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Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems
Aquaculture 230 (2004) 475 – 491 www.elsevier.com/locate/aqua-online
Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems Kirsten Engell-Sørensen a,*, Josianne G. Støttrup b, Martin Holmstrup c Department of Plankton Ecology, Bio/consult as, Johs. Ewalds Vej 42-44, DK-8320 A˚byhøj, Denmark b Department for Marine Ecology and Aquaculture, Danish Institute for Fisheries Research, Charlottenlund Castle, DK-2920 Charlottenlund, Denmark c Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, Postbox 314, DK-8600 Silkeborg, Denmark a
Received 6 February 2003; received in revised form 13 June 2003; accepted 13 June 2003
Abstract A low-technology rearing system was implemented for rearing juvenile flounder for stock enhancement in a Danish fjord, the Limfjord. Each year during 1996 – 2002, between 13,000 and 153,000 juveniles were reared from the yolk-sac stage until metamorphosis in outdoor ponds relying on phyto- and zooplankton blooms as their main food source. In contrast to other similar systems, the blooms in this system are closely monitored and, to a certain extent, regulated. The zooplankton blooms consisted mainly of calanoid copepods, dominated by the species Temora longicornis and Centropages hamatus. Most juveniles produced (>99.5%) were normally pigmented with average yearly survival rates from hatch to metamorphosis varying from 7 F 9% to 48 F 18%, lowest in the first years of production. D 2004 Elsevier B.V. All rights reserved. Keywords: Flatfish culture; Flounder; Platichthys flesus; Calanoid copepods
1. Introduction In nature, different stages of copepods constitute the majority of prey of marine fish larvae (Last, 1978a,b, 1979, 1980; Purcell and Grover, 1990). In larviculture, most marine fish larvae depend on live food for first feeding (Black and Pickering, 1998). Shortage of live food has been considered the main cause of mortality during the period of transition * Corresponding author. Tel.: +45-86-251811; fax: +45-86-258173. E-mail address: [email protected] (K. Engell-Sørensen). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/S0044-8486(03)00437-X
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from endogenous to external feeding (Kamler, 1992). Although the suitability of copepods as live feed for fish larvae is now well established, their use in aquaculture is not (Støttrup, 2000; Treece and Davis, 2000). Instead of copepods, the more readily available rotifers and Artemia nauplii are widely used. These are cultured in the hatcheries and enriched by lipids, minerals and vitamins to change their composition towards nutritionally more suitable live feeds (Black and Pickering, 1998). Copepods contain highly different levels of essential fatty acids as a percentage of total fatty acids than Artemia enriched with various additives such as Super Selco (INVE Aquaculture, Belgium), DHA Selco (INVE Aquaculture, Belgium) and spray dried microalgae (Evjemo and Olsen, 1997; Shields et al., 1999; Evjemo et al., in press; Helland et al., 2003). The percentage of essential fatty acids in the prey is important for larval fish development (Witt et al., 1984; Navarro et al., 1995; Shields et al., 1999) and copepods are considered to be superior to enriched Artemia as food for marine fish larvae in terms of survival and growth as well as normal pigmentation and eye development (Witt et al., 1984; Shields et al., 1999). In some cases, successful rearing of marine fish larvae on rotifers and Artemia has not been possible without a supplement of copepods (MangorJensen et al., 1998; Rimmer, 1998). Various abnormalities (e.g. malpigmentation) have been observed in species reared solely on enriched rotifers and/or Artemia nauplii (Black and Pickering, 1998; Mangor-Jensen et al., 1998). Also, stress tolerance during the juvenile stage can be related to the larval diet (Howell, 1997). Postrelease survival is a major attribute to successful stock enhancement. It is therefore important to ensure that the juveniles to be released are normally pigmented, have similar morphology and possess similar attributes and tolerances as in wild fish. The extensive system is a method that closely imitates the natural environment. In the present study, such a system was established for the production of European flounder fry involving a certain degree of control of the prey organisms. In Denmark, turbot and European flounder are successfully reared on copepods, but several other marine fish species may also be reared in similar systems. This paper aims at describing a semiextensive method for rearing juvenile flounder carried out over a 7-year period (1996 –2002). This system developed for rearing flounder is based on the concrete pond systems described by Paulsen (1985a,b) and Urup (1994) for rearing turbot. In these papers, the population dynamics of calanoid copepods in tanks (Paulsen 1985a,b) and large-scale production of calanoid copepods for rearing turbot (Urup 1994) are described. Details of this system diverging from other described systems are highlighted and the advantages and limitations discussed.
2. Materials and methods 2.1. Breeding facilities and broodstock The breeding facilities consist of an indoor controlled egg incubation section, outdoor ponds of approximately 1200 m3 (25 30 1.5– 2 m) for larval rearing and outdoor/ indoor facilities for on-growing (April to May).
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Adults caught by local fishermen were used for broodstock. From 1998 and onwards, the F1 generation reared to maturity at an age of 2 –3 years were also used. The adults were stripped and the eggs fertilised. Average fertilisation was approximately 80%, lower in the beginning and end of the breeding season. 2.2. Egg incubation The larvae were hatched in thermoregulated, recirculated, active charcoal and sand filtered water, sterilised by the use of UV radiation. The eggs were incubated in cylindrical incubators with a conical bottom, with top inflow, mid-water outflow and bottom air diffusers. This system is similar to that described elsewhere (e.g. Rappen et al., 1987). Salinity was adjusted to about 30 x , where eggs were floating. The larvae were transferred to production lagoons as newly hatched yolk-sac larvae. 2.3. Larval rearing Until 2001, three outdoor ponds were used for larval rearing and zooplankton production. From 2001 and onwards, the larval rearing facilities were expanded to six outdoor ponds. A filtering system (Hydrotech, Norway) provided fresh 40 Am filtered seawater from the Limfjord. Zooplankton could be filtered from the lagoon water and transferred between all lagoons as needed during the production. Zooplankton was fractionated in two fractions (50 –250 Am and particles larger than 250 Am) (UNIK filtersystems, Norway). Production took place in April, May and June each year. A phytoplankton bloom mimicking a natural bloom was desired and therefore the nutrient levels at the onset of the production was similar to those in the natural environment prior to a moderate plankton bloom (in Danish coastal waters several hundred Ag total nutrient-N l 1 and tens of Ag orthophospate-P l 1). Once the phytoplankton bloom had begun, precultured copepods were added to the ponds. The precultured copepods were allowed to grow over several generations before transfer to the larvae ponds to prevent problems with potential fish parasites having copepods as the intermediate host. In some cases, cultures were derived from resting eggs that had survived the winter in the rearing ponds. 2.4. Monitoring of nutrient levels During the initial years of rearing, data on nutrients (ammonia-N, nitrate-N, nitrite-N and orthophosphate-P) were obtained from local authorities bordering the Limfjord. During the later years of production, the same nutrients were monitored at the onset of pond production to make sure that the levels of ammonia, nitrate – nitrite and orthophosphate were sufficiently high to result in a moderate plankton bloom. 2.5. Monitoring of oxygen, temperature and salinity Oxygen and temperature were measured (Handymac II, Oxygard, Denmark) twice a day (early morning and mid afternoon) at two depths (2 – 3 cm above bottom, 10 cm below
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surface). Salinity was measured at the onset of production by use of a refraktometer (ATAGO, Japan). 2.6. Phyto- and zooplankton sampling and monitoring Mixed samples of phytoplankton, ciliates and mesozooplankton were taken from two fixed points and two depths (0.5 and 1 m below surface) in each production lagoon. Phytoplankton and ciliate species determination and biomass determination were done once, twice or three times a week. Mesozooplankton species, stage and concentration were monitored at least three times a week in each production lagoon. ¨ termo¨hl, Phytoplankton and ciliates were monitored by use of inverted microscopy (U 1958). Size parameters were determined and volume and carbon content calculated according to Edler (1979). Results were calculated using proprietary software based on standard methods for calculation of volume and biomass of phytoplankton (Olrik, 1991). Identification was done using 100 , 200 and 320 magnification. All algae, except for flagellates with a diameter less than 10 Am, were determined to species. Flagellates were grouped in size classes. Clearance rates were calculated according to Andersen (1988) using temperatureand volume-specific filtration rates of the different groups of mesozooplankton (Hansen et al., 1997).
Table 1 Yield of larvae in different batch productions year 1996 to year 2002 Year
Batch no.
No. of released larvae
Yield
Survival (%)
1996
1 2 1 2 3 1 2 1 2 1 2 1 2 3 4 1 2 3 4
100,000 100,000 100,000 100,000 70,000a 100,000 100,000 100,000 100,000 90,000 100,000 100,000 100,000 100,000 60,000 100,000 100,000 100,000 100,000
13,000 0 20,000 17,800 22,000 23,500 56,500 60,000 35,000 22,500 38,500 19,300 18,900 21,200 10,800 26,000 41,500 37,700 47,900
13 0 20 18 31 24 57 60 35 25 39 19 19 21 18 26 42 38 48
1997
1998 1999 2000 2001
2002
a
Yearly yield
Average survival (%)
13,000
6.5
59,800
22.1
80,000
40.0
95,000
47.5
61,000
32.1
70,200
19.5
153,100
38.3
Larvae produced from eggs where hatching was delayed due to decreased temperatures.
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2.7. Larval sampling and monitoring Flounder larvae were sampled approximately twice a week either in the corners of the lagoons using a beaker or with a 500 Am plankton net. Size parameters of live specimens were measured under a light microscope (notochord length, height and width). The mouth width, yolk-sac volume and development of eyes, gut and gut/stomach contents were also determined. Notochord length of flounder larvae, Platichthys flesus, was measured to the nearest 1/100 mm. Dry weight of metamorphosed flounder larvae were determined to nearest 1/100 mg after drying the larvae at 85 jC for 24 h. 2.8. Calculation of larval growth rate and consumption rates Dry weight of flounder larvae was calculated from length (mm) using a relationship between length and dry weight (mg) of 177 individual fish larvae, where dry weight = 0.009 length2.328 (nonlinear regression), R2 = 0.63, p < 0.001, n = 177. Growth rate ( G) related to day-degrees from hatch of the flounder larvae was estimated from the exponential equations: GL ¼ lnðLt Þ lnðL0 Þ=Dt GW ¼ lnðWt Þ lnðW0 Þ=Dt where Lt = length at time t (t measured in day-degrees); Wt = dry weight at time t (t measured in day-degrees); t = time measured in day-degrees (day jC) from hatch; W0 = dry weight at t = 0; L0 = length at t = 0; and SGR (%) = 100 [(expG)-1].
Fig. 1. Phytoplankton biomass during lagoon production of the second batch in 1998, distributed in different taxonomic groups.
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Fig. 2. Oxygen levels during lagoon production of the second batch in 1998.
Theoretical daily food consumption by the flounder larvae was calculated from agespecific growth rates for different prey sizes (nauplii with a length of 100 and 200 Am, copepodites with a length of 500 Am and copepods with a length of 1000 Am) using a
Fig. 3. Temperature levels during lagoon production of the second batch in 1998.
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production/consumption rate of 0.2. Consumption rate of a given prey was only calculated when the type of prey was actually found in the stomach/gut of flounder larvae of that length. 2.9. On-growth After 4– 6 weeks, depending on temperature, the juveniles were caught by slowly dragging a gillnet from one end of the pond towards the opposite, collecting the juveniles in a small area and transferring them from the pond to land-based (indoor or outdoor) 2-m3 tanks for on-growth. The juveniles were weaned from live
Fig. 4. Concentration levels of zooplankton during lagoon production of the second batch in 1998.
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food to dry food (Perla Marin Respons, Skretting, Italy) immediately after catch without the use of Artemia in the weaning period. The flounder were released in the Limfjord, when they had reached a length of 2, 3 – 4 or 10 – 15 cm, respectively.
3. Results During the years 1996 to 2002, a total of 533,000 juveniles were produced. The yearly production figures are given in Table 1. Most juveniles were successfully weaned. More than 95% were weaned directly to a dry food diet. In the following, results from the second batch of fish during 1998 are given as an example of a relatively good production run. This particular production was initiated on April 6, 1998 (day 0) and metamorphosed larvae were caught on June 12, 1998 (day 36). Dynamics of the phytoplankton through the production of the second batch in 1998 are shown in Fig. 1. The initial phytoplankton bloom was not very strong. Phytoplankton was dominated by small unidentified flagellates during most of the production period. Cryptophyceae were very abundant in the first weeks of production. Later on, during the last weeks of production a bloom of diatoms (Bacillariophyceae) and Haptophyceae occurred. Oxygen levels were never below saturation (Fig. 2). Through the entire production oxygen levels were lowest in the morning and highest in the afternoon. Oxygen levels were generally lower in the surface waters than the bottom waters and were highest during the initial plankton bloom in the first days of production and also during the bloom of phytoplankton in the last two weeks of production.
Fig. 5. Clearance pressure from different zooplankton groups during the production period of the second batch from 1998.
Table 2 Development of flounder larvae and contents of stomach and gut contents during the production period of the second batch in 1998 Day-degrees
Mean (F Std.) larval length [mm]
Mean (F Std.) yolk-sac volume [mm3]
Mean (F Std.) mouth width [mm]
n
Development of digestive tract
Eyes
Stomach and gut contents (number of prey items per larvae)
Eyes white Eyes light brown Iris developed in some, not in others Iris developed Iris developed
Empty Empty
– –
Empty
–
0 4
0 28
2.98 F 0.17 3.95 F 0.21
0.140 F 0.010 0.139 F 0.054
0.00 F 0.00 0.00 F 0.00
9 9
Anus open Gut straight
6
40
4.06 F 0.18
0.051 F 0.037
0.02 F 0.05
8
Gut straight
10 12
73 93
4.39 F 0.30 4.89 F 0.32
0.008 F 0.012 0
0.19 F 0.07 0.26 F 0.04
5 7
Gut straight Gut straight with bulge
24
242
7.75 F 0.07
0
0.50 F 0.00
2
Gut-winded, metamorphosed
34
387
10.26 F 0.03
0
0.67 F 0.05
10
42
527
9.91 F 0.93
0
–
20
Empty Phytoplankton, chlorophyll remains 0.5 copepod nauplii 3.5 copepod nauplii, 0.5 copepodite and 5.5 podon 21.7 copepod nauplii, 0.3 barnacle nauplii, 3 harp. copepods, 5 calanoid copepods, 0.8 copepod eggs, 0.1 Artemia
Length of prey [mm]
– 0.005 – 0.2
0.10 – 0.55
0.09 – 1.29
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Days from hatch
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Temperatures were generally rising through the whole production period (Fig. 3). Temperature increased from about 6 jC at the outset of the production to about 20 jC at the time of fry harvest. Fig. 4 shows the development of zooplankton through the production period. Adult copepods (less than 2 l 1) were introduced to the lagoon in the very beginning of the production along with some nauplii (less than 20 l 1). After a few days, the adult females started to produce eggs. The eggs hatched and the concentration of nauplii increased to maximum levels (approximately 160 l 1) two weeks after hatching of the flounder larvae. The concentration of copepodites remained low (below 6 l 1) through the whole production. Until day 20, mostly Temora longicornis were found among the adult copepods. From this day onwards Centropages hamatus became abundant (10 –50%). Acartia spp. were observed from day 33 ( < 15%). Total mesozooplankton clearance pressure did not exceed 1 per day through the whole production period (Fig. 5), indicating that the mesozooplankton never filtered the water more than once per day. Although the nauplii were by far the most dominating stage of copepods, the nauplii were not responsible for more than at the most 80% of the total mesozooplankton clearance due to the smaller size of the nauplii. Flounder larvae were introduced to the lagoon as yolk-sac larvae. During the production period, larvae from the lagoon were caught and examined. These results are shown in Table 2. Data on length of flounder larvae as a function of day-degrees from hatch (n = 753) for all batch productions from 1996 to 2002 are compiled in Fig. 6. A linear relationship (length = 0.0175 day-degrees + 3.0875) exists between day-degrees from hatch and length of larvae.
Fig. 6. Total length of larvae as a function of day-degrees from hatch for all productions from 1996 to 2002 (n = 753).
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Fig. 7. Weight of larvae as a function of day-degrees from hatch for all productions from 1996 to 2002 (n = 753).
Dry weight of flounder larvae plotted against number of day-degrees from hatch (n = 753) for all batch productions from 1996 to 2002 are shown in Fig. 7. Dry weight as a function of day-degrees can be described by nonlinear regression, where dry weight = 0.715 day-degrees0.0056. The temperature range during productions was 3 –23 jC. The type and size of prey caught by the larvae depended on the size of the larvae. Fig. 8 shows the type of prey found in the digestive tract of the larvae (n>300) through the productions in year 1996 to year 2002. If the specific growth rate of the larvae is calculated for different periods of larvae life (measured in day-degrees from hatch), theoretical consumption of the flounder larvae can
Fig. 8. Prey of different sizes of flounder larvae from productions year 1996 to 2002 (n>300).
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Table 3 Calculated consumption of different prey (number of prey per day per day-degree) Production period [day jC]
Length [mm]
SGR (length) [% day jC 1]
SGR (dry weight) [% day jC 1]
Consumed 100 Am nauplii [day jC 1]
Consumed copepodites [day jC 1]
Consumed copepods [day jC 1]
0 – 100 100 – 200 200 – 300 300 – 400 400 – 500 500 – 600 600 – 700
3.1 – 4.8 4.8 – 6.6 6.6 – 8.3 8.3 – 10.1 10.1 – 11.8 11.8 – 13.6 13.6 – 15.3
0.45 0.31 0.24 0.19 0.16 0.14 0.12
0.56 0.56 0.56 0.56 0.56 0.56 0.56
– 48 64 84 111 147 195
– 10 14 18 24 31 42
– – – 7 10 13 17
SGR = specific growth rate. It is assumed that (1) the larvae consumes only one type of prey at a time. (2) 20% of the consumed energy is used for growth. (3) Nauplii with a length of 100 Am has a dry weight of 0.64 Ag, a copepodite has a dry weight of 3 Ag, and a copepod has a dry weight of 7.5 Ag. Values are average values from Danish coastal waters obtained during several years (Bio/consult, unpublished data).
be calculated. Table 3 shows the theoretical consumption of different sizes of prey. The size distribution used for this calculation reflected that found in the digestive tract of flounder larvae of the same size (Fig. 8). Table 3 can be used to calculate daily needs of prey of the flounder larvae. For example, if a larvae of a length of 6.6 –8.3 mm, 200 –300 day-degrees from hatch should fulfil its metabolic needs at 20 jC entirely on nauplii with a length of 100 A, it would have to consume 20 64 nauplii day 1 = 1280 nauplii day 1. At 10 jC, the same larvae would need only 640 nauplii day 1.
4. Discussion The present study has shown that successful larval survival depends on a good timing between the consumption needs of the larvae and the onset/magnitude of microalgae bloom and the copepod nauplii production. The first food items ingested after the yolk-sac stage were microalgae (Table 2; Fig. 8). These food items were ingested for three to five days prior to a switch to copepod nauplii. The presence of microalgae in the guts of marine fish larvae prior to feeding on zooplankton has been previously observed in other species of flatfish and is reportedly important for their subsequent growth and survival (Last 1978a; Reitan et al., 1991). Calculated consumption needs (Table 3) suggest that larvae must search for food at a high rate. For example, at the time during and just after first feeding, between 100 and 200 day-degrees from hatch, a larva must consume 480 nauplii day 1 at 10 jC to fulfil its requirements for optimal growth and would have to capture and eat one nauplii every second minute during a 16-h daylight period. Although this may seem a high search rate, it is consistent with observations of individual flounder larvae. In the period just before catch, 600 – 700 day-degrees from hatch, assuming a temperature of 20 jC, the necessary number of prey has risen to 3900 nauplii, 840 copepodites or 340 copepods day 1 (Table 3). Assuming that a larva at this time preys
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solely on copepodites and that it can hunt for food 16 h day 1, the larva must capture and eat one copepodite every 1.1 min. Assuming that the copepodites are grazed to a level where life of the larvae cannot be sustained, the larvae will capture and eat prey with a higher frequency (increase its swimming activity) and decrease its prey-size selectivity (Munk, 1995). If the same larvae had to depend solely on nauplii with a size of 100 Am, it would have to eat 1 nauplius every 15 s. When estimating the minimum density of food items to fulfil the requirements for optimal growth of the flounder larvae, food search potential (Blaxter and Staines, 1971), feeding success (Blaxter and Staines, 1971), competition between larvae (e.g. density of fish larvae), prey size, temperature and possible patchy distribution of fish larvae and prey (Paulsen, 1985a) have to be taken into consideration. The fact that the flounder eggs are fertilised 10 to 20 days before the flounder larvae will start to eat nauplii necessitates a good control of peak occurrence of suitable food items. Thus, the main feature of the system described here is timing the onset of a new generation of copepod nauplii with first-feeding fish larvae. Newly hatched larvae are transferred to the outdoor ponds at a time when the zooplankton population has reached copepodite/adult copepod stage. By the time they are ready to start ingesting live prey, about a week later (depending on temperature), the new generation of copepod nauplii must be developing to ensure enough food for the fish larvae. As the flounder larvae grow and develop in the lagoons, the nauplii that were not eaten develop into larger stages, thereby meeting the prey-size requirements of the growing flounder larvae. A high concentration of prey in the system is not necessarily the best because the zooplankton may graze down the microalgae in the water column (Fig. 5). The clearance rate depends on temperature and concentration of microalgae (Kiørboe et al., 1982; Støttrup and Jensen, 1990; Mauchline, 1998). If the total clearance rate is too high, the microalgae may be grazed to a level that is insufficient for survival of the copepods causing a collapse of the copepod culture. Our experience suggests that zooplankton clearance, without addition of microalgae biomass to the system, should be kept below 1 to prevent a collapse of the zooplankton culture. Grazing by copepods will shift the microalgae community towards flagellates, which are not effectively grazed by the different stages of copepods (small flagellates with a diameter less than 2 –4 Am). It is therefore useful to monitor the microalgae community by microscopy (species and concentration) during a production. This will also make it possible to give an early warning of a possible harmful microalgal bloom. The overall survival rate of larvae in the described production system was 30% over the 7 years of the study. Moreover, less than 0.5% malpigmentation was observed and no other developmental abnormalities. Improved survival rates from year 1996 to year 1998 were observed, mainly due to an adjustment of the semiextensive method (e.g. timing between the development of the flounder larvae and the onset of phytoplankton bloom and egg production of the copepods). From 1999 to 2002, the average survival rates remained high except in 2001, where survival were lower due to unusually cold weather in the early spring and thereby a slow development of copepods. The growth rates of flounder reported here were similar to growth rates found for reared cod, winter flounder and silver hake (Buckley et al., 1993) or less than half of recorded growth rates of turbot larvae (Paulsen, 1985b).
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To utilise cultured calanoid copepods by the semiextensive method, it is necessary to have large, outdoor water volumes of filtered seawater. Additional filter and pumping systems are needed to fractionate and distribute different stages of copepods during the production. A culture of calanoid copepods may be initiated using calanoid copepods from surrounding seawater. This assures that the cultured copepods are well adapted to the water used to breed the copepods. Several generations of copepods should be bred before the copepods are used as food for marine fish larvae to ensure that there is no transfer of parasites from the copepods to the fish. Different species of copepods can be used. Most important is the production potential of the specific copepod species. Also important is the size of the nauplii or adult. Adult copepods in good condition (well fed with microalgae) are introduced into the lagoons or tanks. If the algal biomass is sufficiently high, then females will start to produce eggs. The egg production rate is controlled by a number of factors, the principal one being the availability of food (Mauchline, 1998). The development time of the eggs to hatching is species specific, but mainly depends on temperature (Mauchline, 1998). One thousand to five thousand nauplii per litre can be produced per generation under optimal conditions. Several successful attempts have been made to culture marine fish larvae on copepods (for review, see Van der Meeren and Naas, 1997; Støttrup et al., 1998; Shields, 2001). During the last 20 years, Norwegian groups have used enclosed pond systems to produce marine fish larvae. The method is traditionally called the ‘‘extensive’’ Norwegian method. The ponds are operated to maintain a high production of zooplankton used for larval fish. Most often, the ponds were treated with rotenone to kill all predators of fish larvae. After the treatment a bloom of copepods would occur in the early spring, mainly as a result of hatching of copepod resting eggs (Næss, 1996) and a bloom of phytoplankton that might be reinforced by the use of fertilizers. The main differences between the system described here and the Norwegian system are: (1) the production is performed in closed, PVC coated ponds that can be cleaned mechanically and treated by disinfectants or simply dried out; (2) the copepod culture is controlled to some degree through culture of several generations of copepods before onset of the larvae production, which will facilitate the availability of copepods of the right stage and condition; and (3) most external parameters that might influence the ecosystem are eliminated and, except for the weather conditions and species of algae in the water filtered from the Limfjord, controlled. This makes the production results more predictable. Nutrients can be added to the system, but this has to be done with great care because excessive phytoplankton that is not exploited by the zooplankton will eventually sink to the bottom and the energy of the algae is thereby most likely lost because the algal biomass has to be mineralised before the nutrients return to the food web. The flow of energy, carbon and nutrients out of the pelagic system is likely to result in a suboptimal production of fish larvae as well as increasing the risk of harmful algal production, oxygen depletion and hydrogen sulphide production. The season can be a limiting factor in temperate or arctic climates. The nauplii production will slowdown in the winter season, especially with decreasing temperatures, because most calanoid copepods tend to produce diapause eggs during periods of decreasing environmental temperatures. Temperature is the primary controlling factor of
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dormancy of diapause eggs, which is exploited when keeping the culture over winter. Low oxygen concentrations can delay embryonic development (Mauchline, 1998). However, diapause eggs are able to survive both low oxygen concentrations and sulphide (Marcus, 1996; Marcus and Lutz, 1998). In Denmark, turbot and European flounder are successfully reared on copepods. Similar systems can probably be used for production of all species of marine fish that depend on different stages of copepods as larval prey. This includes almost all marine fish (Last, 1978a,b, 1979, 1980; Purcell and Grover 1990). Artemia are widely used as a prey item in rearing of marine fish larvae. However, the market price of Artemia has increased considerably in the past years, mainly as a result of reduced catches of some of the natural sources of Artemia. Although the efforts to harvest Artemia have increased, the actual harvest of Artemia has not (Anonymous, 2000, 2002). Therefore, it seems important to improve the availability of prey such as copepods that will constitute an alternative to Artemia. We suggest that the semiextensive method involving copepods provides a good solution to solve the need for live prey for marine fish larvae production. After adjusting the method for a desired fish species, all elements of the production method can be carried out by experienced aquaculturists with some understanding of plankton ecology.
Acknowledgements We sincerely thank the many persons involved for their invaluable support and efforts. Especially we thank Ole Borbjerggaard and Hans Svanborg for their dedicated work. We also thank members of Nordvestjydsk Fritidsfiskerforening, especially Kay Hansen, employees at Venø Fish Farm and colleagues at Bio/consult and the Danish Institute for Fisheries Research. The project was funded by the Danish Marine Coastal Fisheries Management Programme.
References Andersen, P., 1988. The quantitative importance of the ‘‘microbial loop’’ in the marine pelagic: a case study from the North Bering/Chukchi seas. Arch. Hydrobiol. Beih. 31, 243 – 251. Anonymous, 2000. Historical brine shrimp harvest from the Great Salt Lake. Fish Farming Int. 27 (1), 16 – 21. Anonymous, 2002. Artemia harvest ‘‘terrible’’. Fish Farming Int. 29 (11), 1. Black, K.D., Pickering, A.P., 1998. Biology of Farmed Fish. Sheffield Academic Press, Sheffield, UK. Blaxter, J.H.S., Staines, M.E., 1971. Food searching potential in marine fish larvae. Proc. 4th European Marine Biol., 467 – 485. Buckley, L.J., Smigielski, A.S., Halavik, T.A., Burns, B.R., Lawrence, G.C., 1993. Growth and survival of three temperate marine fish species at discrete prey densities: II. Cod (Gadus morhua), winter flounder (Pseudoopleuronectus americanus) and silver hake (Merluccius biliniaris). In: Walter, B.T., Fyhn, H.J. (Eds.), Physiological and Biochemical Aspects of Fish Development. University of Bergen, Zoological Institute, Grafisk hus, Bergen. Edler, L., 1979. Recommendations on methods for marine biological studies in the Baltic Sea. Phytoplankton and Chlorophyll. Baltic Mar. Biol., vol. 5. Dept. of Marine Botany, Univ. of Lund, Sweden. Evjemo, J.O., Olsen, Y., 1997. Lipid and fatty acid content in cultivated live feed organisms compared to marine copepods. Hydrobiologia 358, 159 – 162.
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Evjemo, J.O., Reitan, K.I., Olsen, Y., in press. Copepods as a food source in first feeding of marine fish larvae. In: Hendry, C.I., Van Stappen, G., Wille, M., Sorgeloos, P. (Eds.), Larvi 2001—Fish and Shellfish Larviculture Symposium. Spec. Publ. - Eur. Aquac. Soc., vol. 30. Ghent, Belgium. Hansen, P.J., Bjørnsen, P.K., Hansen, B.W., 1997. Zooplankton grazing and growth: scaling within the 2 – 2000Am body size range. Limnol. Oceanogr. 42, 687 – 704. Helland, S., Terjesen, B.F., Berg, L., 2003. Free amino acids and protein content in the planktonic copepod Temora longicornis compared to Artemia franciscana. Aquaculture 215, 213 – 228. Howell, B.R., 1997. A re-appraisal of the potential of the sole, Solea solea (L.) for commercial cultivation. Aquaculture 155, 355 – 365. Kamler, E., 1992. Early Life History of Fish. An Energetics Approach. Fish Fish. Ser., vol. 4. Chapman & Hall, London, England. Kiørboe, T., Møhlenberg, F., Nicolajsen, H., 1982. Ingestion rate and gut clearance in the planktonic copepod Centropages hamatus (Lilljeborg) in relation to food concentration and temperature. Ophelia 21 (2), 191 – 194. Last, J.M., 1978a. The food of four species of pleuronectiform larvae in the Eastern English Channel and Southern North Sea. Mar. Biol. 45, 359 – 368. Last, J.M., 1978b. The food of three species of gadoid larvae in the Eastern English Channel and Southern North Sea. Mar. Biol. 48, 377 – 386. Last, J.M., 1979. The food of larval turbot Scophthalmus maximus L. from the west central North Sea. J. Cons. Int. Explor. Mer. 38 (3), 308 – 313. Last, J.M., 1980. The food of twenty species of fish larvae in the west-central North Sea. Fish. Res. Tech. Rep., vol. 60. Ministry of Aquaculture, Fisheries and Food, Directorate of Fisheries Research, Lowestoft, England. Mangor-Jensen, A., Harboe, T., Shields, R.J., Gara, B., Naas, K.E., 1998. Atlantic halibut, Hippoglossus hippoglossus L., larvae cultivation literature, including bibliography. Aquac. Res. 29, 857 – 886. Marcus, N.H., 1996. Ecological and evolutional significance of resting eggs in marine copepods: past, present and future studies. Hydrobiologia 320, 141 – 152. Marcus, N.H., Lutz, R.V., 1998. Longevity of subitaneous and diapause eggs of Centropages hamatus (Copepoda: Calanoida) from the northern Gulf of Mexico. Mar. Biol. 1333 (2), 249 – 257. Mauchline, J., 1998. The biology of calanoid copepods. Advances in Marine Biology. Academic Press, San Diego. Munk, P., 1995. Foraging behaviour of larval cod (Gadus morhua) influenced by prey density and hunger. Mar. Biol. 122, 205 – 212. Næss, T., 1996. Benthic resting eggs of calanoid copepods in Norwegian enclosures in mariculture: abundance, species composition and hatching. Hydrobiologia 320, 161 – 168. Navarro, J.C., McEvoy, L.A., Bell, M.V-., Amat, F., Hontoria, F., Sargent, J.R., 1995. Effects of dietary lipids on the lipid composition of fish larvae eyes. In: Lavens, P., Jaspers, Roelants, E. (Eds.), Larvi 95. Fish and Shellfish Larviculture Symposium. Spec. Publ. - Eur. Soc., vol. 24, pp. 196 – 199. European Aquac. Soc., Ghent, Belgium. Olrik, K., 1991. Phytoplankton—Methods. Ministry of the Environment, Denmark, pp. 1 – 108. Danish Environmental Protection Agency, Project No. 187 (In Danish). Paulsen, H., 1985a. Effects on Food Patches on Distribution, Growth and Survival of Fish Larvae—A Summary of Experimental Results. ICES, Copenhagen, Denmark. ICES C.M. 1985/L: 22. Paulsen, H., 1985b. Extensive Rearing of Turbot Larvae (Scophthalmus maximus L.) on Low Concentrations of Natural Plankton. ICES, Copenhagen, Denmark. ICES C.M. 1985/L: 33. Purcell, J.E., Grover, J.J., 1990. Predation and food limitation as causes of mortality in larval herring at a spawning ground in British Columbia. Mar. Ecol., Prog. Ser. 59, 55 – 61. Rappen, H., Jelmert, A., Huse, I., 1987. Production Experiment of Halibut Fry (Hippoglossus hippoglossus L.) in Silos. International Council for the Exploration for the Sea, Copenhagen. C.M. 1986/F: 42. Reitan, K.I., Bolla, S., Olsen, Y., 1991. Ingestion and assimilation of microalgae in yolk-sac larvae of halibut Hippoglossus hippoglossus. Spec. Publ. - Eur. Aquac. Soc. 15, 332 – 334. Ghent, Belgium. Rimmer, M., 1998. Grouper and snapper aquaculture in Taiwan. Austasia Aquac. 12 (1), 3 – 7. Shields, R.J., 2001. Larviculture of marine finfish in Europe. Aquaculture 200, 55 – 88. Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R., Sargent, R.J., 1999. Natural copepods are superior to enriched Artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of
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survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr. 129 (6), 1186 – 1196. Støttrup, J.G., 2000. The elusive copepods: their production and suitability in marine aquaculture. Aquac. Res. 31, 703 – 711. Støttrup, J.G., Jensen, J., 1990. Influence of algal diet on feeding and egg-production of the calanoid copepod Acartia tonsa Dana. J. Exp. Mar. Biol. Ecol. 141, 87 – 105. Støttrup, J.G., Shields, R., Gillispie, M., Gara, M.B., Sargent, J.R., Bell, J.G., Henderson, R.J., Tocher, D.R., Sutherland, R., Næss, T., Mangor Jensen, A., Naas, K., Van der Meeren, T., Harboe, T., Sanchez, F.J., Sorgeloos, P., Dhert, P., Fitzgerald, R., 1998. The production and use of copepods in larval rearing of halibut, turbot and cod. Bull. Aquac. Assoc. Can. 4, 41 – 46. Treece, G.D., Davis, D.A., 2000. Culture of small zooplanktons for the feeding of larval fish. SRAC Publ. 701. 7 pp. Urup, B., 1994. Methods for the production of turbot fry using copepods as food. In: Lavens, P., Remmerswaal, R.A.M. (Eds.), Turbot Culture: Problems and Prospects. Spec. Publ. - Eur. Aquac. Soc., vol. 22. European Aquac. Soc., Ghent, Belgium, pp. 47 – 53. ¨ termo¨hl, H., 1958. Zur vervollkomnung der quantitativen Phytoplankton Metodik. Mitt. Int. Ver. Limnol. 9, U 1 – 38. Van der Meeren, T., Naas, K.E., 1997. Development of rearing techniques using large enclosed ecosystems in the mass production of marine fish fry. Rev. Fish. Sci. 5 (4), 367 – 390. Witt, U., Quantz, G., Kuhlmann, D., Kattner, G., 1984. Survival and growth of turbot larvae Scophthalmus maximus reared on different food organisms with special regard to long chain PUFA. Aquac. Eng. 3, 177 – 190.
Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems Kirsten Engell-Sørensen a,*, Josianne G. Støttrup b, Martin Holmstrup c Department of Plankton Ecology, Bio/consult as, Johs. Ewalds Vej 42-44, DK-8320 A˚byhøj, Denmark b Department for Marine Ecology and Aquaculture, Danish Institute for Fisheries Research, Charlottenlund Castle, DK-2920 Charlottenlund, Denmark c Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, Postbox 314, DK-8600 Silkeborg, Denmark a
Received 6 February 2003; received in revised form 13 June 2003; accepted 13 June 2003
Abstract A low-technology rearing system was implemented for rearing juvenile flounder for stock enhancement in a Danish fjord, the Limfjord. Each year during 1996 – 2002, between 13,000 and 153,000 juveniles were reared from the yolk-sac stage until metamorphosis in outdoor ponds relying on phyto- and zooplankton blooms as their main food source. In contrast to other similar systems, the blooms in this system are closely monitored and, to a certain extent, regulated. The zooplankton blooms consisted mainly of calanoid copepods, dominated by the species Temora longicornis and Centropages hamatus. Most juveniles produced (>99.5%) were normally pigmented with average yearly survival rates from hatch to metamorphosis varying from 7 F 9% to 48 F 18%, lowest in the first years of production. D 2004 Elsevier B.V. All rights reserved. Keywords: Flatfish culture; Flounder; Platichthys flesus; Calanoid copepods
1. Introduction In nature, different stages of copepods constitute the majority of prey of marine fish larvae (Last, 1978a,b, 1979, 1980; Purcell and Grover, 1990). In larviculture, most marine fish larvae depend on live food for first feeding (Black and Pickering, 1998). Shortage of live food has been considered the main cause of mortality during the period of transition * Corresponding author. Tel.: +45-86-251811; fax: +45-86-258173. E-mail address: [email protected] (K. Engell-Sørensen). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/S0044-8486(03)00437-X
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from endogenous to external feeding (Kamler, 1992). Although the suitability of copepods as live feed for fish larvae is now well established, their use in aquaculture is not (Støttrup, 2000; Treece and Davis, 2000). Instead of copepods, the more readily available rotifers and Artemia nauplii are widely used. These are cultured in the hatcheries and enriched by lipids, minerals and vitamins to change their composition towards nutritionally more suitable live feeds (Black and Pickering, 1998). Copepods contain highly different levels of essential fatty acids as a percentage of total fatty acids than Artemia enriched with various additives such as Super Selco (INVE Aquaculture, Belgium), DHA Selco (INVE Aquaculture, Belgium) and spray dried microalgae (Evjemo and Olsen, 1997; Shields et al., 1999; Evjemo et al., in press; Helland et al., 2003). The percentage of essential fatty acids in the prey is important for larval fish development (Witt et al., 1984; Navarro et al., 1995; Shields et al., 1999) and copepods are considered to be superior to enriched Artemia as food for marine fish larvae in terms of survival and growth as well as normal pigmentation and eye development (Witt et al., 1984; Shields et al., 1999). In some cases, successful rearing of marine fish larvae on rotifers and Artemia has not been possible without a supplement of copepods (MangorJensen et al., 1998; Rimmer, 1998). Various abnormalities (e.g. malpigmentation) have been observed in species reared solely on enriched rotifers and/or Artemia nauplii (Black and Pickering, 1998; Mangor-Jensen et al., 1998). Also, stress tolerance during the juvenile stage can be related to the larval diet (Howell, 1997). Postrelease survival is a major attribute to successful stock enhancement. It is therefore important to ensure that the juveniles to be released are normally pigmented, have similar morphology and possess similar attributes and tolerances as in wild fish. The extensive system is a method that closely imitates the natural environment. In the present study, such a system was established for the production of European flounder fry involving a certain degree of control of the prey organisms. In Denmark, turbot and European flounder are successfully reared on copepods, but several other marine fish species may also be reared in similar systems. This paper aims at describing a semiextensive method for rearing juvenile flounder carried out over a 7-year period (1996 –2002). This system developed for rearing flounder is based on the concrete pond systems described by Paulsen (1985a,b) and Urup (1994) for rearing turbot. In these papers, the population dynamics of calanoid copepods in tanks (Paulsen 1985a,b) and large-scale production of calanoid copepods for rearing turbot (Urup 1994) are described. Details of this system diverging from other described systems are highlighted and the advantages and limitations discussed.
2. Materials and methods 2.1. Breeding facilities and broodstock The breeding facilities consist of an indoor controlled egg incubation section, outdoor ponds of approximately 1200 m3 (25 30 1.5– 2 m) for larval rearing and outdoor/ indoor facilities for on-growing (April to May).
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Adults caught by local fishermen were used for broodstock. From 1998 and onwards, the F1 generation reared to maturity at an age of 2 –3 years were also used. The adults were stripped and the eggs fertilised. Average fertilisation was approximately 80%, lower in the beginning and end of the breeding season. 2.2. Egg incubation The larvae were hatched in thermoregulated, recirculated, active charcoal and sand filtered water, sterilised by the use of UV radiation. The eggs were incubated in cylindrical incubators with a conical bottom, with top inflow, mid-water outflow and bottom air diffusers. This system is similar to that described elsewhere (e.g. Rappen et al., 1987). Salinity was adjusted to about 30 x , where eggs were floating. The larvae were transferred to production lagoons as newly hatched yolk-sac larvae. 2.3. Larval rearing Until 2001, three outdoor ponds were used for larval rearing and zooplankton production. From 2001 and onwards, the larval rearing facilities were expanded to six outdoor ponds. A filtering system (Hydrotech, Norway) provided fresh 40 Am filtered seawater from the Limfjord. Zooplankton could be filtered from the lagoon water and transferred between all lagoons as needed during the production. Zooplankton was fractionated in two fractions (50 –250 Am and particles larger than 250 Am) (UNIK filtersystems, Norway). Production took place in April, May and June each year. A phytoplankton bloom mimicking a natural bloom was desired and therefore the nutrient levels at the onset of the production was similar to those in the natural environment prior to a moderate plankton bloom (in Danish coastal waters several hundred Ag total nutrient-N l 1 and tens of Ag orthophospate-P l 1). Once the phytoplankton bloom had begun, precultured copepods were added to the ponds. The precultured copepods were allowed to grow over several generations before transfer to the larvae ponds to prevent problems with potential fish parasites having copepods as the intermediate host. In some cases, cultures were derived from resting eggs that had survived the winter in the rearing ponds. 2.4. Monitoring of nutrient levels During the initial years of rearing, data on nutrients (ammonia-N, nitrate-N, nitrite-N and orthophosphate-P) were obtained from local authorities bordering the Limfjord. During the later years of production, the same nutrients were monitored at the onset of pond production to make sure that the levels of ammonia, nitrate – nitrite and orthophosphate were sufficiently high to result in a moderate plankton bloom. 2.5. Monitoring of oxygen, temperature and salinity Oxygen and temperature were measured (Handymac II, Oxygard, Denmark) twice a day (early morning and mid afternoon) at two depths (2 – 3 cm above bottom, 10 cm below
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surface). Salinity was measured at the onset of production by use of a refraktometer (ATAGO, Japan). 2.6. Phyto- and zooplankton sampling and monitoring Mixed samples of phytoplankton, ciliates and mesozooplankton were taken from two fixed points and two depths (0.5 and 1 m below surface) in each production lagoon. Phytoplankton and ciliate species determination and biomass determination were done once, twice or three times a week. Mesozooplankton species, stage and concentration were monitored at least three times a week in each production lagoon. ¨ termo¨hl, Phytoplankton and ciliates were monitored by use of inverted microscopy (U 1958). Size parameters were determined and volume and carbon content calculated according to Edler (1979). Results were calculated using proprietary software based on standard methods for calculation of volume and biomass of phytoplankton (Olrik, 1991). Identification was done using 100 , 200 and 320 magnification. All algae, except for flagellates with a diameter less than 10 Am, were determined to species. Flagellates were grouped in size classes. Clearance rates were calculated according to Andersen (1988) using temperatureand volume-specific filtration rates of the different groups of mesozooplankton (Hansen et al., 1997).
Table 1 Yield of larvae in different batch productions year 1996 to year 2002 Year
Batch no.
No. of released larvae
Yield
Survival (%)
1996
1 2 1 2 3 1 2 1 2 1 2 1 2 3 4 1 2 3 4
100,000 100,000 100,000 100,000 70,000a 100,000 100,000 100,000 100,000 90,000 100,000 100,000 100,000 100,000 60,000 100,000 100,000 100,000 100,000
13,000 0 20,000 17,800 22,000 23,500 56,500 60,000 35,000 22,500 38,500 19,300 18,900 21,200 10,800 26,000 41,500 37,700 47,900
13 0 20 18 31 24 57 60 35 25 39 19 19 21 18 26 42 38 48
1997
1998 1999 2000 2001
2002
a
Yearly yield
Average survival (%)
13,000
6.5
59,800
22.1
80,000
40.0
95,000
47.5
61,000
32.1
70,200
19.5
153,100
38.3
Larvae produced from eggs where hatching was delayed due to decreased temperatures.
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2.7. Larval sampling and monitoring Flounder larvae were sampled approximately twice a week either in the corners of the lagoons using a beaker or with a 500 Am plankton net. Size parameters of live specimens were measured under a light microscope (notochord length, height and width). The mouth width, yolk-sac volume and development of eyes, gut and gut/stomach contents were also determined. Notochord length of flounder larvae, Platichthys flesus, was measured to the nearest 1/100 mm. Dry weight of metamorphosed flounder larvae were determined to nearest 1/100 mg after drying the larvae at 85 jC for 24 h. 2.8. Calculation of larval growth rate and consumption rates Dry weight of flounder larvae was calculated from length (mm) using a relationship between length and dry weight (mg) of 177 individual fish larvae, where dry weight = 0.009 length2.328 (nonlinear regression), R2 = 0.63, p < 0.001, n = 177. Growth rate ( G) related to day-degrees from hatch of the flounder larvae was estimated from the exponential equations: GL ¼ lnðLt Þ lnðL0 Þ=Dt GW ¼ lnðWt Þ lnðW0 Þ=Dt where Lt = length at time t (t measured in day-degrees); Wt = dry weight at time t (t measured in day-degrees); t = time measured in day-degrees (day jC) from hatch; W0 = dry weight at t = 0; L0 = length at t = 0; and SGR (%) = 100 [(expG)-1].
Fig. 1. Phytoplankton biomass during lagoon production of the second batch in 1998, distributed in different taxonomic groups.
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Fig. 2. Oxygen levels during lagoon production of the second batch in 1998.
Theoretical daily food consumption by the flounder larvae was calculated from agespecific growth rates for different prey sizes (nauplii with a length of 100 and 200 Am, copepodites with a length of 500 Am and copepods with a length of 1000 Am) using a
Fig. 3. Temperature levels during lagoon production of the second batch in 1998.
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production/consumption rate of 0.2. Consumption rate of a given prey was only calculated when the type of prey was actually found in the stomach/gut of flounder larvae of that length. 2.9. On-growth After 4– 6 weeks, depending on temperature, the juveniles were caught by slowly dragging a gillnet from one end of the pond towards the opposite, collecting the juveniles in a small area and transferring them from the pond to land-based (indoor or outdoor) 2-m3 tanks for on-growth. The juveniles were weaned from live
Fig. 4. Concentration levels of zooplankton during lagoon production of the second batch in 1998.
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food to dry food (Perla Marin Respons, Skretting, Italy) immediately after catch without the use of Artemia in the weaning period. The flounder were released in the Limfjord, when they had reached a length of 2, 3 – 4 or 10 – 15 cm, respectively.
3. Results During the years 1996 to 2002, a total of 533,000 juveniles were produced. The yearly production figures are given in Table 1. Most juveniles were successfully weaned. More than 95% were weaned directly to a dry food diet. In the following, results from the second batch of fish during 1998 are given as an example of a relatively good production run. This particular production was initiated on April 6, 1998 (day 0) and metamorphosed larvae were caught on June 12, 1998 (day 36). Dynamics of the phytoplankton through the production of the second batch in 1998 are shown in Fig. 1. The initial phytoplankton bloom was not very strong. Phytoplankton was dominated by small unidentified flagellates during most of the production period. Cryptophyceae were very abundant in the first weeks of production. Later on, during the last weeks of production a bloom of diatoms (Bacillariophyceae) and Haptophyceae occurred. Oxygen levels were never below saturation (Fig. 2). Through the entire production oxygen levels were lowest in the morning and highest in the afternoon. Oxygen levels were generally lower in the surface waters than the bottom waters and were highest during the initial plankton bloom in the first days of production and also during the bloom of phytoplankton in the last two weeks of production.
Fig. 5. Clearance pressure from different zooplankton groups during the production period of the second batch from 1998.
Table 2 Development of flounder larvae and contents of stomach and gut contents during the production period of the second batch in 1998 Day-degrees
Mean (F Std.) larval length [mm]
Mean (F Std.) yolk-sac volume [mm3]
Mean (F Std.) mouth width [mm]
n
Development of digestive tract
Eyes
Stomach and gut contents (number of prey items per larvae)
Eyes white Eyes light brown Iris developed in some, not in others Iris developed Iris developed
Empty Empty
– –
Empty
–
0 4
0 28
2.98 F 0.17 3.95 F 0.21
0.140 F 0.010 0.139 F 0.054
0.00 F 0.00 0.00 F 0.00
9 9
Anus open Gut straight
6
40
4.06 F 0.18
0.051 F 0.037
0.02 F 0.05
8
Gut straight
10 12
73 93
4.39 F 0.30 4.89 F 0.32
0.008 F 0.012 0
0.19 F 0.07 0.26 F 0.04
5 7
Gut straight Gut straight with bulge
24
242
7.75 F 0.07
0
0.50 F 0.00
2
Gut-winded, metamorphosed
34
387
10.26 F 0.03
0
0.67 F 0.05
10
42
527
9.91 F 0.93
0
–
20
Empty Phytoplankton, chlorophyll remains 0.5 copepod nauplii 3.5 copepod nauplii, 0.5 copepodite and 5.5 podon 21.7 copepod nauplii, 0.3 barnacle nauplii, 3 harp. copepods, 5 calanoid copepods, 0.8 copepod eggs, 0.1 Artemia
Length of prey [mm]
– 0.005 – 0.2
0.10 – 0.55
0.09 – 1.29
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Days from hatch
483
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Temperatures were generally rising through the whole production period (Fig. 3). Temperature increased from about 6 jC at the outset of the production to about 20 jC at the time of fry harvest. Fig. 4 shows the development of zooplankton through the production period. Adult copepods (less than 2 l 1) were introduced to the lagoon in the very beginning of the production along with some nauplii (less than 20 l 1). After a few days, the adult females started to produce eggs. The eggs hatched and the concentration of nauplii increased to maximum levels (approximately 160 l 1) two weeks after hatching of the flounder larvae. The concentration of copepodites remained low (below 6 l 1) through the whole production. Until day 20, mostly Temora longicornis were found among the adult copepods. From this day onwards Centropages hamatus became abundant (10 –50%). Acartia spp. were observed from day 33 ( < 15%). Total mesozooplankton clearance pressure did not exceed 1 per day through the whole production period (Fig. 5), indicating that the mesozooplankton never filtered the water more than once per day. Although the nauplii were by far the most dominating stage of copepods, the nauplii were not responsible for more than at the most 80% of the total mesozooplankton clearance due to the smaller size of the nauplii. Flounder larvae were introduced to the lagoon as yolk-sac larvae. During the production period, larvae from the lagoon were caught and examined. These results are shown in Table 2. Data on length of flounder larvae as a function of day-degrees from hatch (n = 753) for all batch productions from 1996 to 2002 are compiled in Fig. 6. A linear relationship (length = 0.0175 day-degrees + 3.0875) exists between day-degrees from hatch and length of larvae.
Fig. 6. Total length of larvae as a function of day-degrees from hatch for all productions from 1996 to 2002 (n = 753).
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Fig. 7. Weight of larvae as a function of day-degrees from hatch for all productions from 1996 to 2002 (n = 753).
Dry weight of flounder larvae plotted against number of day-degrees from hatch (n = 753) for all batch productions from 1996 to 2002 are shown in Fig. 7. Dry weight as a function of day-degrees can be described by nonlinear regression, where dry weight = 0.715 day-degrees0.0056. The temperature range during productions was 3 –23 jC. The type and size of prey caught by the larvae depended on the size of the larvae. Fig. 8 shows the type of prey found in the digestive tract of the larvae (n>300) through the productions in year 1996 to year 2002. If the specific growth rate of the larvae is calculated for different periods of larvae life (measured in day-degrees from hatch), theoretical consumption of the flounder larvae can
Fig. 8. Prey of different sizes of flounder larvae from productions year 1996 to 2002 (n>300).
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Table 3 Calculated consumption of different prey (number of prey per day per day-degree) Production period [day jC]
Length [mm]
SGR (length) [% day jC 1]
SGR (dry weight) [% day jC 1]
Consumed 100 Am nauplii [day jC 1]
Consumed copepodites [day jC 1]
Consumed copepods [day jC 1]
0 – 100 100 – 200 200 – 300 300 – 400 400 – 500 500 – 600 600 – 700
3.1 – 4.8 4.8 – 6.6 6.6 – 8.3 8.3 – 10.1 10.1 – 11.8 11.8 – 13.6 13.6 – 15.3
0.45 0.31 0.24 0.19 0.16 0.14 0.12
0.56 0.56 0.56 0.56 0.56 0.56 0.56
– 48 64 84 111 147 195
– 10 14 18 24 31 42
– – – 7 10 13 17
SGR = specific growth rate. It is assumed that (1) the larvae consumes only one type of prey at a time. (2) 20% of the consumed energy is used for growth. (3) Nauplii with a length of 100 Am has a dry weight of 0.64 Ag, a copepodite has a dry weight of 3 Ag, and a copepod has a dry weight of 7.5 Ag. Values are average values from Danish coastal waters obtained during several years (Bio/consult, unpublished data).
be calculated. Table 3 shows the theoretical consumption of different sizes of prey. The size distribution used for this calculation reflected that found in the digestive tract of flounder larvae of the same size (Fig. 8). Table 3 can be used to calculate daily needs of prey of the flounder larvae. For example, if a larvae of a length of 6.6 –8.3 mm, 200 –300 day-degrees from hatch should fulfil its metabolic needs at 20 jC entirely on nauplii with a length of 100 A, it would have to consume 20 64 nauplii day 1 = 1280 nauplii day 1. At 10 jC, the same larvae would need only 640 nauplii day 1.
4. Discussion The present study has shown that successful larval survival depends on a good timing between the consumption needs of the larvae and the onset/magnitude of microalgae bloom and the copepod nauplii production. The first food items ingested after the yolk-sac stage were microalgae (Table 2; Fig. 8). These food items were ingested for three to five days prior to a switch to copepod nauplii. The presence of microalgae in the guts of marine fish larvae prior to feeding on zooplankton has been previously observed in other species of flatfish and is reportedly important for their subsequent growth and survival (Last 1978a; Reitan et al., 1991). Calculated consumption needs (Table 3) suggest that larvae must search for food at a high rate. For example, at the time during and just after first feeding, between 100 and 200 day-degrees from hatch, a larva must consume 480 nauplii day 1 at 10 jC to fulfil its requirements for optimal growth and would have to capture and eat one nauplii every second minute during a 16-h daylight period. Although this may seem a high search rate, it is consistent with observations of individual flounder larvae. In the period just before catch, 600 – 700 day-degrees from hatch, assuming a temperature of 20 jC, the necessary number of prey has risen to 3900 nauplii, 840 copepodites or 340 copepods day 1 (Table 3). Assuming that a larva at this time preys
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solely on copepodites and that it can hunt for food 16 h day 1, the larva must capture and eat one copepodite every 1.1 min. Assuming that the copepodites are grazed to a level where life of the larvae cannot be sustained, the larvae will capture and eat prey with a higher frequency (increase its swimming activity) and decrease its prey-size selectivity (Munk, 1995). If the same larvae had to depend solely on nauplii with a size of 100 Am, it would have to eat 1 nauplius every 15 s. When estimating the minimum density of food items to fulfil the requirements for optimal growth of the flounder larvae, food search potential (Blaxter and Staines, 1971), feeding success (Blaxter and Staines, 1971), competition between larvae (e.g. density of fish larvae), prey size, temperature and possible patchy distribution of fish larvae and prey (Paulsen, 1985a) have to be taken into consideration. The fact that the flounder eggs are fertilised 10 to 20 days before the flounder larvae will start to eat nauplii necessitates a good control of peak occurrence of suitable food items. Thus, the main feature of the system described here is timing the onset of a new generation of copepod nauplii with first-feeding fish larvae. Newly hatched larvae are transferred to the outdoor ponds at a time when the zooplankton population has reached copepodite/adult copepod stage. By the time they are ready to start ingesting live prey, about a week later (depending on temperature), the new generation of copepod nauplii must be developing to ensure enough food for the fish larvae. As the flounder larvae grow and develop in the lagoons, the nauplii that were not eaten develop into larger stages, thereby meeting the prey-size requirements of the growing flounder larvae. A high concentration of prey in the system is not necessarily the best because the zooplankton may graze down the microalgae in the water column (Fig. 5). The clearance rate depends on temperature and concentration of microalgae (Kiørboe et al., 1982; Støttrup and Jensen, 1990; Mauchline, 1998). If the total clearance rate is too high, the microalgae may be grazed to a level that is insufficient for survival of the copepods causing a collapse of the copepod culture. Our experience suggests that zooplankton clearance, without addition of microalgae biomass to the system, should be kept below 1 to prevent a collapse of the zooplankton culture. Grazing by copepods will shift the microalgae community towards flagellates, which are not effectively grazed by the different stages of copepods (small flagellates with a diameter less than 2 –4 Am). It is therefore useful to monitor the microalgae community by microscopy (species and concentration) during a production. This will also make it possible to give an early warning of a possible harmful microalgal bloom. The overall survival rate of larvae in the described production system was 30% over the 7 years of the study. Moreover, less than 0.5% malpigmentation was observed and no other developmental abnormalities. Improved survival rates from year 1996 to year 1998 were observed, mainly due to an adjustment of the semiextensive method (e.g. timing between the development of the flounder larvae and the onset of phytoplankton bloom and egg production of the copepods). From 1999 to 2002, the average survival rates remained high except in 2001, where survival were lower due to unusually cold weather in the early spring and thereby a slow development of copepods. The growth rates of flounder reported here were similar to growth rates found for reared cod, winter flounder and silver hake (Buckley et al., 1993) or less than half of recorded growth rates of turbot larvae (Paulsen, 1985b).
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To utilise cultured calanoid copepods by the semiextensive method, it is necessary to have large, outdoor water volumes of filtered seawater. Additional filter and pumping systems are needed to fractionate and distribute different stages of copepods during the production. A culture of calanoid copepods may be initiated using calanoid copepods from surrounding seawater. This assures that the cultured copepods are well adapted to the water used to breed the copepods. Several generations of copepods should be bred before the copepods are used as food for marine fish larvae to ensure that there is no transfer of parasites from the copepods to the fish. Different species of copepods can be used. Most important is the production potential of the specific copepod species. Also important is the size of the nauplii or adult. Adult copepods in good condition (well fed with microalgae) are introduced into the lagoons or tanks. If the algal biomass is sufficiently high, then females will start to produce eggs. The egg production rate is controlled by a number of factors, the principal one being the availability of food (Mauchline, 1998). The development time of the eggs to hatching is species specific, but mainly depends on temperature (Mauchline, 1998). One thousand to five thousand nauplii per litre can be produced per generation under optimal conditions. Several successful attempts have been made to culture marine fish larvae on copepods (for review, see Van der Meeren and Naas, 1997; Støttrup et al., 1998; Shields, 2001). During the last 20 years, Norwegian groups have used enclosed pond systems to produce marine fish larvae. The method is traditionally called the ‘‘extensive’’ Norwegian method. The ponds are operated to maintain a high production of zooplankton used for larval fish. Most often, the ponds were treated with rotenone to kill all predators of fish larvae. After the treatment a bloom of copepods would occur in the early spring, mainly as a result of hatching of copepod resting eggs (Næss, 1996) and a bloom of phytoplankton that might be reinforced by the use of fertilizers. The main differences between the system described here and the Norwegian system are: (1) the production is performed in closed, PVC coated ponds that can be cleaned mechanically and treated by disinfectants or simply dried out; (2) the copepod culture is controlled to some degree through culture of several generations of copepods before onset of the larvae production, which will facilitate the availability of copepods of the right stage and condition; and (3) most external parameters that might influence the ecosystem are eliminated and, except for the weather conditions and species of algae in the water filtered from the Limfjord, controlled. This makes the production results more predictable. Nutrients can be added to the system, but this has to be done with great care because excessive phytoplankton that is not exploited by the zooplankton will eventually sink to the bottom and the energy of the algae is thereby most likely lost because the algal biomass has to be mineralised before the nutrients return to the food web. The flow of energy, carbon and nutrients out of the pelagic system is likely to result in a suboptimal production of fish larvae as well as increasing the risk of harmful algal production, oxygen depletion and hydrogen sulphide production. The season can be a limiting factor in temperate or arctic climates. The nauplii production will slowdown in the winter season, especially with decreasing temperatures, because most calanoid copepods tend to produce diapause eggs during periods of decreasing environmental temperatures. Temperature is the primary controlling factor of
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dormancy of diapause eggs, which is exploited when keeping the culture over winter. Low oxygen concentrations can delay embryonic development (Mauchline, 1998). However, diapause eggs are able to survive both low oxygen concentrations and sulphide (Marcus, 1996; Marcus and Lutz, 1998). In Denmark, turbot and European flounder are successfully reared on copepods. Similar systems can probably be used for production of all species of marine fish that depend on different stages of copepods as larval prey. This includes almost all marine fish (Last, 1978a,b, 1979, 1980; Purcell and Grover 1990). Artemia are widely used as a prey item in rearing of marine fish larvae. However, the market price of Artemia has increased considerably in the past years, mainly as a result of reduced catches of some of the natural sources of Artemia. Although the efforts to harvest Artemia have increased, the actual harvest of Artemia has not (Anonymous, 2000, 2002). Therefore, it seems important to improve the availability of prey such as copepods that will constitute an alternative to Artemia. We suggest that the semiextensive method involving copepods provides a good solution to solve the need for live prey for marine fish larvae production. After adjusting the method for a desired fish species, all elements of the production method can be carried out by experienced aquaculturists with some understanding of plankton ecology.
Acknowledgements We sincerely thank the many persons involved for their invaluable support and efforts. Especially we thank Ole Borbjerggaard and Hans Svanborg for their dedicated work. We also thank members of Nordvestjydsk Fritidsfiskerforening, especially Kay Hansen, employees at Venø Fish Farm and colleagues at Bio/consult and the Danish Institute for Fisheries Research. The project was funded by the Danish Marine Coastal Fisheries Management Programme.
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