture to production unit can be considerably reduced by the use of larger numbers of cysts. The use of cysts is also highly recommended to prevent contamination. Cysts can easily be treated before hatching in order to ensure start cultures free from bacteria and ciliates. The resting eggs could be disinfected with heavy doses of antibiotics, so that the emerging rotifers are essentially bacteria free. The resting eggs can also resist short exposure to disinfectants such as NaOCl or glutaraldehyde. Student s activity Monitoring rotifer populations Check all rotifer cultures daily for both quantitative and qualitative evaluations. From each vessel, flask, bag and tank, take a 1 ml sample and observe under the stereoscopic microscope. Counting and Observations Each day, collect a sample from the rotifer culture for observation and enumeration. It is important to note that rotifers are phototactic and have the ability to migrate through the water column, so take care to ensure that the culture is well mixed prior to sampling. 35

12 Initially, a sample of live rotifers should be examined under a dissecting microscope Measure the following parameters: qualitative parameters: repletion (presence of food in the stomach, note 0 for empty, + for medium full, ++ when full), healthy rotifers should appear full of food motility or activity (++ active, + slow, 0 absent), healthy rotifers should appear very active. Slow or sluggish rotifers may indicate that water quality parameters are not ideal, and that corrective action is needed. filtration (activity of the ciliated corona) The overall cleanliness of the culture, (0 when clean, + for medium contamination, ++ large contamination) the presence or absence of other micro-organisms, such as protozoa, fungi, bacterial flocks, etc. should be noted. While it is common to have some ciliated protozoans in rotifer cultures, they compete with rotifers for oxygen and food. An overabundance of protozoan contaminants often correlates with excessively high levels of dissolved organic matter, a condition that is not ideal for rotifer culture. High numbers of ciliates can be abated by collecting rotifers in a 55-µm harvest bag, rinsing thoroughly and restarting the culture. presence of foam at the culture surface or sediment on the wall and bottom of the container Quantitative parameters: After observing the live sample, collect a 1 ml sub-sample with a pipette and load a Sedgwick-Rafter slide. Then add one to two drops of formalin or Lugol s solution to immobilize the rotifers. Place the Sedgwick-Rafter slide onto a microscope under 40x magnification and count o the total number of rotifers per ml o the number of rotifers with eggs. Number of rotifers with eggs o The percentage of rotifers with eggs = 100 Total number of rotifers o total number of eggs per ml Samples may need to be diluted prior to loading the Sedgwick-Rafter slide if rotifer densities approach 1000/ml. The percentage of rotifers with eggs is a useful indicator of the health of a population. o A rotifer population with 30 percent of individuals with eggs is consistent with a healthy culture. o A drop in this percentage to 15 percent to 20 percent is still acceptable, but growth of the culture will be reduced. o A decrease to <15 percent often indicates that conditions in the culture tank are sub-optimal, likely due to feed rate or water quality. In this case, corrective action should be taken immediately. 36

13 Egg percentage or fertility is the best indicator of whether or not your system is in equilibrium and it the best leading indicator of how well the culture will do over the next day of two. We measure egg percentage as following. total number of eggs % of eggs = 100 total number of rotifers o A good egg percentage is 35% to 50% depending on your harvest rate and the temperature of your water. o If your rotifer density is too high for the amount of feed egg percentages will drop to 20%-30%. o If your rotifer density is to low for the amount of feed egg percentages will rise to 70%-90%. o A high number of detached eggs is a sign of rotifer stress usually from low oxygen, high free ammonia levels or excess sheer stress from pumping or aeration. Another option for making a quick estimate of rotifer density involves collecting a sample from the culture tank with a 1.0 ml pipette with 0.1ml gradations. Count the number of rotifers in 0.1 ml and multiply by 10. All counts should be conducted two to three times for each culture and then used to calculate an average. The reproductive rates (G) or reproductive potential can be calculated from counts made on the first and last day of the experiment (suppose 3 days) as following: 1 N ln t G = T N o where T = days of the experiment. N o = the initial number of rotifers and the eggs they carry in each flask N t = the final number of rotifers and the eggs they carry in each flask 37