6.1.1. Biology and life cycle of Daphnia
6.1.2. Nutritional value of Daphnia
6.1.3. Feeding and nutrition of Daphnia
6.1.4. Mass culture of Daphnia
6.1.5. Production and use of resting eggs
6.1.6. Use of Moina
Daphnia is a frequently used food source in the freshwater larviculture (i.e. for different carp species) and in the ornamental fish industry (i.e. guppies, sword tails, black mollies and plattys etc.)
Daphnia belongs to the suborder Cladocera, which are small crustaceans that are almost exclusively living in freshwater. The carapace encloses the whole trunk, except the head and the apical spine (when present). The head projects ventrally and somewhat posteriorly in a beak-like snout. The trunk appendages (five or six pairs) are flattened, leaf-like structures that serve for suspension feeding (filter feeders) and for locomotion. The anterior part of the trunk, the postabdomen is turned ventrally and forward and bears special claws and spines to clean the carapace (Fig. 6.1.). Species of the genus Daphnia are found from the tropics to the arctic, in habitats varying in size from small ponds to large freshwater lakes. At present 50 species of Daphnia are reported worldwide, of which only six of them normally occur in tropical lowlands.
The adult size is subjected to large variations; when food is abundant, growth continues throughout life and large adults may have a carapace length twice that of newly-mature individuals. Apart from differences in size, the relative size of the head may change progressively from a round to helmet-like shape between spring and midsummer. From midsummer to fall the head changes back to the normal round shape. These different forms are called cyclomorphs and may be induced, like in rotifers, by internal factors, or may be the result from an interaction between genetic and environmental conditions.
Normally there are 4 to 6 Instar stages; Daphnia growing from nauplius to maturation through a series of 4-5 molts, with the period depending primarily on temperature (11 days at 10°C to 2 days at 25°C) and the availability of food. Daphnia species reproduce either by cyclical or obligate parthenogenesis and populations are almost exclusively female. Eggs are produced in clutches of two to several hundred, and one female may produce several clutches, linked with the molting process. Parthenogenetic eggs are produced ameiotically and result in females, but in some cases males can appear. In this way the reproductive pattern is similar to rotifers, where normally parthenogenetic diploid eggs are produced. The parthenogenetic eggs (their number can vary from 1 to 300 and depends largely upon the size of the female and the food intake) are laid in the brood chamber shortly after ecdysis and hatch just before the next ecdysis. Embryonic development in cladocerans occurs in the broodpouch and the larvae are miniature versions of the adults. In some cases the embryonic period does not correspond with the brood period, and this means that the larvae are held in the brood chamber even after the embryonic period is completed, due to postponed ecdysis (environmental factors). For different species the maturation period is remarkably uniform at given temperatures, ranging from 11 days at 10°C to only 2 days at 25°C.
Factors, such as change in water temperature or food depreviation as a result of population increase, may induce the production of males. These males have one or two gonopores, which open near the anus and may be modified into a copulatory organ. The male clasps the female with the first antennae and inserts the copulatory processes into the single, median female gonopore. The fertilized eggs are large, and only two are produced in a single clutch (one from each ovary), and are thick-shelled: these resting or dormant eggs being enclosed by several protective membranes, the ephippium. In this form, they are resistant to dessication, freezing and digestive enzymes, and as such play an important role in colonizing new habitats or in the re-establishment of an extinguished population after unfavourable seasonal conditions.
The nutritional value of Daphnia depends strongly on the chemical composition of their food source. However, since Daphnia is a freshwater species, it is not a suitable prey organism for marine organisms, because of its low content of essential fatty acids, and in particular (n-3) HUFA. Furthermore, Daphnia contains a broad spectrum of digestive enzymes such, as proteinases, peptidases, amylases, lipases and even cellulase, that can serve as exo-enzymes in the gut of the fish larvae.
The filtering apparatus of Daphnia is constructed of specialized thoracic appendages for the collection of food particles. Five thoracic limbs are acting as a suction and pressure pump. The third and fourth pair of appendages carry large filter-like screens which filter the particles from the water. The efficiency of the filter allows even the uptake of bacteria (approx. 1µm). In a study on the food quality of freshwater phytoplankton for the production of cladocerans, it was found that from the spectrum blue-greens, flagellates and green algae, Daphnia performed best on a diet of the cryptomonads, Rhodomonas minuta and Cryptomonas sp., containing high levels of HUFA (more than 50% of the fatty acids in these two algae consisted of EPA and DHA, while the green algae were characterized by more 18:3n-3). This implies that the long-chained polyunsaturated fatty acids are important for a normal growth and reproduction of Daphnia. Heterotrophic microflagellates and ciliates up to the size of Paramecium can also be used as food for Daphnia. Even detritus and benthic food can be an important food source, especially when the food concentration falls below a certain threshold. In this case, the water current produced by the animals swimming on the bottom whirls up the material which is eventually ingested. Since daphnids seem to be non-selective filter feeders (i.e., they do not discriminate between individual food particles by taste) high concentrations of suspended material can interfere with the uptake of food particles.
Figure 6.1. Schematic drawing of the internal and external anatomy of Daphnia.
6.1.4.1. General procedure for tank culture
6.1.4.2. Detrital system
6.1.4.3. Autotrophic system
6.1.4.4. General procedure for pond culture
6.1.4.5. Contamination
Daphnia is very sensitive to contaminants, including leaching components from holding facilities. When plastic or other polymer containers are used, a certain leaching period will be necessary to eliminate toxic compounds.
The optimal ionic composition of the culture medium for Daphnia is unknown, but the use of hard water, containing about 250 mg.l-1 of CO32-, is recommended. Potassium and magnesium levels should be kept under 390 mg.l-1 and 30-240 µg. l-1, respectively. Maintenance of pH between 7 to 8 appears to be important to successful Daphnia culture. To maintain the water hardness and high pH levels, lime is normally added to the tanks. The optimal culture temperature is about 25°C and the tank should be gently aerated to keep oxygen levels above 3.5 mg.l-1 (dissolved oxygen levels below 1.0 mg.l-1 are lethal to Daphnia). Ammonia levels must be kept below 0.2 mg.l-1.
Inoculation is carried out using adult Daphnia or resting eggs. The initial density is generally in the order of 20 to 100 animals per litre.
Normally, optimal algal densities for Daphnia culture are about 105 to 106 cells. ml-1 (larger species of Daphnia can support 107 to 109 cells.ml-1). There are two techniques to obtain the required algal densities: the detrital system and the autotrophic system:
The “stable tea” rearing system is a culture medium made up of a mixture of soil, manure and water. The manure acts as a fertilizer to promote algal blooms on which the daphnids feed. One can make use of fresh horse manure (200 g) that is mixed with sandy loam or garden soil (1 kg) in 10 l pond water to a stable stock solution; this solution diluted two to four times can then be used as culture medium. Other fertilizers commonly used are: poultry manure (4 g.l-1) or cow-dung substrates. This system has the advantage to be self-maintaining and the Daphnia are not quickly subjected to deficiencies, due to the broad spectrum of blooming algae. However, the culture parameters in a detrital system are not reliable enough to culture Daphnia under standard conditions, i.e. overfertilization may occur, resulting in anoxic conditions and consequently in high mortalities and/or ephippial production.
Autotrophic systems on the other hand use the addition of cultured algae. Green water cultures (105 to 106 cells.ml-1) obtained from fish pond effluents are frequently used but these systems show much variation in production rate mainly because of the variable composition of algal species from one effluent to another. Best control over the culture medium is obtained when using pure algal cultures. These can be monocultures of e.g. algae such as Chlorella, Chlamydomonas or Scenedesmus, or mixtures of two algal cultures. The problem with these selected media is that they are not able to sustain many Daphnia generations without the addition of extra vitamins to the Daphnia cultures. A typical vitamin mix is represented in Table 6.1.
Table 6.1. A vitamin mix for the monospecific culture of Daphnia on Selenastrum, Ankistrodesmus or Chlamydomonas. One ml of this stock solution has to be added to each litre of algal culture medium (Goulden et al., 1982).
|
Nutrient |
Concentration of stock solution
(µg.1-1) |
|
Biotin |
5 |
|
Thiamine |
100 |
|
Pyridoxine |
100 |
|
Pyridoxine |
3 |
|
Calcium Panthothenate |
250 |
|
B12 (as mannitol) |
100 |
|
Nicotinic acid |
50 |
|
Nicotinomide |
50 |
|
Folic acid |
20 |
|
Riboflavin |
30 |
|
Inositol |
90 |
Mass cultivation of Daphnia magna can also be achieved on cheap agro-industrial residues, like cotton seed meal (17 g.l-1), wheat bran (6.7 g.l-1), etc. Rice bran has many advantages in comparison to other live foods (such as microalgae): it is always available in large quantities, it can be purchased easily at low prices, it can be used directly after simple treatment (micronisation, defatting), it can be stored for long periods, it is easy to dose, and it has none of the problems involved in maintenance of algal stocks and cultures.
In addition to these advantages, there is also the fact that rice bran has a high nutritional value; rice bran (defatted) containing 24% (18.3%) crude protein, 22.8% (1.8%) crude fat, 9.2% (10.8%) crude fibre, and being a rich source of vitamins and minerals. Daphnia can be grown on this food item for an unlimited number of generations without noticeable deficiencies.
Defatted rice bran is preferred above raw rice bran because it prevents hydrolysis of the fatty acids present and, consequently, rancidity of the product. Micronisation of the bran into particles of less than 60 µm is generally carried out by treating an aqueous suspension (50 g.l-1) with a handmixer and filtering it through a 60 µm sieve, or by preparing it industrially by a dry mill process. The suspension is administered in small amounts throughout a 24 h period: 1 g of defatted rice bran per 500 individuals for two days (density: 100 animals.l-1). The food conversion ratio has an average of 1.7, which implies that with less than 2 kg of dry rice bran approximately 1 kg wet daphnid material can be produced (with a 25% water renewal per week; De Pauw et al., 1981).
Daphnia can also be produced in ponds of at least 60 cm in height. To produce 1 ton of Daphnia biomass per week, a 2500 m3 culture pond is required. The pond is filled with 5 cm of sun-dried (for 3 days) soil to which lime powder is added at a rate of 0.2 kg lime powder per ton soil. After this the pond is then filled with water up to 15 cm. Poultry manure is added to the ponds on the 4th day at a rate of 0.4 kg.m-3 to promote phytoplankton blooms. Fertilization of the pond with organic manure instead of mineral fertilizers is preferred because cladocerans can utilize much of the manure directly in the form of detritus. On day 12 the water level is raised to 50 cm and the pond is fertilized a second time with poultry manure (1 kg.m-3). Thereafter, weekly fertilization rates are maintained at 4 kg poultry manure per m-3. In addition, fresh cow dung may also be used: in this instance a suspension is prepared containing 10 g.l-1, which is then filtered through a 100 µm sieve. During the first week a 10 l extract is used per day per ton of water; the fertilization increasing during the subsequent weeks from 20 l.m-3.day-1 in the second week to 30 l.m-3.day-1 in the following weeks.
The inoculation of the ponds is carried out on the 15th day at a rate of 10 daphnids per litre. One month after the inoculation, blooms of more than 100 g.m-3 can be expected. To maintain water quality in these ponds, fresh hard water can be added at a maximum rate of 25% per day. Harvesting is carried out by concentrating the daphnids onto a 500 µm sieve. The harvested biomass is concentrated in an aerated container (< 200 daphnids.l-1). In order to separate the daphnids from unfed substrates, exuviae and faecal material, the content of the container is brought onto a sieve, which is provided with a continuous circular water flow. The unfed particles, exuviae and faeces will collect in the centre on the bottom of the sieve, while the daphnids remain in the water column. The unwanted material can then be removed by using a pipette or sucking pump. Harvesting can be complete or partial; for partial harvesting a maximum of 30% of the standing crop may be harvested daily.
Daphnia cultures are often accidentally contaminated with rotifers. In particular Brachionus, Conochilus and some bdelloids may be harmful, (i.e. B. rubens lives on daphnids and hinders swimming and food collection activities). Brachionus is simply removed from the culture by flushing the water and using a sieve of appropriate mesh size as Daphnia is much bigger than Brachionus. Conochilus, on the other hand, can be eliminated by adding cow dung to the culture (lowering the oxygen levels). Bdelloids are more difficult to remove from the culture since they are resistant to a wide range of environmental conditions and even drought. However, elimination is possible by creating strong water movements, which bring the bdelloids (which are bottom dwellers) in the water column, and then removing them by using sieves.
Resting eggs are interesting material for storage, shipment and starting of new Daphnia cultures. The production of resting eggs can be initiated by exposing a part of the Daphnia culture to a combination of stressful conditions, such as low food availability, crowding of the animals, lower temperatures and short photoperiods. These conditions are generally obtained with aging populations at the end of the season. Collection of the ephippia from the wild can be carried out by taking sediment samples, rinsing them through a 200 µm sieve and isolating the ephippia under a binocular microscope. Normally, these embryos remain in dormancy and require a diapause inhibition to terminate this status, so that they can hatch when conditions are optimal. Possible diapause termination techniques are exposing the ephippia to low temperatures, darkness, oxygen and high carbon dioxide concentrations for a minimal period of several weeks (Davison, 1969).
There is still no standard hatching procedure for Daphnia. Generally the hatching process is stimulated by exposing the ephippia to higher temperatures (17-24°C), bright white light (70 W.m-2), longer photoperiods and high levels of dissolved oxygen. It is important, however, that these shocks are given while the resting eggs are still in the ephippium. After the shock the eggs may be removed from the ephippium. The hatching will then take place after 1-14 days.
Moina also belongs to the Cladocera and many of the biological and cultural characteristics that have been discussed for Daphnia can be applied to Moina.
Moina thrives in ponds and reservoirs but primarily inhabits temporary ponds or ditches. The period to reach reproductive maturity takes four to five days at 26°C. At maturity clear sexual dimorphic characteristics can be observed in the size of the animals and the antennule morphology. Males (0.6-0.9 mm) are smaller than females (1.0-1.5 mm) and have long graspers which are used for holding the female during copulation. Sexually mature females carry only two eggs enclosed in an ephippium which is part of the dorsal exoskeleton.
Moina is of a smaller size than Daphnia, with a higher protein content, and of comparable economic value. Produced biomass is successfully used in the larviculture of rainbow trout, salmon, striped bass and by tropical fish hobbyists who also use it in a frozen form to feed over sixty fresh and salt water fish varieties. The partial replacement of Artemia by Moina micrura was also reported to have a positive effect during the larviculture of the freshwater prawn Macrobrachium rosenbergii (Alam, 1992).
Enrichment of Moina can be carried out using the direct method, by culturing them on baker’s yeast and emulsified fish or cuttlefish liver oils. Experiments have shown that Moina takes up (n-3) HUFA in the same way, although slower, than rotifers and Artemia nauplii, reaching a maximum concentration of around 40% after a 24 h-feeding period.
