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ICES Journal of Marine Science: Journal du Conseil 2006 63(2):335-339; doi:10.1016/j.icesjms.2005.10.007
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© 2005 International Council for the Exploration of the Sea

Metabolic cost of feeding in Atlantic Cod (Gadus morhua) larvae using microcalorimetry

Artie McColluma, Jessica Geubtnerb and Ione Hunt von Herbinga,*

a School of Marine Science, University of Maine 5741 Libby Hall, Room 214, Orono, ME 04469, USA
b American Fisheries Society 5410 Grosvenor Lane, Bethesda, MD 20814, USA

*Correspondence to I. Hunt von Herbing: tel: +1 207 581 9019; fax: +1 207 581 4388. e-mail: ihuntvon{at}nsf.gov.

A microcalorimeter that measures total heat output (µW) was used to determine total metabolic rate (aerobic and anaerobic) and the cost of feeding (specific dynamic action, SDA) in larval Atlantic cod (Gadus morhua) from hatching to 4 weeks post-hatch at 10°C. Total heat output increased throughout development from 2.14 µW at first-feeding to 23.72 µW at 4 weeks post-hatch. SDA was determined by comparing the total heat output among unfed larvae and fed larvae simultaneously. Total heat output increased in the first 2 h after feeding with rotifers (Brachionus sp.) and Artemia, remained high for up to 10 h, was significantly higher in fed larvae than in unfed larvae, and ranged from 16.56 µW at first-feeding to 47.84 µW at 4 weeks post-hatch. The differences in total heat output between unfed and fed larvae were 14.42 µW and 24.12 µW, representing an increase in metabolic cost of feeding by a factor of 1.67 over the first 4 weeks of larval life. That the metabolic cost of feeding increased with development and remained elevated suggests that cod larvae allocate a large part of their energy budget to growth in order to meet the demands of their fast growth rates.

Keywords: Atlantic cod, larvae, specific dynamic action (SDA)

Received 13 June 2004; accepted 28 October 2005.


   Introduction
Top
 Introduction
Material and methods
 Results
 Discussion
 References
 
Variation is the template of natural selection, and high levels of individual level variation have been measured in fish larvae that are thought to be related to their high mortality rates and low recruitment (Chambers and Leggett, 1996). Fish larval growth rates are much higher, sometimes reaching more than 50% of body weight per day, than those of their juvenile and adult counterparts. In order to determine the energy allocated from the total energy budget to growth in such rapidly developing and growing stages, reliable measurements of metabolic rate at different larval feeding states must be made. Often, metabolic rates in larval fish have been determined by measurements of oxygen uptake in a respirometer (indirect calorimetry) (Hunt von Herbing and Boutilier, 1996). Recently, with the increased sensitivity of new microcalorimeters (direct calorimetry), which can measure total (aerobic and anaerobic) metabolism in the form of enthalpy or heat output released by a living organism, reliable measurements have been made for fish and invertebrate larvae (Gnaiger, 1983; Finn et al., 1996).

In the past, studies using respirometers have measured oxygen uptake in the early developmental stages of both fish and invertebrates for a variety of purposes, e.g. to study metabolic adaptation and acclimation to environmental factors; to determine the effects of metabolic cost of swimming on growth; to quantify the amount of energy associated with nutrient assimilation and production under the effects of environmental contaminants; and to determine total biological metabolic demand in water quality control (Pamatmat, 1983). In all these studies, sample sizes consisted of multiples of tens or hundreds of larvae for each experimental run. Large sample sizes were necessary owing to the fact that an individual small fish does not produce enough heat to be measured reliably. Accurate measurement of individual larval stages was difficult to achieve because continuously measuring oxygen electrodes (the most commonly used type) used such a large amount of oxygen to make each measurement that the relatively small amount of oxygen utilized by a single larva was undetectable in comparison (Gnaiger, 1983).

Over the past 20 years, direct measurement of the total rate of heat dissipation (Q) or total heat output (H) in samples that contained from one to five larvae has been possible using a Thermal Activity Monitor (TAM) or microcalorimeter with sensitivities of ±0.15 µW. These measurements of total heat output represent total (aerobic and anaerobic) metabolism, while respirometers only measure aerobic metabolism. Measurements using the microcalorimeter have been made for embryonic and larval turbot (Scophthalmus maximus) (Finn et al., 1996); metabolic regulation during dormancy in Artemia embryos (Hand and Gnaiger, 1988); metabolic responses of aquatic oligochaetes to anoxia/hypoxia (Gnaiger and Staudigl, 1987); and the effects of anoxia on activity and metabolism in mussels (Shick et al., 1986). The present study provides new first-time information on individual measurements of total heat output in larval Atlantic cod (Gadus morhua). Furthermore, changes in metabolic rates attributable to feeding (specific dynamic action, SDA) or food-induced thermogenesis (FIT) were measured in an individual larval fish, also using a microcalorimeter. Results from the present study will provide the first estimates of total metabolism and SDA measured as total heat output in a microcalorimeter throughout the first 4 weeks of development in fish larvae. These estimates may be valuable in helping us understand the factors underlying the extremely rapid early growth rates of cod and possibly contribute to improving productivity in cold-water marine fish aquaculture.


   Material and methods
Top
 Introduction
 Material and methods
Results
 Discussion
 References
 
Fertilized cod eggs were obtained from Aqua-Ventures in Newfoundland, Canada in June 2000. All fertilized eggs were incubated and transported to the Aquaculture Research Center (ARC) at the University of Maine in Orono, Maine, USA, at 10°C. All eggs were disinfected in 200 mg l–1 glutaraldehyde mixed in seawater for 10 min, then transported to 25-l incubation/rearing tanks. Eggs were incubated in the dark in artificial seawater (Crystal Sea®, Marine Enterprises International) at a salinity of 32 and at 10°C until hatching. One day before hatching, eggs were again disinfected in 200 mg l–1 glutaraldehyde mixed in seawater for 5 min, counted volumetrically, and returned to the incubation tanks (Baskerville-Bridges and Kling, 2000). Eggs were held in constant aeration and water flow of 850 ml min–1, and light intensity was held at 1000 lux for 24 h. Temperature, salinity, dissolved oxygen (YSI 85 probe, YSI Inc., Yellow Spring, Ohio, USA), pH (Hach EC30 pH meter, Hach Company, Loveland, Colorado, USA), and ammonia and nitrite (Hach Permachem reagents, Hach Company, Loveland, Colorado, USA) levels were monitored, and tanks were also siphoned daily to remove dead eggs. Beginning at 1 day post-hatch (dph), larvae were fed ten rotifers ml–1 (Brachionus plicatilis) enriched daily with DHA Selco (INVE), Algamac 2000 (Bio-Marine, Inc., Hawthorne, California, USA), and feedings occurred six times per day at 07:00, 10:00, 13:00, 16:00, 19:00, and 22:00.

Prior to each experimental run, 20 larvae were removed from their nursery tanks and held overnight without feeding in two 200-ml glass beakers (ten larvae per beaker) at 10°C. On the subsequent morning, larvae were separated into four groups of five larvae (two replicates for the control, unfed treatment, and two replicates for the experimental, fed treatment) and moved carefully to separate clean glass beakers. Larvae in the control unfed group were left to acclimate, while the larvae in the experimental, fed treatment group were provided ten rotifers ml–1 and allowed to eat for 30 min while being videotaped. At the end of the feeding period, a fed larva was carefully pipetted into a stainless steel 10-ml microcalorimeter ampoule, and one control, unfed larva, was carefully pipetted into a second ampoule. Then, the ampoules were filled completely with artificial seawater, and a 0.45-µm Millipore filter was inserted at the top of the ampoule before sealing the ampoule. Sample ampoules were inserted into one of the four dual chambers in the microcalorimeter; at the same time, an ampoule filled with seawater without a larva was also inserted as a reference. All ampoules remained in the microcalorimeter for 12 h, and data were recorded automatically every 10 s of the 12-h duration. The dual chamber design allows an experimental sample to be placed in one ampoule (larva – either unfed or fed – and filtered seawater), while the second chamber contained only the reference (filtered seawater). The microcalorimeter determined the heat output of each larva by comparing the values between the sample and a reference chamber.

At the termination of each experiment, each larva was anaesthetized with 5% tricane methanesulfonate (MS-222) and documented through image capture using an Olympus SZH10 research stereomicroscope fitted with a Hitachi HV-C20 camera and with Optimus 6.1 (©Optimus, 1994) software. To determine developmental stage, total length (LT), and dry weight, ten additional larvae were removed from the nursery tanks and placed in screw top vials with formalin in seawater (10%) for preservation, and five additional larvae were rinsed of saltwater and placed in a snap-top plastic tube and then frozen at –80°C prior to dry weight determination.

To determine the effects of larval age, dry weight, developmental stage, and feeding on total heat output, univariate and multivariate repeated measures tests were run for each experiment using SYSTAT 11.0.


   Results
Top
 Introduction
 Material and methods
 Results
Discussion
 References
 
Cod larvae grew well throughout the experiment. They grew from 5 to 8 mm and dry weight increased threefold from 0.25 to 0.75 mg at 10°C (Table 1).


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  		Table 1 Biometric and gravimetric data of the cod larvae reared at 10°C.

 
Mean total heat output generally increased with age and dry weight (Table 2). There was, however, a large degree of variation in mean total heat output for each individual larva and among larvae. This was evident when daily data were combined to show mean weekly patterns in total heat output.


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  		Table 2 Mean heat output (µW) per hour (for hours 2–10) for four larvae represented in Figure 1. The first hour was not included because of machine calibration. The four larvae are 4 dph, 13 dph, 21 dph, and 28 dph. This represents larval heat output from 4 dph to 4 weeks post-hatch. This table shows the SDA index (mean heat output of fed larvae – the mean heat output of the unfed larvae). This gives an approximate amount of energy available for growth in the fed fish compared with the unfed fish.

 
SDA was determined by comparing the total heat output among unfed larvae and fed larvae simultaneously. Total heat output increased after feeding, compared to the heat output of unfed larvae, and is considered in the present study to be an index of specific dynamic action, SDA. Results are shown for four comparisons of total heat output for unfed and fed individual larvae throughout the 4 weeks of larval development, from first-feeding at 4 dph to 28 dph (Figure 1).


Figure 1
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  		Figure 1 Comparison of heat output between fed and unfed larval cod at 10°C for (a) 4 dph, (b) 13 dph, (c) 21 dph, and (d) 28 dph. Each point represents mean (±s.e.) heat outputs per hour for fish reared at 10°C. This figure is a representation of larval heat output from 4 dph to 4 weeks post-hatch.

 
For each larva tested, total heat output increased rapidly in the first 2 h after feeding and remained elevated for many hours after feeding. The duration of elevated total heat output decreased with increasing age, from 8 h at 4 dph to 6 h at 13 dph, 6 h at 21 dph, and 5 h at 28 dph (Table 2). An index of SDA was calculated for each of the larvae tested and total heat output appeared to almost double over the 4-week developmental period, increasing from 16.35 µW at 4 dph to 27.55 µW at 28 dph (Table 2).


   Discussion
Top
 Introduction
 Material and methods
 Results
 Discussion
References
 
This study presents for the first-time measurements of total heat output obtained using a microcalorimeter for individual Atlantic cod larvae from first-feeding at 4 dph to 28 dph, and documents the existence of specific dynamic action (SDA), or feeding-induced thermogenesis as early as first-feeding in cod larvae. While metabolism in larval Atlantic cod has been measured and reported in many studies in the past (Fyhn and Seristag, 1987; Finn et al., 1995a, b, 2002; Hunt von Herbing and Boutilier, 1996; Hunt von Herbing et al., 2001), most measurements were obtained by monitoring oxygen uptake (or the decrease of oxygen concentration in a closed vessel over time) by respirometry. Respirometers are able to record only oxygen uptake and do not record the anaerobic component of metabolism. Furthermore, most chambers are large, and in order for oxygen uptake of the larvae to be distinguished from that of the oxygen electrode, many larvae (ten or more) have been used at one time. This introduces errors in the oxygen uptake measurements relating to crowding and increased bacterial contamination and often obscures the real values. Measurements of individual embryos and larvae have been rare, and in one recent study on zebrafish (Brachadanio rerio), measurements on individual embryos lead to high variance in the rates of oxygen uptake among specimens (Bang et al., 2004).

While total metabolism (aerobic + anaerobic metabolism) was successfully and repeatedly measured in Atlantic cod larvae using a microcalorimeter, a previous study was also able to successfully measure total heat output and was able to separate it into both its metabolic components (aerobic and anaerobic) (Finn et al., 1995a). Using turbot larvae, Finn et al. (1996) measured total heat output by using the same type of microcalorimeter as used in the present study. Oxygen uptake (aerobic metabolism) was measured simultaneously by connecting a respirometer to the calorimeter, creating a microcalorespirometer. Aerobic metabolism was then subtracted from total heat output to determine anaerobic metabolism. The results obtained from Finn et al. (1996) suggest that metabolism was mostly aerobic during the first 12 days after hatching in turbot larvae and that glycogen was the sole metabolic fuel during the first 3 weeks post-hatch. In the present study, comparison of heat output with published oxygen consumption values for cod larvae at the same age and temperature (10°C) suggests that metabolism for the first 4 weeks post-hatch may also be primarily aerobic, at least in non-actively swimming larvae (Hunt von Herbing and Boutilier, 1996). Larvae were considered to be not actively swimming in the ampoules as they were held in the dark in the microcalorimeter chamber.

Calculations show that a 2-week-old cod larva with a dry weight of approximately 0.20 mg had a mean rate of oxygen uptake of approximately 0.99 mg O2 h–1 (Hunt von Herbing and Boutilier, 1996) and a mean rate of total heat output of 12.22 µW (Table 1). The rate of oxygen uptake of cod larvae from Hunt von Herbing and Boutilier (1996) was converted to heat output, assuming no anaerobic component, using the oxycaloric equivalence of 13.96 J (Elliot and Davison, 1975). It equalled about 4 µW at 2 weeks post-hatch. While this calculated value is lower than that measured in the present study (12.22 µW), the estimates are close, especially considering the inherent high variability in metabolic rate in larval fish. This suggests that future studies on larval fish metabolism would benefit from considering alternatives to respirometry, such as calorimetry, in determination of metabolic rates of small organisms such as vertebrate and invertebrate larvae. More studies using alternative methods of measuring metabolic rate would help determine the best methods for accurate measurement of metabolic rates in small aquatic organisms.

In the present study, increases in metabolic rates were recorded in individual larvae after feeding, and subsequently, these values were compared to values of total heat output in unfed larvae. Increases in metabolic rates as a function of feeding were considered to be representative of specific dynamic action, SDA (Finn et al., 2002). Results from the present study suggest that the magnitude of SDA increased with larval age and dry weight. The duration of SDA, or the time that the metabolic rate remained elevated after feeding, decreased from 8 to 5 h with age and size. This suggests that older, larger larvae may allocate more energy to growth for a shorter period of time directly after feeding, thereby achieving high growth rates sooner than at younger, smaller stages and making more energy available for other activities such as swimming.

While several other studies have estimated larval fish SDA (e.g. Dabrowski, 1986; Kiorboe et al., 1987; Rombough, 1994), there have been several confounding factors inherent in these studies that indicate that the larval fish SDA values should be treated with caution. A key shortcoming is that previous studies compared larval metabolism at different feeding levels with that of unfed, anaesthetized larvae, but did not measure gut contents (Kiorboe et al., 1987). In the present study, the intestine of each larva was inspected to make sure that food was present before SDA measurements were made and the number of prey consumed was monitored per larva, thus making future estimations for energy budgets possible. Furthermore, our study determined that it was necessary to measure individual larval values in order to estimate values of SDA.

In our study, the increase in heat output as a function of feeding was considered to be an index of SDA and could be measured in individual cod larvae. Measurable SDA occurs as early as 4 days post-hatching in larval cod and increases with age and size. SDA in fed larvae can reach values as high as twice that for heat output rates in unfed cod larvae. As it is difficult to measure metabolic scope in larval fish, it is difficult to determine how much of the total scope for activity SDA occupies. This should be determined in future studies. In terms of the duration of SDA, rates of heat output increased in the first 2 h after feeding, which is as rapid as in juvenile cod (see Hunt von Herbing and White, 2000). Heat output rates remained elevated in fed larvae for up to 8 h in the early stages after feeding and this is within the range of the duration in juvenile cod (Hunt von Herbing and White, 2000). High increases in SDA as a function of feeding, and long SDA duration, suggest that if SDA represents the amount of the total energy budget that is allocated for protein synthesis and growth, then a large percentage of energy available from food is directed to growth for several hours after feeding. In this way, fish larvae may be able to maintain their high growth rates, increasing their chance for survival because they can develop rapidly from a stage of high mortality (larval stage) to one of lower mortality (juvenile stage). Future work at different temperatures and feeding regimes for individual larval fish will be important in determining how the metabolic cost of feeding (SDA) is related to growth efficiency (conversion of food energy to somatic growth); and how SDA and growth efficiency change when the growth rate decreases, and larvae transform into juveniles and adults.


   Acknowledgements
 
We thank Nick Chacos from Allied Chemical Technologies and Nathan Hesse from Thermometric, Inc. for valuable technical assistance on the microcalorimeter. We are also grateful to the students and staff of the Aquaculture Research Center at the University of Maine. This research was funded by a Hatch grant (# 5538302) and a USDA grant (# 5535670) to I. Hunt von Herbing.


   References
Top
 Introduction
 Material and methods
 Results
 Discussion
 References
 

    Bang A., Grønkjær P., Malte H. (2004) Individual variation in the rate of oxygen consumption by zebrafish embryos. Journal of Fish Biology 64:1285–1296.[CrossRef][Web of Science]

    Baskerville-Bridges B. and Kling L.J. (2000) Development and evaluation of microparticulate diets in the early weaning of Atlantic cod Gadus morhua larvae. Aquaculture Nutrition 6:171–182.[CrossRef][Web of Science]

    Chambers R.C. and Leggett W.C. (1996) Maternal influences on variation in egg sizes in temperate marine fishes. American Zoologist 36:180–196.[Web of Science]

    Dabrowski K. (1986) A new type of metabolism chamber for the determination of active and postprandial metabolism of fish, and consideration of results for coregonid and salmon juveniles. Journal of Fish Biology 28:105–117.[CrossRef][Web of Science]

    Elliot J.M. and Davison W. (1975) Energy equivalents of oxygen consumption in animal energetics. Oecologia 19:195–201.[CrossRef][Web of Science]

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    Finn R.N., Hendersen J.R., Fyhn H.J. (1995) Physiological energetics of developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua). II. Lipid metabolism and enthalpy balance. Marine Biology 124:371–379.[CrossRef]

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    Finn R.N., Widdows J., Fyhn H.J. (1996) Calorespirometry of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus). Marine Biology 122:157–163.

    Fyhn H.J. and Seristag B. (1987) Free amino acids as energy substrate in developing eggs and larvae of the cod (Gadus morhua). Marine Biology 96:335–341.[CrossRef]

    Gnaiger E. (1983) Simultaneous direct and indirect calorimetry. In Gnaiger E. and Forstner H. (Eds.). Polarographic Oxygen Sensors: Aquatic and Physiological Applications(Springer-Verlag, Berlin) pp. 134–165 370 pp.

    Gnaiger E. and Staudigl I. (1987) Aerobic metabolism and physiological responses of aquatic oligochaetes to environmental anoxia: heat dissipation, oxygen consumption, feeding and defecation. Physiological Zoology 60:659–677.

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