5.2.1. Introduction
5.2.2. Life cycle
5.2.3. Biometrics
5.2.4. Nutritional quality
5.2.5. Culture techniques
5.2.6. Use of resting eggs
5.2.7. Applications in larviculture
Numerous studies have demonstrated that copepods may have a higher nutritional value than Artemia, as the nutritional profile of copepods appear to match better the nutritional requirements of marine fish larvae. Furthermore, they can be administered under different forms, either as nauplii or copepodites at startfeeding and as ongrown copepods until weaning. Moreover, their typical zigzag movement, followed by a short gliding phase, is an important visual stimulus for many fish which prefer them over rotifers. Another advantage of the use of copepods, especially benthos-type species like Tisbe, is that the non-predated copepods keep the walls of the fish larval rearing tanks clean by grazing on the algae and debris.
Several candidate species belonging to both the calanoid and the harpacticoid groups have been studied for mass production. Calanoids can be easily recognized by their very long first antennae (16-26 segments), while the harpacticoids have only a short first antennae (fewer than 10 segments).
· calanoids:- Acartia tonsa
- Eurytemora affinis
- Calanus finmarchicus & C. helgolandicus
- Pseudocalanus elongatus· harpacticoids:
- Tisbe holothuriae
- Tigriopus japonicus
- Tisbenta elongata
- Schizopera elatensis
Although some success has been reported when using cultured copepods as live food in fish larviculture, it should be pointed out that the economic feasibility (or not) of copepod culture may be the main bottleneck for its routine application. Infrastructure and labour costs for the production of sufficient quantities of live copepods for commercial hatchery operations may indeed be prohibitive.
The Copepoda are the largest class of crustaceans forming an important link between phytoplankton and higher trophic levels in most aquatic ecosystems. Most adult copepods have a length between 1 and 5 mm. The body of most copepods is cylindriconical in shape, with a wider anterior part. The trunk consists of two distinct parts, the cephalothorax (the head being fused with the first of the six thoracic segments) and the abdomen, which is narrower than the cephalothorax. The head has a central naupliar eye and unirameous first antennae, that are generally very long.
Planktonic copepods are mainly suspension feeders on phytoplankton and/or bacteria; the food items being collected by the second maxillae. As such, copepods are therefore selective filter-feeders. A water current is generated by the appendages over the stationary second maxillae, which actively captures the food particles.
The male copepods are commonly smaller than the females and appear in lower abundance then the latter. During copulation the male grasps the female with his first antennae, and deposits the spermatophores into seminal receptacle openings, where they are glued by means of a special cement. The eggs are usually enclosed by an ovisac, which serves as a brood chamber and remains attached to the female’s first abdominal segment. Calanoids shed their eggs singly into the water. The eggs hatch as nauplii and after five to six naupliar stages (moltings), the larvae become copepodites. After five copepodite moltings the adult stage is reached and molting is ceased. The development may take from less than one week to as long as one year, and the life span of a copepod ranging from six months to one year.
Under unfavourable conditions some copepod species can produce thick-shelled dormant eggs or resting eggs. Such cysts can withstand desiccation and also provide means for dispersal when these are carried to other places by birds or other animals. In more northern regions a diapause stage is present in the development of the copepods so as to survive adverse environmental conditions, such as freezing; such a diapause usually taking place between the copepodite stage II to adult females and recognised by an empty alimentary tract, the presence of numerous orange oil globules in the tissue and an organic, cyst-like covering. The major diapause habitat is the sediment, although a minor part of the diapausing individuals may stay in the planktonic fraction, the so-called “active diapause”.
The size of copepods depends on the species as well as on the ontogenetic stage. Various copepod sizes are used for specific larviculture applications, assuring an efficient uptake by the target predator at any time during its larval rearing.
The harpacticoid Tisbe holothuriae grows from a nauplius size of 55 µm to an adult size of more than 180 µm, Schizopera elatensis from 50 to 500 µm, and Tisbentra elongata from 150 to more than 750 µm. Sizes for Eurytemora sp. (Calanoidea) are on an average 220 µm, 490 µm, and 790 µm for nauplii, copepodites, and adults, respectively.
The nutritional quality of copepods is generally accepted to be very good for marine fish larvae, and believed to be of a higher quality than the commonly used live food Artemia. In general copepods have a high protein content (44-52%) and a good amino acid profile, with the exception of methionine and histidine (Table 5.4.).
The fatty acid composition of copepods varies considerably, since it reflects the fatty acid composition of the diet used during the culture. For example, the (n-3)HUFA content of individual adult Tisbe fed on Dunaliella (low (n-3)HUFA content) or Rhodomonas algae (high (n-3)HUFA content) is 39 ng, and 63 ng respectively, and corresponds to 0.8% and 1.3% of the dry weight. Within nauplii, the levels are relatively higher; (i.e. around 3.9% and 3.4%, respectively). Specific levels of EPA and DHA are respectively 6% and 17% in adults fed Dunaliella, and 18% and 32% in adults fed Rhodomonas. In nauplii the levels of EPA, DHA and (n-3)HUFA are high, (i.e. around 3.5%, 9.0% and 15%, respectively). The fatty acid profiles of Tigriopus japonicus cultured on baker’s yeast or Omega-yeast are shown in Table 5.5. and their respective nutritional value for flatfish larvae is shown in Table 5.6.
Differences in the biochemical composition, and in particular the HUFA content, are not the only advantages of copepods over Artemia when offered as food to marine fish larval. For example, copepods (copepodites and adults) are believed to contain higher levels of digestive enzymes which may play an important role during larval nutrition.
As mentioned previously, the early stages of many marine fish larvae do not have a well-developed digestive system and may benefit from the exogenous supply of enzymes from live food organisms. Evidence that copepods may be preferable to Artemia in this respect comes from Pederson (1984) who examined digestion in first-feeding herring larvae, and found that copepods passed more quickly through the gut and were better digested than Artemia.
Table 5.4 Amino acid composition of Tigropus brevicornis cultured on different types of food (g.100g -1 crude protein) (Vilela, pers.comm.).
|
T. brevicornis cultured on Platymona sueceica
with different additives: |
||||
|
Amino acid |
+ yeast |
+ rice bran |
+ wheat |
+ fish food |
|
Aspartic acid |
7.30 |
6.98 |
7.08 |
7.63 |
|
Threonine |
3.35 |
3.09 |
3.53 |
3.74 |
|
Serine |
3.37 |
2.98 |
3.39 |
3.59 |
|
Glutamic acid |
12.05 |
12.00 |
11.90 |
10.62 |
|
Proline |
5.13 |
4.49 |
6.56 |
4.82 |
|
Glycine |
4.40 |
4.24 |
4.31 |
4.71 |
|
Alanine |
5.44 |
5.45 |
5.97 |
5.87 |
|
Cystine |
0.39 |
0.84 |
1.23 |
1.27 |
|
Valine |
4.52 |
4.30 |
4.21 |
4.71 |
|
Methionine |
1.78 |
1.75 |
1.64 |
1.81 |
|
Isoleucine |
3.35 |
3.21 |
3.28 |
3.48 |
|
Leucine |
4.79 |
4.71 |
6.24 |
6.73 |
|
Tyrosine |
3.89 |
3.99 |
3.21 |
3.87 |
|
Phenylalanine |
2.64 |
2.67 |
3.37 |
3.44 |
|
Histidine |
1.94 |
1.75 |
1.78 |
1.33 |
|
Lysine |
4.81 |
4.65 |
4.81 |
4.92 |
|
Arginine |
6.52 |
6.34 |
5.76 |
6.11 |
|
Total |
75.67 |
73.44 |
78.27 |
78.65 |
|
Protein (%) |
51.1 |
48.6 |
43.9 |
46.5 |
|
FA |
Baker’s yeast |
Omega-yeast |
||||||
|
Total |
TG |
FFA |
PL |
Total |
TG |
FFA |
PL |
|
|
14:0 |
0.6 |
0.8 |
0.7 |
0.6 |
1.2 |
1.8 |
1.7 |
0.5 |
|
15:0 |
1.8 |
1.7 |
0.8 |
0.5 |
0.8 |
0.6 |
0.6 |
0.4 |
|
16:0 |
7.1 |
8.2 |
8.1 |
13.2 |
9.1 |
10.1 |
9.9 |
13.2 |
|
16:1n-7 |
13.9 |
22.3 |
12.8 |
3.2 |
6.5 |
7.2 |
6.6 |
2.3 |
|
18:0 |
2.5 |
0.8 |
2.1 |
6.6 |
2.6 |
1.3 |
2.5 |
6.8 |
|
18:1n-9 |
23.7 |
31.6 |
20.6 |
15.7 |
22.1 |
32.4 |
21.8 |
14.2 |
|
18:2n-6 |
2.9 |
2.9 |
2.4 |
2.2 |
1.5 |
1.4 |
1.7 |
1.2 |
|
18:3n-3 |
4.4 |
5.3 |
3.8 |
1.2 |
0.9 |
0.7 |
0.7 |
0.5 |
|
18:4n-3 |
1.1 |
0.8 |
0.8 |
2.3 |
9.1 |
11.5 |
5.6 |
3.7 |
|
20:1 |
1.4 |
0.8 |
0.8 |
2.3 |
9.1 |
11.5 |
5.6 |
3.7 |
|
20:4n-3 |
2.1 |
1.6 |
2.0 |
0.8 |
0.7 |
0.4 |
0.5 |
0.3 |
|
20:5n-3 |
6.0 |
2.9 |
13.1 |
8.1 |
4.7 |
3.2 |
7.9 |
6.4 |
|
22:1 |
0.3 |
0.7 |
0.5 |
0.1 |
5.4 |
5.9 |
3.3 |
2.2 |
|
22:5n-3 |
1.1 |
0.8 |
0.7 |
1.0 |
0.9 |
0.7 |
0.6 |
0.4 |
|
22:6n-3 |
13.8 |
5.2 |
16.8 |
33.2 |
20.9 |
15.8 |
26.2 |
38.8 |
|
(n-3) HUFA |
23.0 |
10.5 |
32.6 |
43.1 |
27.2 |
20.1 |
35.2 |
45.9 |
|
|
Survival rate |
Total length |
Body weight |
Condition factor |
|
Baker’s yeast |
96.1 |
23.3 |
90.9 |
7.1 |
|
91.4 |
22.3 |
87.8 |
7.8 |
|
|
Omega yeast |
97.0 |
23.7 |
102.5 |
7.7 |
|
97.4 |
23.3 |
104.0 |
8.1 |
5.2.5.1. Calanoids
5.2.5.2. Harpacticoids
In general, it may be stated that harpacticoid copepods are less sensitive and more tolerant to extreme changes in environmental conditions (i.e. salinity: 15-70 g.l-1; temperature: 17-30°C) than calanoids and thus are easier to rear under intensive conditions. Moreover, harpacticoids have a higher productivity than calanoids and can be fed on a wide variety of food items, such as microalgae, bacteria, detritus and even artificial diets. However, as mentioned previously, care should be taken in this respect as the lipid and (n-3) HUFA composition of the copepods is largely dependent on that of the diet fed.
A continuous production system for the calanoid copepod Acartia tonsa has been described by Støttrup et al. (1986). It consists of three culture units: basis tanks, growth tanks and harvest tanks. The Acartia tonsa are isolated from natural plankton samples or reared from resting eggs onwards (see 5.2.6. Surface-disinfection of resting eggs).
The basis tanks (200 l grey PVC tanks: 1500 × 50cm) are run continuously, regardless of production demands, and the eggs produced are used to adjust population stocks. These tanks are very well controlled and kept under optimal hygienic conditions: using filtered (1 µm) seawater (salinity 35 g.l-1) and fed with Rhodomonas algae (8.108.days-1) produced under semi-sterile indoor conditions. Temperatures are kept at 16-18°C and a gentle aeration from the bottom is provided. Adult concentrations with a ratio of 1:1 males to females are maintained at less than 100.l-1 by adjusting once a week with stage IV-V copepodites. Approximately 10 l of the culture water is siphoned daily from the bottom of the tanks (containing the eggs), and replaced by new, clean seawater. Eggs are collected from the effluent waters by the use of a 40 µm sieve; production averaging 95,000 eggs.day-1, and corresponding to a fecundity rate of 25 eggs.female-1.day-1. The basis cultures are emptied and cleaned two to three times per year, by collecting the adults on a 180 µm sieve and transferring them to cleaned and disinfected tanks.
Collected eggs are transferred to the growth tanks where maximal densities reach 6000.1-l. The nauplii start to hatch after 24 h with hatching percentages averaging 50% after 48 h incubation. Initially Isochrysis is given at a concentration of 1000 cells.ml-1 and after 10 days a mixture of Isochrysis and Rhodomonas administered at a concentration of 570 and 900 cells.ml-1, respectively. The generation time (period needed to reach 50% fertilised females) is about 20 days with a constant mortality rate of about 5%.day-1.
After 21 days, the adults are collected using a 180 µm sieve and added either to the basis or harvest tanks. Harvesting tanks are only in use once the fish hatchery starts to operate. Cultures are maintained in 450 l black tanks under the same conditions as described above. Each tank receives a daily amount of 16.108 Rhodomonas cells, harvested from bloom cultures. These tanks are emptied and cleaned more regulary than stock tanks. To facilitate the harvesting of solely nauplii or copepodites of a specific stage (depending on the requirements), eggs are harvested daily and transferred to the hatching tanks; the aeration levels within these tanks being increased to maintain 80% oxygen saturation. Nauplii of appropriate size (and fed on Isochrysis) are harvested on a 45 µm screen and by so doing cannibalism by the copepod adults is also minimized.
The scaling up of the operation to a production of 250,000 nauplii.day-1 usually requires three harvest tanks and a culture period of about two months.
All species investigated to date have several characteristics in common, including:
· high fecundity and short generation time· extreme tolerance limits to changes in environmental conditions: i.e. salinity ranges of 15-70 mg.g-1 and temperature ranges of 17-30°C.
· a large variety of foods can be administered to the cultures; rice bran or yeast even facilitating a higher production than algae
· potential to achieve high biomass densities: i.e. Tigriopus fed on rice bran increasing rapidly from 0.05 to 9.5 ind.ml-1 in 12 days
The culture can be started by isolating 10-100 gravid female copepods in 2 to 40 l of pure filtered (1 µm) seawater. The culture is then maintained at a density of at least one copepod per ml at a temperature of 24-26°C. No additional lighting is needed; if outdoor cultures are used, partial shading should be provided. The main culture tanks contain 500 l of filtered seawater (100 µm). Optimal culture densities are 20-70 copepods.ml-1, with a population growth rate of approximately 15%.day-1. Since high densities are used, it is advisable to use (semi) flow-through conditions instead of batch systems so as to avoid deterioration and eutrophication of the culture medium; the main problem here is the clogging of the fine-mesh screen. Food concentrations are maintained at 5.104 to 2.105 cells.ml-1 of Chaetoceros gracilis corresponding to a water transparency level of 7-10 cm. Faster growth and higher fecundity can be obtained by using dinoflagellates (Gymnodinium splendens) or flagellated green phytoplankton.
The generation time under optimal conditions is about 8-11 days at 24-26°C. E. acutifrons having 6 naupliar stages and 6 copepodite stages (including the adult); the newly hatched nauplii (N1) measuring 50 × 50 × 70 µm, and the copepodites C6 measuring 150 × 175 × 700 µm.
Before harvesting the copepods, the biomass and carrying capacity of the population must be calculated. To achieve this three samples of 2 ml should be taken daily and the different development stages counted under a binocular microscope. With these data the required harvest volume can therefore be estimated. N1 can be collected from the culture medium on a 37 µm sieve and separated from the other nauplii using a 70 µm sieve and the copepodites can be concentrated on a 100 µm screen.
With the exception of the culture of Tigriopus japonicus, copepod culture should always be free from rotifers. If rotifers should start to take over the culture, then a new stock culture should be started with gravid females as described previously. Check always for rotifers during sampling. In some cases, T. japonicus is batch cultured in combination with the rotifer Brachionus plicatilis (Fukusho, 1980) using baker’s yeast or Omega-yeast as a food source (although the cultures are always started with Chlorella algae). A bloom of this alga is first induced in big outdoor tanks which are subsequently seeded with rotifers and Tigriopus, at concentrations of 15-30 animals.l-1. In this way a total amount of 168 kg live weight of Tigriopus can be harvested during 89 days at maximal densities of 22,000 animals.l-1; the amount of yeast used for a 1 kg production of Tigriopus being 5 to 6 kg.
Many temperate copepods produce resting eggs as a common life-cycle strategy to survive adverse environmental conditions, which is analogous to Artemia and Brachionus sp. Experiments have shown that resting eggs can tolerate drying at 25°C or freezing down to -25°C and that they are able to resist low temperatures (3-5°C) for as long as 9 to 15 months. These characteristics make the eggs very attractive as inoculum for copepod cultures.
Since copepod resting eggs are generally obtained from sediments, they need to be processed prior to their use. Samples of sediments rich in resting eggs can be stored in a refrigator at 2-4°C for several months. When needed, the sediment containing the resting eggs is brought in suspension and sieved through 150 µm and 60 µm sieves. The size-fraction containing the resting eggs is then added to tubes containing a 1:1 solution of sucrose and distilled water (saturated solution) and centrifuged at 300 rpm for 5 min and the supernatants then washed through a double sieve of 100 µm and 40 µm. The 40 µm sieve with the resting eggs is then immersed in the disinfectant, (i.e. FAM-30 or Buffodine); surface-disinfection being needed to eliminate contaminating epibiotic micro-organisms. Successful experiments have been undertaken with the surface disinfection of resting eggs of Acartia clausi and Eurytemora affinis (Table 5.7.). After disinfection, the eggs are then washed with 0.2 µm filtered sterile seawater and transferred to disinfected culture tanks (see above) or stored under dark, dry and cool conditions.
Before starting the surface-disinfection procedure attention must be paid to the physiological type of resting eggs. Some marine calanoids are able to produce two kinds of resting eggs, i.e. subitanous and diapause eggs. Since subitanous eggs only have a thin vitelline coat covering the plasma membrane, they are more susceptive to disinfectants than the diapause eggs which are enveloped by a complex four-layer structure.
Table 5.7. Effect of various disinfectant procedures on hatching percentage, survival at day 5, and percentage of eggs on which bacterial growth was found after 6 weeks for Acartia clausi and Eurytemora affinis (modified from Naess & Bergh, 1994).
|
|
Disinfectant |
Control |
|||
|
Glutardialdehyde |
FAM-30 |
Buffodine |
|||
|
Concentration |
250 mg.1-1(v/v) |
1% (v/v) |
1% (v/v) |
- |
|
|
Application time |
3 min |
10 min |
10 min |
10 min |
|
|
Hatching percentage (%) |
|||||
|
A. clausi |
95.8 |
95.8 |
100 |
100 |
|
|
E. affinis |
79.2 |
37.2 |
83.3 |
91.7 |
|
|
Survival at Day 5 (%) |
|||||
|
A. clausi |
0 |
78.3 |
70.8 |
79.2 |
|
|
E. affinis |
73.7 |
0 |
100 |
86.4 |
|
|
Bacterial growth (%) on culture media MB and TSB |
|||||
|
A.clausi |
MB |
16.7 |
16.7 |
54.2 |
100 |
|
|
TSB |
4.2 |
0 |
33.3 |
100 |
|
E. affinis |
MB |
8.3 |
20.8 |
25.0 |
100 |
|
|
TSB |
12.5 |
12.5 |
12.5 |
100 |
|
Glutardialdehyde from Merck (Germany) Fam-30 and Buffodine from Evab Vanodine (Preston, UK) |
|||||
Cultured copepods have been successfully used in the larviculture of various flatfish larvae. 30 days-old larvae of the mud dab were fed T. japonicus cultured on baker’s yeast or Omega-yeast, and showed excellent survival and growth rates (Fukusho et al., 1980). For turbot, Nellen et al. (1981) demonstrated that the larvae at startfeeding showed a preference for copepod nauplii over Brachionus plicatilis; after 14 days culture their feeding preference shifting towards adult copepods. The survival of the larvae was high (50%), and the fry reached 12 mg DW (17 mm TL) at day 26.
Kuhlmann et al. (1981) successfully used 7.5 to 10% harvests of 24 m³ Eurytemora cultures for feeding turbot larvae. Population densities after 4-6 weeks of culture approximated to several hundred adults and copepodites, and several thousand nauplii per litre. Despite these good results, these authors were not able to stabilize production at such levels or to develop a reliable method, and therefore had to add rotifers in addition to the copepod supply. Although the culture was not fully controlled, Kuhlmann et al. (1981) estimated the capacity of his 24 m³ copepod culture and came to the conclusion that this capacity should be sufficient to feed a batch of 4000 freshly-hatched turbot larvae until metamorphosis.
