Potential phasic and tonic muscles express a common set of fast and slow myosin light chains and fast tropomyosin during early development of chick embryo

We investigated the expression of myosin light chains and tropomyosin subunits during chick embryonic development of the anterior (ALD) and posterior (PLD) parts of the latissimus dorsi muscles. As early as day 8 in ovo, both muscles accumulate a common set of myosin light chains (LC) in similar ratios (LC1F : 55 per cent; LC2S : 25 per cent; LC2F : 12 per cent ; LC1S : 8 per cent) and a common set of tropomyosin (TM) subunits (β², β¹, α²_F).Later during development, the slow components of the LC regularly disappear in the PLD and the fast components of the LC and the α²_FTM disappear in the ALD, so that the adult pattern is almost established at the time of hatching.Thus, early in development, the two muscles accumulate a common set of fast and slow myosin light chains and fast tropomyosin and some isoforms are repressed at a later stage during development. These data might suggest that during development, the regulatory mechanisms of muscle specific isoform expression differ from one contractile protein to another.     

A new muscle phenotype is expressed by subcultured quail myoblasts isolated from future fast and slow muscles

Differentiation of quail myoblasts, isolated from thigh pectoralis and anterior latissimus dorsi muscle, was analyzed in primary cultures and in cultures obtained following repeated subculturing. Our study shows that quail myoblasts can survive many generations without losing their ability to form myotubes. However, during these subcultures the cells progressively express a new phenotype. This phenotype is characterized by a mixture of myosin light chains such that LC1F, LC2F, and LC2S are present in roughly equimolar amounts, each accounting for 25 to 30% of the total light chain synthesis while LC1S accounts for the remaining 10 to 15%, and by a mixture of fast and slow alpha tropomyosin in which alpha S accounts for 10 to 15% of the alpha subunits synthesis. Clonal analysis indicates that all cells in the population express this phenotype which is also characteristic of subcultures obtained from both future fast and slow muscles. Relationships between this phenotype and muscle development are discussed.     

Influence of neurons on the occurrence of fibre types and myosin light chains in cultured presumptive slow and fast myoblasts

Myoblasts from 9-day-old quail embryo slow anterior latissimus dorsi (ALD) and fast posterior and latissimus dorsi (PLD) muscles were co-cultured with neurons. The presence of neurons allowed ALD-derived muscle fibres to express characteristic features of a slow muscle (occurrence of alpha' and of beta' fibres and predominance of slow myosin light chains). On the contrary, PLD-derived fibres did not differentiate into normal fast fibres (occurrence of alpha'-like fibres and absence of LC3f). These results are compared with the differentiation of ALD and PLD myoblasts in aneural condition. It is suggested that neurons can modify some phenotypic expression of presumptive slow or fast myoblasts.     

Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit striated muscles.

We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The β subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the α subunit of tropomyosin gradually increases so that in adult skeletal muscles the α subunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the α subunit which remains invariant with development. Two subspecies of the α subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC₃ light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles.     

Differentiation of slow and fast muscles in chickens

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.     

Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers

Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.     

Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres.

Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide ''maps'' published in Cleveland. Fischer, Kirschner & Laemmli [(1977) J. Biol. Chem. 252, 1102-1106], allowed a classification of muscle fibres into four classes, corresponding to histochemical types I, IIA, IIB and IIC. Type I fibres with a pure slow-twitch-type of myosin were found to be characterized by a unique set of isoforms of troponins I, C and T, in agreement with the immunological data of Dhoot & Perry [(1979) Nature (London) 278, 714-718], by predominance of the beta-tropomyosin subunit and by the presence of a small amount of an additional tropomyosin subunit, apparently dissimilar from fast-twitch-fibre alpha-tropomyosin subunit. The myofibrillar composition of type IIB fast-twitch white fibres was the mirror image of that found for slow-twitch fibres in that the fast-twitch-fibre isoforms only of the troponin subunits were present and the alpha-tropomyosin subunit predominated. Type IIA fast-twitch red fibres showed a troponin subunit composition identical with that of type IIB fast-twitch white fibres. On the other hand, a unique type of myosin heavy chains was found to be associated with type IIA fibres. Furthermore, the myosin light-chain composition of these fibres was invariably characterized by a small amount of LC3F light chain and by a pattern that was either a pure fast-twitch-fibre light-chain pattern or a hybrid LC1F/LC2F/LC3F/LC1Sb light-chain pattern. By these criteria type IIA fibres could be distinguished from type IIC intermediate fibres, which showed coexistence of fast-twitch-fibre and slow-twitch-fibre forms of myosin light chains and of troponin subunits.     

Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in rat.

The histochemical ATPase activity and the myosin light chains of a rat fast muscle (extensor digitorum longus, EDL) and a rat slow muscle (soleus) during development have been investigated. Both muscles initially synthesize fast myosin light chains and show the intense histochemical ATPase activity characteristic of adult fast muscle fibers. After birth, the soleus begins to accumulate slow fibers with their characteristic low histochemical ATPase activity, and slow myosin light chains begin to appear. Sciatic neurectomy prevents the development of slow fibers and the synthesis of slow myosin light chains in the soleus, while the EDL is unaffected. Similarly, cordotomy of an adult rat results, in the soleus, in the appearance of fibers with more intense staining for ATPase and an increase in fast myosin light chains. The EDL is unchanged by cordotomy. As a result, we suggest that slow muscle development, but not fast muscle development, is dependent upon the functional activity of the nervous system.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.     

Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle

Isoforms of C-protein in adult chickens which differ in fast (pectoralis major, PM) and slow (anterior latissimus dorsi, ALD) skeletal muscles can be distinguished immunochemically with monoclonal antibodies (McAbs) specific for the respective fast (MF-1) and slow (ALD-66) protein variants (Reinach et al., 1982 and 1983). The expression of these C-proteins during chick muscle development in vivo has been analyzed by immunoblot and immunofluorescence procedures. Neither MF-1 nor ALD-66 reacted with whole-cell lysates or myofibrils from PM of 12-day-old embryos. However, both McAbs bound to peptides of 145 kDa in PM from late embryonic and young posthatched chickens. All of the myofibers in these muscles reacted with both antibodies, but the binding of the anti-slow McAb (ALD-66) diminished progressively with age and was completely negative with PM by 2 weeks after hatching. In contrast, the ALD muscle from 17 days in ovo thru adulthood only reacted with ALD-66; no binding of MF-1 could be detected at these stages. Since both fast and slow myosin light chains (LC) coexist within embryonic pectoralis and ALD muscles (e.g., G. F. Gauthier, S. Lowey, P. A. Benfield, and A. W. Hobbs, 1982, J. Cell Biol.92, 471–484) yet segregate to specific fast and slow muscle fibers at different stages of development, the temporal transitions of C-protein and myosin LC were compared during myogenesis. “Slow-type” C-protein appeared after the disappearance of slow myosin light chains, whereas the accumulation of the “fast-type” light chains occurred before the expression of “fast-type” C-protein. The pattern of isoform transitions appears to be far more complex than previously suspected.     

Changes in tropomyosin subunit pattern in chronic electrically stimulated rabbit fast muscles.

The tropomyosin subunit ratio of rabbit fast muscle (α:β = 80:20) changes to that characteristic of skeletal slow muscles (α:β = 55:45) on continuous (10 Hz) stimulation for 3 weeks. The altered myosin light chain pattern and histochemical ATPase stain also show clear changes of fast → slow transformation. However, the rate of changes in the light chain patterns of myosin are slower than those of tropomyosin subunits. These results do not support the previous finding (Amphlett et al., Nature $\text{257}$, 602, 1975) that the tropomyosin subunit pattern remains unaltered during transformation of skeletal muscles and the conclusion that the genetic expression of tropomyosin is regulated under separate control from other myofibrillar proteins. Rather, our results suggest that the polymorphic patterns of all myofibrillar proteins in skeletal muscles undergo changes in a temporal manner during skeletal muscle transformation.     

Developmental changes in myosin isoforms from slow and fast latissimus dorsi muscles in the chicken

In the course of muscle differentiation, changes in fibre-type population and in myosin composition occur. In this work, the expression of native myosin isoforms in developing fast-twitch (posterior latissimus dorsi; PLD) and slow-tonic (anterior latissimus dorsi; ALD) muscles of the chick was examined using electrophoresis under nondissociating conditions. The major isomyosin of 11-day-old embryonic PLD comigrated with the adult fast myosin FM3. Two additional components indistinguishable from adult fast FM2 and FM1 isomyosins appeared successively during the embryonic development. The relative proportion of these latter isoforms increased with age, and the adult pattern was established by the end of the 1st month after hatching. Between day 11 and day 16 of embryonic development, PLD muscle fibres also contained small amounts of slow isomyosins SM1 and SM2. This synthesis of slow isoforms may be related to the presence of slow fibres within the muscle. At all embryonic and posthatch stages, ALD was composed essentially of slow isomyosins that comigrated with the two slow components SM1 and SM2 identified in adult. Several studies have reported that the SM1:SM2 ratio decreases progressively throughout embryonic and posthatching development, SM2 being predominant in the adult. In contrast, we observed a transient increase in SM1:SM2 ratio at the end of embryonic life. This could reflect a transitional neonatal stage in myosin expression. In addition, the presence in trace amounts of fast isomyosins in developing ALD muscle could be related to the presence of a population of fast fibres within this muscle.     

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of mysoin light chains in addition to α and β forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, α-fast, α-slow, and β-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, β-tropomyosin and the α-fast form of tropomyosin, are found. In addition, β-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.     

Characterization of human muscle myosins with respect to the light chains.

Isolated myosins from human predominantly fast and slow muscles, human neonatal and foetal muscle were examined for light chain composition by one- and two-dimensional electrophoresis. The LC1F, LC2F and LC3F light chains were identical with their counterparts from rabbit fast myosin. Human LC1S was identified by correlative criteria as a single component having a molecular weight slightly lower than, but an electric charge similar to, that of rabbit LC1Sb. Consequently, human LC1S appears to be much less heterogeneous relative to LC1F than is the case with other mammalian species. A high immunological cross-reactivity was likewise observed, with antibody specific to rabbit LC1F, between the isolated myosins from several human mixed muscles and rabbit fast myosin, though reactivity was highest with foetal myosin (having a pure-fast-light-chain pattern).     

Biphasic expression of slow myosin light chains and slow tropomyosin isoforms during the development of the human quadriceps muscle

Using a two-dimensional electrophoresis technique coupled with sensitive silver staining, we have investigated the chronology of appearance of the myosin light chain and tropomyosin isoforms during early stages of human quadriceps development. Our results show that slow myosin light chains and the slow tropomyosin isoform are not detected at 6 weeks of gestation. These isoforms transiently appear between 12.5 weeks and 15 weeks of gestation and then disappear. The slow myosin light chains are re-expressed at 31 weeks of gestation and the slow tropomyosin isoform later at 36 weeks of gestation, and normally remained expressed into the adulthood. Our study thus reveals a biphasic expression of the slow myosin light chains and the slow tropomyosin isoform in developing human quadriceps muscle.     

Calcium regulation of muscle contraction.

Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.     

Posthatching changes in levels and molecular forms of acetylcholinesterase in slow and fast muscles of the chicken: Effects of denervation and direct electrical stimulation

The evolution of acetylcholinesterase (AChE) activity and AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of chickens 2-18 days of age. In ALD as well as in PLD muscles, the AChE-specific activity increased transiently from day 2 to day 4; the activity then decreased more rapidly in PLD muscle. During this period asymmetric AChE forms decreased dramatically in ALD muscle and the globular forms increased. In PLD muscle, the most striking change was the decline in A8 form between days 2 and 18 of development. Denervation performed at day 2 delayed the normal decrease in AChE-specific activity in PLD muscle, whereas little change was observed in ALD muscle. Moreover, A forms in these two muscles were virtually absent 8 days after denervation. Direct electrical stimulation depressed the rise in AChE-specific activity in denervated PLD muscle and prevented the loss of the A forms. Furthermore, the different molecular forms varied according to the stimulus pattern. In ALD muscle, electrical stimulation failed to prevent the effect of denervation. This study emphasizes the differential response of denervated slow and fast muscles to electrical stimulation and stresses the importance of the frequency of stimulation in the regulation of AChE molecular forms in PLD muscle during development.     

Myofibrillar proteins in skeletal muscles of parr,smolt and adult atlantic salmon (Salmo salarl.). Comparison with another salmonid,the arctic charr Salvelinus alpinus (l.)

The heavy and light subunits of myosin from white and red muscles of Atlantic salmon parr, smolt and adult individuals were analyzed by SDS-PAGE and two-dimensional electrophoresis. Tropomyosin was identified by comigration with rat tropomyosins in two-dimensional gels in the presence and absence of urea. These myofibrillar proteins were compared to those of Arctic charr.

• 1.1. The myosin heavy chain from Atlantic salmon red muscles was associated with two types of light chain, 1S and 2S, that comigrated with the light chains 1S and 2S of Arctic charr.
• 2.2. As in the Arctic charr, four myosin light chain spots were detected in white muscles: two fast myosin light chains type 1, one of which comigrated with its analogous in the Arctic charr; one fast myosin light chain type 2, differing slightly in isoelectric point from that of Arctic charr; and one fast myosin light chain type 3 with higher electrophoretic mobility than that of Arctic charr.
• 3.3. Three tropomyosin spots were detected. White muscles contained only one type of β-tropomyosin and red muscles two types of α-tropomyosin. These three tropomyosin spots comigrated with those of Arctic charr.
• 4.4. Two myosin heavy chain bands were observed in red muscles of salmon parrs but only one in the rest of the red muscles analyzed.
• 5.5. Only one myosin heavy chain band was detected in white muscles by SDS-glycerol-polyacrylamide gel electrophoresis. Alfa-chymotryptic peptide mapping of these white myosin heavy chain bands revealed differences attributed to the presence of a new type of myosin heavy chain first detected several months after smoltification.