Muscle growth and development in atlantic cod larvae (Gadus morhua L.) related to different somatic growth rates

To increase their chances of survival, teleost fish larvae use most of their energy resources in developing organs associated with food intake and locomotion (Osse et al., 1997). In newly hatched larvae of the common carp (Cyprinus carpio), the axial musculature constitutes approximately 20 % of the total tissue volume, and from day 4 to day 18 after hatching the muscle mass increases by 28 % per day (Alami-Durante, 1990). In other studies of the common carp, the axial musculature has been reported to constitute approximately 40 % of the body mass in the early life stages, increasing to 60 % in adults (Osse and van den Boogaart, 1995). The axial musculature is therefore the largest and most rapidly growing tissue in larval and juvenile fishes.

The axial musculature of fish larvae differs from that of adult fishes in many ways. In the adult fish, a lateral zone of red fibres at the level of the horizontal septum is separated from the white bulk of the muscle mass by a pink or intermediate zone (Johnston, 1977). In yolk-sac larvae, there is an inner presumptive white muscle mass surrounded by a superficial red muscle monolayer. During larval development, the distribution pattern of adult muscle fibre types, the expression of adult myosin isoforms and the specialisation of metabolism in the different fibre types occurs gradually (Hinterleitner et al., 1987).

Numerous reports exist concerning muscle growth mechanisms in juvenile and adult fishes (Weatherley, 1990). A number of studies have investigated the effects of temperature on muscle development and growth in embryonic and larval fishes (Stickland et al., 1988; Vieira and Johnston, 1992; Brooks and Johnston, 1993; Usher et al., 1994; Nathanailides et al., 1995; Johnston and McLay, 1997; Johnston et al., 1998; Galloway et al., 1998). Slow growth rates in larvae of the common carp can be induced by hormone treatment or by incubation temperature and are associated with a decreased contribution of hyperplasia (the addition of new muscle fibres) to muscle growth (Alami-Durante et al., 1997). However, few studies have focused on the effects of nutrition on muscle growth in larval fishes. Different diets given to African catfish larvae (Clarias gariepinus) resulted in different growth rates but, at a given length, the larvae had similar numbers and sizes of white muscle fibres (Akster et al., 1995). Indeed, there is generally a positive correlation between body length and ontogenetic state within a fish species (Laurence, 1979; Fuiman and Higgs, 1997). This is important for individual-based models of population dynamics, which often use body length as a measure for developmental changes in growth and mortality (Rice et al., 1993; Rose and Cowan, 1993). Standard length, rather than larval age, was therefore used as the dimension for describing muscular growth in the present study.

Although the Atlantic cod (Gadus morhua L.) is commercially important, muscle growth has only been described in adult cod (Greer-Walker, 1970, 1971). Several studies have focused on the somatic growth of larval cod in the wild (Bolz and Lough, 1984; Meekan and Fortier, 1996), in enclosures (Pedersen et al., 1989; OtterÅ, 1993; van der Meeren and Næss, 1993; van der Meeren et al., 1994), in intensive rearing systems (Rosenlund et al., 1993) and in the laboratory (Gamble and Houde, 1984; Hunt von Herbing et al., 1996; E. Kjørsvik, K. Hoehne, J. Rainuzzo, K. I. Reitan, T. F. Galloway and P. Makridis, in preparation). In these studies, the effects of different environmental or nutritional factors on larval somatic growth were investigated, but the cellular basis for differences in growth was not the main focus. Recently, Galloway et al. (1998) found that cod eggs incubated at temperatures close to their lower thermal tolerance limit (1 °C) produced smaller, less-viable larvae that had fewer functional muscle fibres at the time of first feeding than larvae from eggs incubated at 5 or 8 °C. Literature on muscle fibre morphology in first-feeding cod larvae is scarce (Morrison, 1993; Galloway et al., 1998), but it has been shown that muscle growth in early life may have an effect on the ultimate size and also on the rate of growth in later life stages of many fish species (Weatherley, 1990; Johnston et al., 1998).

The objectives of the present study were (1) to describe the morphological development of the axial swimming musculature, (2) to quantify growth in terms of muscle fibre cross-sectional area, fibre number and the volume fraction of myofibrils in each fibre, and (3) to investigate how different growth rates, induced as a result of different ratios of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) in the feed, may affect the relative contributions of hyperplasia and hypertrophy to muscle growth in first-feeding cod larvae.

The wild-caught broodstock of Atlantic (Northeast Arctic) cod (Gadus morhua L.) was kept in pens at Lofilab A/S at Leknes, Lofoten, Norway, where several females and males spawned naturally at 4–5 °C. The eggs were incubated at 5 °C. Four days prior to hatching, the eggs were transported by air to Brattøra Research Centre at the Norwegian University of Science and Technology in Trondheim, Norway. The eggs were disinfected for 10 min in 400 p.p.m. glutaraldehyde and rinsed thoroughly in running filtered (5 μm) sea water (34 ‰) before incubation (Salvesen and Vadstein, 1995). The eggs were then transferred to each of six rearing units (180 l) at stocking densities of approximately 40 l⁻¹, and incubated at 8 °C in darkness, with stagnant water and gentle aeration. Day 0 (D0) of the experiment was defined as the time when 50 % of the eggs had hatched. The larvae were reared according to the method of Rosenlund et al. (1993), with a gradual increase in temperature to 12 °C between D0 and D4, continuous illumination of each rearing unit by a 60 W light bulb from D4 onwards and a water exchange that started on D6 and increased gradually to 200 % day⁻¹. The salinity was 34 ‰, and the water was first filtered through a sand filter and then through 25, 10 and 5 μm cartridge filters before entering the tanks. Dead larvae were removed 2–3 times each week.

From D4 to D20, the algae Isochrysis galbana and Tetraselmis sp. were added to the tanks at a final concentration of 1 mg algal carbon l⁻¹ (Reitan et al., 1993). Rotifers were added from D5 to D20 at a constant density of 5 ml⁻¹ in the tanks. The rotifers were long-term-enriched to obtain a high (1.4; treatment 1) or low (0.2; treatment 2) ratio of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA). Otherwise the fat content and composition of the enrichments were identical (E. Kjørsvik, K. Hoehne, J. Rainuzzo, K. I. Reitan, T. F. Galloway and P. Makridis, in preparation). From D17 to D31, short-term-enriched Artemia nauplii (DHA:EPA ratio 0.7) were added to both treatments until a constant density of 1.5 ml⁻¹ was reached in the tanks. Both treatments had three replicates. The experiment was terminated on D31.

SGR is the specific growth rate and DW₁ and DW₂ are the mean larval dry masses at sampling times t₁ and t₂, respectively.

For light and electron microscopy, larvae were sampled, anaesthetised with metomidate hydrochloride (Wildlife Laboratories, Inc., CO 80524, USA) and fixed at regular intervals according to the method of Galloway et al. (1998). Standard length (SL; from tip of snout to end of notochord) and myotome height (MH) were measured on 10 larvae from each treatment at each sampling point (Fig. 1A). The onset of metamorphosis was determined by the first observation of fin rays in the dorsal and ventral unpaired fins. From each treatment, three fixed larvae at D5, D17 and D31 were selected randomly for further processing. The larvae were first measured individually for SL and MH and then embedded in Epon as described by Galloway et al. (1998). Transverse semi-thin (1 μm) and ultrathin (60 nm) sections were cut immediately posterior to the anus (see Fig. 1A). This region was selected to allow comparison with previous studies.

Serial semi-thin sections were stained with Toluidine Blue, Weigert’s eisenhaematoxylin (Romeis, 1968) or p-phenylenediamine (PPDA) (Hollander and Vaaland, 1968). The total cross-sectional area of red fibres in one epaxial quadrant of the myotome was measured directly on the sections (Fig. 1B) using a Leica Dialux microscope (magnification 40×) linked to a Leitz ASM 64K semi-automatic image-analysis system. The number of red fibres in the same cross-sectional area was counted, and the mean cross-sectional area of individual red fibres was calculated. Because of the large variations in the SL of the sectioned larvae, all muscle data are presented as a function of larval SL rather than larval age.

The outlines of all white muscle fibres in one epaxial quadrant of the myotome (see Fig. 1B) (magnification100×) were traced using a camera lucida. The resulting drawings were used to count the number of fibres and to measure the individual cross-sectional areas of the white fibres in this part of the myotome using a Leitz ASM 64K semi-automatic image-analysis system. These data were used to calculate the total cross-sectional area of white fibres. Since the number of new small fibres increased rapidly in germinal zones at the dorsal and ventral apices of the myotome, a distinction was made between areas consisting of small and large white fibres (see Fig. 6). The evaluation of fibre size proceeded from the notochord towards the layer of red fibres, and a fibre was considered to belong to the small fibre zone if its cross-sectional area was half or less of that of the fibre lying medial to it. The number of fibres was counted, and the mean cross-sectional area of individual fibres and total cross-sectional area of each fibre category were calculated. The relative contributions of hyperplasia and hypertrophy to growth of the small fibre zone were calculated using the method of Alami-Durante et al. (1997). The number of nuclei in the white muscle mass was counted directly on the sections (magnification 100×).

The ultrathin sections were contrasted with 2 % lead citrate and observed in a JEOL 1200EX electron microscope. Electron micrographs were analysed at a final magnification of 5000×. A square lattice grid (Weibel, 1979) was superimposed on the micrographs, and the number of points falling on myofibrils, mitochondria and other organelles was counted. From these data, the volume density of the respective organelle was calculated. Five fibres of each type from each larva were randomly chosen and analysed, in total 15 red and 15 white fibres from each treatment at each sampling point. At D31, the volume density of organelles was also measured in five fibres from the dorsal germinal zones in each of the three larvae, totalling 15 fibres from each treatment.

The results are expressed as means ± S.D. Data on dry mass, standard length and myotome height were compared using Student’s t-test. Data on total cross-sectional area, number and mean cross-sectional area of individual muscle fibres, number of nuclei and volume density of organelles were compared using a general factorial analysis of covariance (ANCOVA), with treatment type as a fixed factor and standard length as a covariate. All statistical comparisons were made at the 0.05 significance level and were performed using the program SPSS for Windows (SPSS Inc., Chicago, USA).

Larvae fed rotifers with a high DHA:EPA ratio (treatment 1) had a higher growth rate than larvae in the other group (treatment 2) during the whole experiment (Table 1) and hence a significantly greater dry mass on D17 and D31 (N=10–13, P<0.005) (Fig. 2). At hatching, the mean standard length (SL) was 4.0±0.4 mm (N=10) and the myotome height (MH) was 0.3±0 mm (N=10). There were no differences between treatments in SL or MH during the experiment. From the first day of feeding (D5) until D31, SL increased from 4.5±0.3 to 7.4±1.0 mm, while MH increased from 0.3±0 to 0.7±0.2 mm (N=10). From D17 onwards, the length-dependent larval dry mass in treatment 1 was significantly greater than in treatment 2 (N=10–13, P<0.001) (Fig. 2). Large variations in the SL of sectioned larvae were observed from D17 onwards.

The muscle fibre morphology was similar in larvae from both treatment groups during the experiment. On the first day of feeding, when the larvae were approximately 4.5 mm long, 7–8 layers of deep white muscle fibres were covered by a single layer of superficial red fibres (Fig. 3A). The white fibres contained bundles of myofibrils (60 % of fibre cross-sectional area) that encircled some mitochondria (10 %) and, occasionally, the nucleus (Fig. 3A). The red fibres contained numerous mitochondria (35 % of fibre cross-sectional area) and a bundle of myofibrils (30 %) located basally to the nucleus. Some presumptive myoblasts, with a heterochromatic nucleus, little sarcoplasm and few other organelles (definition of Veggetti et al., 1990), were wedged between the white and red fibres at this stage. After the onset of first feeding, the number of recruitment fibres increased in the dorsal, ventral and lateral germinal zones in the white muscle mass (see Fig. 6B–D). These fibres were relatively immature (Veggetti et al., 1990) and contained numerous myofibrils (approximately 50 % of cross-sectional area), mitochondria (10 %) and some peripherally located heterochromatic nuclei (Fig. 3B). Between the recruitment fibres were large intercellular spaces (Fig. 3B). Some small fibres could also be seen close to the notochord, and at the end of the experiment the white muscle mass consisted of 11–15 layers of fibres. The fibres in the deepest layers had peripherally located nuclei, and the bulk of each muscle fibre consisted of myofibrils (78 % of cross-sectional area) (Fig. 3C). At this stage, a single layer of laterally flattened red fibres still covered the white muscle mass, except at the level of the horizontal septum, where there were 2–4 layers of red fibres. The red fibres contained 35–40 % myofibrils and 45 % mitochondria (as a percentage of cross-sectional area).

The white fibres constituted approximately 80 % of the total muscle cross-sectional area at first feeding and 90 % at the end of the experiment, in both treatment groups. The total cross-sectional area of white fibres in one epaxial quadrant of the myotome increased at a significantly higher rate in larvae from the high-DHA:EPA treatment group (treatment 1) than in larvae from the low-DHA:EPA treatment group (treatment 2) (N=9, P=0.019) (Fig. 4). In larvae 8.5 mm long, the calculated total cross-sectional area of white fibres for the treatment 2 group was approximately 75 % of that for the treatment 1 group (Fig. 4).

At the onset of first feeding, when the larvae were approximately 4.5 mm long, the white muscle fibres ranged from 5 to 200 μm² in cross-sectional area, but only a few fibres were larger than 90 μm² (Fig. 5A). As the larvae grew, there was an increase in the size range of white fibres, and by D17 (SL 5.0±0.6 mm), there was a small population of fibres larger than 200 μm² and a slight increase in the number of fibres with a cross-sectional area of approximately 30 μm² (Fig. 5B,C). At the end of the experiment, there was a unimodal distribution of white fibre sizes around a median value of 28 μm² and an increased population of fibres larger than 200 μm² (Fig. 5D,E). The larvae in treatment 1 (Fig. 5D) apparently had a greater proportion of small fibres than the larvae in treatment 2 (Fig. 5E), but the standard deviations were large because of the variability in SL of the sectioned larvae, resulting in no significant difference in fibre cross-sectional area distribution between treatments. Fig. 6 shows the spatial distribution of differently sized white fibres in a transverse section of one epaxial quadrant of the myotome in larvae from treatment 1. The fibre cross-sectional area gradually increased away from the germinal zones. At the onset of first feeding, a few small recruitment fibres could be seen at the dorsal (Fig. 6A) and ventral borders of the white muscle mass. As the larvae grew, the number of small recruitment fibres increased at the dorsal, ventral and lateral borders of the white muscle mass and adjacent to the horizontal septum (Fig. 6B–D).

The total cross-sectional area of the small fibre zone increased at a significantly higher rate in larvae from treatment 1 than in larvae from treatment 2 (N=9, P=0.009) (Fig. 7A). At an SL of 8.5 mm, the small white fibres covered a cross-sectional area of approximately 28×10³ μm² in larvae from treatment 1 and 18×10³ μm² in larvae from treatment 2. These were 60 and 50 % of the total white cross-sectional area in the respective treatments. The number of small fibres also increased at a significantly higher rate in larvae from treatment 1 than in larvae from treatment 2 (N=9, P=0.031) (Fig. 7B): at 8.5 mm SL, the larvae from treatment 1 had approximately 850 small fibres per epaxial quadrant, while larvae from treatment 2 had approximately 630 (Fig. 7B). The mean cross-sectional area of small white fibres increased with increasing larval length, but there was no effect of treatment on this variable (Fig. 7C). The relative contributions of hyperplasia and hypertrophy to growth of the small fibre zone (calculation method of Alami-Durante et al., 1997) were 83 and 17 %, respectively, in the treatment 1 group and 82 and 18 %, respectively, in the treatment 2 group. The increase in total cross-sectional area of the large fibre zone was entirely due to hypertrophy. During the experiment, the number of large white fibres fluctuated around 60–65 in both treatments (Fig. 7E). No differences between treatments were found in the total large fibre zone cross-sectional area and the mean large fibre cross-sectional area (Fig. 7D,F, respectively).

There were no significant differences between treatments in the total cross-sectional area, number and mean cross-sectional area of red muscle fibres. The total cross-sectional area of red fibres in one epaxial quadrant increased significantly with increasing length of the larvae (N=18, P<0.001) from 1200±200 μm² at 4.5 mm SL to 3900±500 μm² at 8.5 mm SL.

The number of red fibres increased from 27±2 to 34±2 and the mean red fibre cross-sectional area increased from 44±8 to 118±10 μm² within the same larval size range.

The absolute number of white fibre nuclei counted in one epaxial quadrant increased significantly with increasing larval length (N=18, P<0.001), from approximately 20 at 4.5 mm SL to 200 at 8.5 mm SL, but no differences were found between the two treatment groups. On D17 and D31, the highest numerical density of nuclei was found in the dorsal and ventral germinal zones.

In the present study, an exponential increase in dry mass was observed from 5 mm SL onwards in Atlantic cod larvae (Fig. 2), as was also found in cod larvae reared in a sea enclosure (Pedersen et al., 1989). Furthermore, a low DHA:EPA ratio during the rotifer-feeding phase resulted in a substantially lower somatic growth rate than when rotifers with a high DHA:EPA ratio were fed to the larvae. This effect was not found when a diet with low DHA:EPA ratio was given during the Artemia phase (E. Kjørsvik, K. Hoehne, J. Rainuzzo, K. I. Reitan, T. F. Galloway and P. Makridis, in preparation). The somatic growth rate obtained with the high-DHA:EPA ratio diet (treatment 1) was comparable with the highest rate reported from other studies of Atlantic cod larvae (Kjørsvik et al., 1991; Rosenlund et al., 1993; van der Meeren and Næss, 1993).

At the end of the experiment, the larvae from treatment 1 had a significantly larger body mass than the larvae from treatment 2, although no differences in SL and MH were found between the two treatment groups. Thus, SL and MH alone do not seem to be adequate variables for describing differences in growth between cod larval groups. The results presented in Fig. 2 in the present study suggest that, in Atlantic cod larvae with low somatic growth rates, higher priority is put into reaching a certain length than into increasing muscle mass. Fish larval length is positively correlated with ontogenetic stage (Laurence, 1979; Fuiman and Higgs, 1997), and it is probably energetically beneficial for the cod larvae to increase rapidly in length because the ratio between inertial and viscous forces acting on swimming fish larvae (Reynolds number) is length-dependent (Fuiman and Webb, 1988; Osse and van den Boogaart, 1995).

In herring, there are three distinct phases of white muscle formation (Johnston et al., 1998): (1) a population of undifferentiated embryonic myoblasts arises prior to hatching; germinal zones of myoblasts arise at the dorsal and ventral borders of the myotome at 15 mm total length (TL; from tip of snout to end of finfold) and become depleted at 22–25 mm TL; and (3) myoblasts/satellite cells scattered in the myotome are activated after 25 mm TL. A similar sequence of muscle fibre recruitment seems to be present in larvae of the Atlantic cod, but it starts at a smaller size and earlier developmental stage than in herring larvae. Cod larvae hatch at 4.0 mm SL with relatively undifferentiated white fibres that mature during the yolk-sac stage (Galloway et al., 1998). At around the onset of first feeding (4.5 mm SL), new white fibres were recruited in germinal zones at the dorsal and ventral borders of the myotome (present study, Fig. 6A), indicating that the onset of muscle hyperplasia was triggered by endogenous energy resources, probably as a preparation for the rapid somatic growth observed after the onset of exogenous feeding. In addition, the cod larvae recruited white fibres laterally between the red and white muscle masses shortly after the onset of first feeding. Such lateral germinal zones have also been found in sea bass Dicentrarchus labrax (Veggetti et al., 1990), gilthead sea bream Sparus aurata (Rowlerson et al., 1995) and plaice Pleuronectes platessa (Brooks and Johnston, 1993). The larval fibre recruitment pattern mentioned above was found in the Atlantic cod up to D31, which was after the onset of metamorphosis. In juvenile and adult cod, the white muscle mass has a mosaic appearance (Greer-Walker, 1970), suggesting that the larval germinal zones are depleted during metamorphosis or in the period shortly after.

Species-specific differences seem to exist with respect to the relationship between hyperplasia and hypertrophy of muscle fibres in fish larvae. In herring larvae (Clupea harengus L.), hypertrophy is the predominant mechanism for increasing muscle mass from hatching (8–9 mm TL), recruitment of new white muscle fibres starting at 15 mm TL (Johnston et al., 1998). In yolk-sac larvae of the Atlantic salmon (Salmo salar L.), some authors have reported hypertrophy as the predominant growth mechanism in axial muscle (Stickland et al., 1988; Usher et al., 1994; Nathanailides et al., 1995), while others have reported a substantial contribution of hyperplasia to muscle growth at this stage (Johnston and McLay, 1997). These seemingly conflicting results may be related to genetic differences or to different methods of calculation. Irrespective of cod larval size in the present study, there were approximately 60 large white fibres per epaxial quadrant, the same number as in newly hatched cod larvae (Galloway et al., 1998). Growth of the large fibre zone was thus entirely due to hypertrophy. The small fibre zone did, however, grow both by hyperplasia and by hypertrophy. The small fibre zone constituted 60 % of the total white cross-sectional area in larvae of 8.5 mm SL from the treatment 1 group, and the relative contribution of hyperplasia to this was 83 %, so that the relative contribution of hyperplasia to the total white cross-sectional area was 50 %. Similarly, this value would be 41 % for treatment 2 larvae. This shows that high somatic growth rates in first-feeding cod larvae are associated with an increased contribution of hyperplasia to axial muscle growth. Increased somatic growth rates have been related to increased contributions of hyperplasia to muscle growth in larvae and juveniles of several other teleost species (Weatherley and Gill, 1987; Akster et al., 1995; Alami-Durante et al., 1997; Johnston et al., 1998), and hypertrophy has also been reported to be affected by somatic growth rate in several fish larval species (Hanel et al., 1996; Alami-Durante et al., 1997; Johnston et al., 1998).

In conclusion, short-term dietary deficiencies during the first-feeding stage of Atlantic cod larvae influenced the somatic growth rate, and length increase was given priority over muscle mass increase in the slower-growing larvae. Muscle growth occurred both by hypertrophy and by hyperplasia, and increased somatic growth rates were associated with increased white fibre hyperplasia. The subsequent growth potential may thus be affected by inadequate early larval feeding. As a result of species-specific differences, alternative methods for describing muscle hypertrophy and hyperplasia may be required, depending on the prevailing growth mechanism at each ontogenetic stage.

The study was financed by the Norwegian Research Council, within the programme for Rearing of Marine Species (project no. 103754/100). T. Bardal and G. K. Totland are thanked for practical help and fruitful discussions during and following the experiment. Two anonymous referees are also thanked for their thorough revision and constructive criticism of the manuscript.