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Ecophysiology,
growth
and
development
of larvae
and
juveniles for aquaculture G.JOAN HOLT
University of Texas Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373 USA (
[email protected]) SUMMARY:The rearing of early life stages is often the most difficult aspect of fish cultivation. Lack of informationon ecophysiological requirements for development and growth hinders the design of appropriate rearing systems and feeding protocols. Larvae are often adapted to specific temperature, salinity and oxygen regimes. Such environmental data from natural habitats can provide insight into optimal rearing conditions but findingsmay be counter intuitive. The dependence of most marine larvae on live prey also presents difficulties in larvalrearing. Biochemical and physiological studies are fundamental to the development of weaning diets for larvae. Data on ontogenetic changes in larval feeding and physiological responses to environmental parameters are especially important for cultivating fish in high density closed systems. Appropriate or optimum rearing conditions can be evaluated with the use of condition measures developed for assessing the status of wildpopulations of fish larvae and juvenile fishes. KEY WORDS: larvae,
temperature,
salinity,
rotifers,
microdiet,
digestive
enzymes,
RNA/DNA
INTRODUCTION
Fishpopulationsare declining on a worldwide basis as a resultof over-fishing, habitat degradation and pollution. Many of the world's fish stocks are considered fully exploited,over exploited, depleted or in the process of rebuildingafter a period of depletion. If production is to keep up with global demands for fisheries products, aquacultureproduction must be increased. Rapid growth of aquaculture could compensate for the declines in catchesby supplying alternate sources of fish products. Constraints on aquaculture development are environ mental(i.e. lack of suitable sites, pollution and exposure to natural and man induced hazards), biotechnological (dependenceon wild stocks, inadequate feeds and disease outbreaks),and socioeconomic (user conflicts in coastal zones, limited demand and market saturation). Some solutions to these problems lie in research and developmentactivities focused on fish rearing, diets and nutrition, and quality control. The growth of the aquacultureindustry is also constrained by the production ofjuvenilesfor grow-out because the early life stages are the most difficult to culture. Numerous physical, chemical and biological factors can potentially affect growthand survival rates during the larval and juvenile stages. For aquaculture to succeed it is important to understandthe ecophysiological requirements during the early life stages. The same information required for understandingnatural population dynamics is needed for successfulculture. Understanding how the environment acts on the physiology of fish larvae and juveniles to promotesurvival, growth and development will form the basisfor successful aquaculture of the species. Recent research
to identify and modify the factors that limit or alter larval fish growth and development is increasing juvenile production of many marine species. ENVIRONMENTAL AND LARVAE Natural
habitats
REQUIREMENTS
and environmental
OF EGGS
conditions
Larvae are generally adapted to specific temperature, and salinity regimes. Estuarine dependent species occur in habitats that exhibit a wide range of environmental conditions, while open-ocean and reef species generally experience fairly constant environments. Culture of a species should mimic natural conditions such as photoperiod and light levels since larvae can successfully feed and grow under these conditions. Data associated with field collected larvae provide insight into the requirements for larva culture but generalizations may be misleading. Paradigms based on data collected sporadically in the field may lead to false conclusions. For example eggs and larvae of spotted seatrout Cynoscion nebulosus have been collected from salinities ranging from 20 to 45 ppt (Holt, S.A., unpubl. data, 2000.) suggesting that they are euryhaline. To the contrary, laboratory studies have shown that the eggs and larvae have a restricted salinity tolerance that is based on the salinity regime of the estuary in which the adults spawn. Egg size varies with spawning salinity (Fig. 1). Egg wet weight but not dry weight decreases with spawning salinity while neutral buoyancy of the eggs increases with increasing salinity1).Spotted seatrout have
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a general ability to adapt to changes in salinity such that they produce buoyant eggs. However this adaptability may be limited at high salinity. Adults from a high salinity estuary that were spawned in the laboratory in 20
supersaturated
ppt were unable to produce buoyant are limits to their adaptation.
cycles
eggs indicating
there
by
as
area.
varies
much An
faster
to Do or
cold
stable
fronts
through
growth
and
larvae/juveniles
is and
the
intensive produce
effect
the
these
condition in
conditions
what
the
is how
environment
and
Daily changes
pass
environmental
growth
dawn.
aquaculture
larval
more
the
for
before occasionally
on
survival
the of
young.
Systems conditions
nursery ocellatus
impact a
just and
when
fluctuating
of
2mg/l
issue
slower
released
of
3-4 •Ž
10 •Ž
might
sturdiness
Since larvae are especially sensitive to alterations in environmental conditions, changes in salinity can result in morphological deformities, low hatching rates, and decreased larval survival. In acute salinity tolerance tests, spotted seatrout larvae produced by adults from a low salinity estuary survived sudden drops but not increases in salinity. Larvae produced by adults from a high salinity bay were significantly more tolerant of increases in salinity2). Data on the effects of salinity on egg size, buoyancy, and larval survival suggests that spotted seatrout may be adapted to the prevailing salinity regime of an estuary and that this adaptation affects the tolerance of the early life stages to changes in salinity. This type of information is especially critical for stock enhancement programs to insure that spawning stocks are kept separate and juveniles are stocked into appropriate sites.
as
by
important
compared culture.
Fig. 1 Egg diameters of spotted seatrout Cynoscion nebulosus spawned at various salinities in the lab or from field collected data. N=40.
to a low
temperature
for
laboratory
studies
of
environmental
Experimental studies of larval fish tolerances to environmental variables are fundamental to successful intensive culture. For example determining optimal and tolerable levels of temperature, salinity, ammonia, light, etc. are needed for the design of appropriate rearing systems and feeding protocols. In order to test these environmental parameters in my lab, a series of self -contained 150 1 tanks with internal biofilters are used4). The larvae can be reared under varying conditions in recirculating tanks with water exchange beginning on the first day of feeding (Fig. 2). These independent experimental tanks have water circulated through an internal biofilter that is inoculated with a commercial culture of nitrifying bacteria5). Small mesh netting (72 micrometers) on the outflow prevents the loss of liveprey from the tank. A larger mesh size (200 micrometers)is placed on the outflow at night to remove uneaten prey. The tanks are blue inside to provide contrast so that the prey are visible to the fish larvae. Photoperiod and light levels are set to match the conditions found in the natural habitat of the larval species under study. Evaluation of optimum temperature, salinity, prey concentration and turbulence for a wide variety of species are assessedwith these systems.
Larvae of many species of fish utilize estuarine grounds. For example, red drum Sciaenops larvae recruit and settle to estuarine nursery
grounds after approximately 3 weeks in the plankton. In South Texas and Florida these sites are seagrass beds, assumed to provide ample food and protection from predators and benign environmental conditions. Recent measurements using data loggers set in the seagrass beds to collect environmental data every half hour showed that temperature and oxygen levels are temporally variable as a result of daily and tidal cycles as well as stochastic weather events3). Daily oxygen levels range from
Figure
2. A recirculating
water
system
for experimental
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studies of larval fish. Open ocean
or
reef
species
require
stable,
pristine water quality. Once feeding is initiated such conditions are hard to maintain making it difficult to rear many of these species under intensive culture conditions. The dilemma is how to provide appropriate environmen tal conditions along with sufficient densities of prey to ensure that larvae encounter and feed on the prey. These difficulties have been overcome by using small chambers for the fish larvae that are suspended in a larger tank system. Water is slowly recirculated to maintain the same water quality within the chambers as in the large tank and prey is concentrated incidence of feeding
in the small chamber 6)
improving
the
FEEDING LIVE PREY
In culture, enriched rotifers are the usual first food for marinelarvae. Among the enrichments typically used are microalgaeand commercial products with high levels of highlyunsaturatedfatty acids (HUFA). The marine food chain provides high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and these HUFAhave been shown to be essential for the culture of marinefish larvae7,8). Changes in rotifer fatty acid composition,particularly in the amounts of DHA and EPA depend upon enrichment since rotifers do not naturallycontain high levels of these n-3 HUFA and reflectthe fatty acid profiles of their food. The highest concentrationsare produced with live or inert diets that containhigh levels of HUFA (Table 1).
Another possible explanation for the difference is that higher levels of HUFA are needed by open ocean or reef species compared to fish larvae that can tolerate brackish conditions. HUFA nutrition was examined in larvae from two different families (Sciaenidae and Lutjanadae). Red drum, a sciaenid that is transported to estuaries as young larvae, grows well on rotifers with n-3 HUFA of 1.5 % dry weight and DHA/EPA -1.09). Larvae fed low HUFA rotifers (low EPA and essentially no DHA) grew at significantly slower rates but the fatty acid composition of the 10 d larvae did not differ from larvae fed high HUFA rotifers suggesting that red drum larvae have a limited capacity to bioconvertl8:3n-3 or EPA to DHA. Similar results have been reported for other marine species. Yellow-tail snapper (Ocyurus chrysurus) a lutjanid found in association with coral reefs apparently requires much higher levels of DHA/EPA. Highest growth rates were measured in yellow-tail snapper larvae fed rotifers enriched with a commercial product (ALGAMAC-2000) high in n-3 HUFA. This product supported the production of rotifers with a DHA/EPA ratio of over 7 (Table 1). Although Isochrysis galbana enriched rotifers were adequate for red drum larvae, yellowtail snapper require a more highly enriched diet. Rotifers enriched with the algae Nannochloris oculata or yeast did not provide adequate nutrition for either species. DEVELOPING Early
Table1. Fatty acid (FA) composition (% area) and total lipidcontent (mg/g dry weight) of rotifers starved or fed Isochrysisgalbana (Iso), Nannochloris oculata (Nanno), yeast,or ALGAMAC-2000* (A2000) for 8 hours.
*Aquafauna
Bio -Marine,
Inc.
While marine species HUFA in their diet for optimum
in culture today need growth and survival the
quantitative requirements vary. Species or families of fishes may vary in their efficiency in using n-3 HUFA:
INERT
DIETS
weaning
Eliminating live food from larval rearing practices would be a major advance in marine aquaculture. Although many species are reported to be weaned only after a functional stomach has developed, there is evidence that this is not the general case. Some species such as red drum4) and more recently, gilthead seabream (Sparus aurata)10) have been successfully weaned to microdiets after several days with live foods. In other species, the live food requirements can be reduced significantly by the addition of micro-diets11).Dry feeds (less than 0.2 mm) are readily accepted at first feeding by many species of marine larvae. Red drum larvae are weaned onto a microbound diet (Kyowa Fry Feed, Kyowa Hakko Kogyo; Tokyo, Japan) at a very precocious age (day 8 post hatch). The standard feeding protocol is to co-feed the microdiet with live enriched rotifers for five days, followed by microdiet only through weaning. Larvae grow as well on this dietary regime as they do on rotifers and artemia4),but they do not survive or grow unless they are fed rotifers for the first few days. When microdiets are fed as the sole food source, larvae barely grow and do not survive more than 10-12 days, but no dietary induced differences in digestive enzyme activities were found compared to treatments with live prey12) Results of these
870
studies and those of Cahu and Infante13) indicate that marine fish larvae have the digestive capability to handle inert diets early in development.
concluded that larvae have low but consistent levels of all necessary digestive enzymes needed for feeding, casting doubt on the idea that fish larvae must depend upon live
Red drum larvae fed only micro-diets but with the addition of Isochrysis galbana completed larval development and transitioned to the juvenile stage14). Growth was not as fast as when larvae were fed rotifers for five days, but survival was high and the elimination of rotifers represents a significant cost savings. The possible mechanism(s) for the ability of larvae to grow and survive on inert diets in the presence of Isochrysis galbana are numerous14).If water quality improvements or enhanced contrast that would improve capture efficiency by the larvae were of importance, then other alga species might provide similar results. When Nannochloris oculata was added to tanks of larvae fed micro-diets there was some improvement in length of survival and growth compared to larvae fed only inert diets. But their growth was significantly less than the Isochrysis galbana treatment and they did not survive through larval development. Larvae that were raised solely with Isochrysis galbana and the inert diet, or rotifers and the inert diet were the only ones surviving to day 32 juveniles (Fig.3). Thus larvae can be successfully raised to juveniles without providing live rotifers or artemia. Red drum larvae are not unusual in their development or nutrient requirements and it is probable that other marine fish species might respond in a similar manner.
prey for digestive enzymes. Measurements of digestive enzymes were used to examine the question of whether microalgae increased the availability of nutrients in inert diets. The presence of algae may have a substantial affect on ingestion and digestion early in development. These studies showed that the presence of algae improved the performance of microdiets by increasing enzyme activity14) and ingestion rates. Work on the development of a diet customized for red drum larvae is continuing with the hope that in the near future they can be cultured without depending upon live rotifers. The goal is to enhance growth and survival of larvae in aquaculture while eliminating zooplankton prey. CONDITION
AND GROWTH
Knowing the state of health or condition of larvae is central to intensive culture. A variety of measures have been applied to the evaluation and condition of natural populations of fish. Many of these condition indices have benefited from lab evaluation and verification that depends upon adequate culture of the fish species of interest. Biochemical measures of condition are sensitive and may indicate future survivability. The ratio of RNA to DNA has been useful as a tool to estimate recent growth in young fishes in response to environmental variables16). High RNA/DNA ratios are indicative of an increased amount of ribosomal activity and protein synthesis17). Since growth is a direct product of protein synthesis, a positive correlation between growth rate and RNA/DNA would be expected. Data on a variety of species have shown that well fed larvae possess significantly higher RNA/DNA ratios than starved or periodically fed larvae 11,18,19)The RNA/DNA ratios of well-fed larvae increase as a function of length, but starving larvae exhibit substantially lower RNA/DNA ratios.
Fig.3 Growth of red drum (Sciaenops ocellatus) larvae fed an inert diet (Kyowa Fry Feed) from first-feeding (solely) or inert diet with the addition of rotifers for five days, or Isochrysis galbana or Nannochloris oculata throughout the larval stage. Digestive
physiology
Basic to diet development studies is knowledge of the digestive physiology of the larvae during ontogeny. Lazo15) assessed the digestive capacity of red drum using histological, biochemical and molecular techniques. He
Temperature also affects larval growth rate but not the relationship of RNA/DNA to length. Growth rates of red drum larvae reared at warm temperatures (28C) were significantly greater than those grown at cooler temperatures (23C). There was no significant temperature effect on RNA/DNA ratios, however, there was a significant effect of temperature on the relationship between RNA/DNA and growth rate20).An increase in growth rate with respect to RNA/DNA was significantly higher at the warmer temperatures presumably due to more efficient utilization of RNA or higher retention of synthesized proteins. In a study of winter flounder (Pseudopleuronectes americanus) Buckley16)reported increased growth rates at higher temperatures and suggested they were not accomplished by an increase in RNA/DNA, but by an increase in growth at a given RNA/DNA. He reported a similar model for eight species
871
of temperate marine species. Therefore, significant differences in RNA/DNA ratios at a given size can be attributed to nutritional condition of the larvae.
Other biochemical measures have been used successfullyto determine nutritional condition of fish larvae including the digestive enzymes trypsin21 and chymotrypsin22,23). Biochemical measures of condition integratefeeding success over time and give an indication of probabilityof survival. These condition measurements couldbe useful for sampling large mesocosms or ponds to assessthe state of health of the larvae and to predict aquaculture production, as well as for evaluating optimum rearing conditions for intensive culture. Producinghigh quality fish larvae and juveniles is the firststep in developing successful aquaculture of marine fish. In summary, understanding how the environment actson the physiology of larvae and juveniles to promote survival,growth and development will form the basis for successfulculture of the species.
REFERENCES
1. Kucera,C.J., ffects of parental spawning salinity on eggs and larvae of spotted seatrout (Cynoscion nebulosus). MS Thesis (Marine Science), University of Texas, Austin, 2001. 2. Faulk, C.K., Kucera, C.J., Holt, G.J. Acute salinity toleranceof spotted seatrout larvae Cynoscion nebulosus C. in relation to spawning salinity. J Fish Biol in review. 3. Perez, R., Holt, G.J. Effects of nursery environmental cycleson larval red drum (Sciaenops ocellatus) growth and survival.Fisheries Sci. Suppl. (this issue). 4. Holt, G.J. Feeding larval red drum on microparticulate diets in a closed recirculating water system. J. World AquacultureSoc. 1993; 24: (2) 225-230. 5. Craig, S.R., Hatch,S.J., Holt, G.J. Biological filter for conicaltanks. Progressive Fish-Culturist 1990; 52: 61-62. 6. Henney, D.C., Holt, G.J., Riley, C.M. Recirculating-water systemfor the culture of marine tropical fish. . Progressive Fish-Culturist 1997; 57: 219-225. 7. Sargent, J.R., McEvoy, L.A., Bell, J.G. Requirements, presentations and sources of polyunsaturated fatty acids in marinefish larval feeds. Aquaculture 1997; 155: 117-127. 8. Watanabe,T., Izquierdo, M.S., Takeuchi, S.S., Kitajima, C. Comparisons between eicosapentaenoic acid and docosahexaenoic acid in terms of essential fatty acid efficacyin larval red sea Bream. Nippon Suisan Gakkaishi 1989;55: 1635-1640. 9. Craig, S.R., Arnold , C.R., Holt, G.J. The effects of enrichinglive foods with highly unsaturated fatty acids on the growth and fatty acid composition of larval red drum Scaienops ocellatus. J. World Aquaculture Soc. 1994; 24: (3) 424-431. 10. Yufera, M., Sarasquete, M.C., Fernandez-Diaz, C. Testing protein-walled microcapsules for the rearing of first feedinggilthead sea bream (Sparus aurata L.) larvae. Mar. Freshwater Res. 1996; 47: 211-216. 11. Kolkovski, S., Tandler, A., Izquierdo, M.S. Effects of live food and dietary digestive enzymes on the efficiency of
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microdiets for seabass (Dicentrarchus labrax) larvae. Aquaculture 1997; 148: 313-322. Lazo, J.P., Holt, G.J., Arnold, C.R. Ontogeny of pancreatic enzymes in larval red drum (Sciaenops ocellatus). Aquaculture Nutrition 2000; 6:183-192. Cahu, C., Infante, J.Z. Substitution of live food by formulated diets in marine fish larvae. Aquaculture 2001; 200: (1-2) 161-180. Lazo, J.P., M.Dinnis, C. Faulk, G.J. Holt, C.R. Arnold. Co-feeding microparticulate diets with algae: Toward eliminating the need for zooplankton at first feeding in red drum. Aquaculture 2000; 188: 339-351. Lazo, J.P. Development of the digestive system in red drum (Sciaenops ocellatus) larvae. PhD Thesis, University of Texas, Austin, 1999. Buckley, L., Caldarone, E., Ong, T.L. RNA-DNA ratio and other nucleic acid-based indicators for growth and condition of marine fishes. Hydrobiol. 1999; 410: 265-277. Bulow, F.J. RNA-DNA ratios of recent growth rates in fish. J. Fish. Res. Bd Can. 1970; 27: 2343-2349. Clemmnsen, C. Laboratory studies on RNA/DNA ratios on starved and fed herring (Clupea harengus) and turbot (Scophthalmus maximus) larvae. J. Cons. Perm. Int. Explor. Mer. 1987: 43: 122-128. Rooker, J.R., Holt, G.J. Application of RNA:DNA ratios to evaluate condition and growth of larval and juvenile red drum (Sciaenops ocellatus). Mar. Freshwat. Res. 1996; 47: 283-290. Fernandez, A.C. The effect of temperature on larval fish growth: changes in RNA:DNA ratios of larval red drum (Sciaenops ocellatus). MA Thesis (Marine Science), University of Texas, Austin, 1997. Ueberschar, B., Clemmesen, C. A comparison of the nutritional condition of herring larvae as determined by two biochemical methods tryptic enzyme activity and RNA/DNA ratio measurements. ICES Journal of Marine Science 1992; 49: 245-249. Applebaum, S.L. Evaluation of the digestive enzyme, chymotrypsin, as an indicator of nutritional condition in larval red drum (Sciaenops ocellatus). MS Thesis (Marine Science), University of Texas, Austin, 2001. Applebaum, S.L., Holt, G.J. The digestive protease, chymotrypsin, as an indicator of nutritional condition in larval red drum (Sciaenops ocellatus). Marine Biology in review.
