Parvalbumin isotypes were isolated by chromatography from trunk white muscle of rainbow trout (Oncorhynchus mykiss W.), brown trout (Salmo trutta L.), and sea bass (Dicentrarchus labrax L.). Five, four and two components were respectively purified and physico-chemically characterized. Expression of the various isotypes was followed in the course of the fish development and, in adult fish, from the anterior to the posterior myotomes. Isotype distribution varies both chronologically and spatially. In trout, parvalbumins occur around hatching; as the fish develop, transitions occur in isotype expression, PA II appearing as the predominant larval form and PA III, IV, or V as the main adult form, as previously observed in Barbus barbus (L.). In the sea bass, the developmental expression pattern is more unexpected: the synthesis of both isotypes (PA II and PA V) is delayed and the larval form PA II remains the principal isotype in adult fish. These observations indicate that the polymorphism of parvalbumins in fish constitutes a subtle mechanism modulating the speed and power of muscle contraction. Our results support the view that each isotype plays a specific role in relation to the muscle activity required in fish at a given developmental stage or a given trunk level in the adult. Published in : Comparative biochemistry and physiology (1996), vol. 113B, n°3, pp. 475-484 DOI: 10.1016/0305-0491(95)02066-7 Status : Postprint (Author’s version)

Developmental changes in myofibrillar protein and parvalbumin isoform composition were investigated in the myotomal muscle of the flatfish Solea solea, characterized by a very brief metamorphic stage. Results were compared with previously obtained data on another pleuronectiform teleost, the turbot (Scophthalmus maximus), displaying prolonged metamorphosis. Electrophoretically measurable changes in myofibrillar proteins and parvalbumins were detected late in the sole, after completion of metamorphosis. In the course of development, muscles showed the usual sequential synthesis of isoforms of the myofibrillar proteins myosin light chain LC2, troponin-T, and troponin-I. An adult parvalbumin isoform (PA III) was found to predominate during sole growth. The two flatfish were characterized by highly species-specific parvalbumin isoforms. Compared with turbot, the profiles of the myofibrillar subunits and parvalbumin isoforms varied little in the course of sole development. The early appearance of adult traits might be correlated with the brevity of metamorphosis of this fish.

Several polyacrylamide gel electrophoresis techniques were used to study developmental changes in myofibrillar protein composition and parvalbumin distribution in the myotomal muscle of Brycon moorei. Two myosin LC2 chains and two troponin I isoforms were successively detected. Up to four troponin T isoforms were synthesized. Slow red‐muscle myofibrils from adult fish showed no common component (except actin) with larval, juvenile or adult fast white‐muscle myofibrils. During growth of B. moorei, two classes of parvalbumin isoforms were sequentially expressed: larval PA I, PA IIa, and PA IIb and adult PA III. In adult fish, the content in Tn T‐2 isoform decreased from the anterior to the posterior myomeres, in favour of Tn T‐1 and Tn T‐4. The parvalbumin content also diminished from the rostral to the caudal muscle. The fast rate of transition from larval to adult isoforms appeared to parallel the extremely fast growth of B. moorei. Sequential expression of these isoforms presumably reflected variations in the contractile properties of the muscle fibres, required by changes in physiological demands of the propulsive musculature.

Eleven parvalbumin isotypes expressed during the development of clariids Heterobranchus longifilis and Clarias gariepinus and claroteid Chrysichthys auratus were purified and electrophoresed on sodium-dodecyl-sulfate polyacrylamide gels. Immunochemical cross-reactions among these proteins were investigated by immunoblotting, using purified antibodies raised against three isotypes chosen at different stages of fish development. Antibodies raised against H. longifilis PA I (larval–juvenile isotype) and against C. gariepinus PA IIIa (juvenile–adult isotype) cross-reacted to a rather similar extent despite a weaker cross-reaction of anti-PA IIIa with larval–juvenile isotypes. On the other hand, antibodies raised against H. longifilis PA IV (an exclusively adult isotype) recognized markedly only H. longifilis PA IV and C. gariepinus PA IIIb. These two adult isotypes most likely belong to the α lineage, and all the others to the β lineage. These results show that parvalbumin isotypes synthesized at different stages of fish growth differ structurally, and that the most marked difference is between larval–juvenile and adult clariid isotypes.

A bstract Expression o f polym orphic myofibrillar and sarcoplasm ic proteins w as investigated in the fish Scophthalm us maximus (L.) undergoing m etam orphosis. A range of electrophoretic techniques w as used to m onitor sequential synthesis o f isoforms from hatching to the adult stage. Two isoforms (larval and adult) o f m yosin light chain LC2 and troponin-I were suc­ cessively detected during turbot growth, in addition to variations in the peptide com position o f m yosin heavy chains. Two isoform s of troponin-T also appeared sequentially, but the first to make its appearance w as not detected until the juvenile stage. The com position o f alkali light chains, actin, tropom yosin, and troponin-C did no t seem to change as the fish progressed through the different stages. Parvalbum in isoform s were isolated and their phy­ sico-chemical param eters defined. As in the other fish exam ined so far, there appeared a succession o f larval (PA Ila and PA lib ) and adult (PA V) parvalbum in isoform s through the life o f the fish. A ll these biochemical changes occurred gradually in the course of turbot development, and did no t appear particularly related to m etam orphosis but rather to phy­ siological needs o f the different growth stages.

The white‐muscle parvalbumin isoforms of Clarias gariepinus, Heterobranchus longifilis and Chrysichthys auratus were purified and their physicochemical parameters determined. The three catfish isoforms are distinct but those of C. gariepinus and H. longifilis are more similar. In the course of development, the successive appearance of larval and adult parvalbumins was observed. Larval isoforms (PA I, PA IIa, PA IIb) displayed a lower isoelectric point (pI) and molecular mass than adult ones (PA IIc, PA IIIa, PA IIIb, PA III, PA IV). The PA IIa isoform appeared as an omnipresent typical larval isoform. PA IIb appeared mostly larval, being insignificant in adult specimens; its physicochemical features were the same in the three catfish species. In Chrysichthys auratus, there were three PA II isoforms, one appearing as an adult isoform (PA IIc). The fact that the two types of parvalbumin isoforms appear at different times should reflect specific physiological needs (mobility, feeding) of different developmental stages.

Developmental changes in myofibrillar protein composition were investigated in the myotomal muscle of the African catfish, Heterobranchus longifilis (Clariidae), by several electrophoretic techniques. The main muscle fibres of larvae and the fast-white muscle fibres of juvenile and adult fish were found to express distinct myosin heavy chain and myosin light chain 2 (LC2) isoforms. Three myosin LC2 chains were successively detected, differing by their isoelectric points. In contrast, the alkali light chains remained qualitatively and quantitatively unchanged during fish growth. Actin, α-tropomyosin, and troponin-C (TN-C) were also similar in larval, juvenile, and adult white muscle, but an additional larval tropomyosin isoform was found in the first developmental stages. Two isoforms of troponin-T (TN-T) and troponin-I (TN-I) were synthesised in the course of fish growth. Transition from the larval to the adult isoform was much faster for TN-T than for TN-I. Slow-red muscle myofibrils from adult H. longifilis showed no common component (except actin) with larval, juvenile, or adult fast-white muscle myofibrils. Red myofibrils displayed a single TN-T and a single TN-I isoform, but two isoforms of TN-C. The myofibrillar protein isoforms synthesised at any given developmental stage almost certainly reflect changes in the functional requirements of swimming muscles in the course of fish development.

At hatching, Dicentrarchus labrax larvae are 3.0 mm long and devoid of any cephalic skeleton. At 3.6 mm, the Meckelian cartilage appears, after which the whole skeleton develops so slowly and gradually that clear-cut stages are impossible to define. Some cephalic elements, however, develop faster than others. Skeletal development is subject to constraints imposed by vital functions such as aquatic respiration and feeding. As the yolk sac shrinks, the branchial parts develop. By the time the vitellus is completely exhausted the mandible, pharyngeal jaws, hyoid bar, and parts of the suspensorium and operculum are present. Though still incomplete, these structures are probably sufficient to allow ingestion of exogenous food. Further development should enable the larvaes to perform suction feeding, as is typical of perciforms, Before the shift to exogenous feeding, the cartilaginous floor of the skull remains open, but the opening is then closed by the parasphenoid and basioccipital, so the brain is completely isolated from the buccal cavity. The cranial vault and ethmoid region develop later: these structures are probably less essential to fry survival than the earlier and more rapidly developing structures.

The inception and development of the cartilaginous cephalis skeleton of Chrysichthys auratus is described from hatching to about 18 days post‐hatching. At hatching, no skeletal structure is present. Not until day 3 do clearly delimited cranial primordia become apparent. As in many siluriforms, the neurocranium is platybasic from the start, the suspensorium constitutes, with Meckel's cartilage and the hyoid bar, a single cartilaginous element, and the junction between the front and rear of the neurocranium is complete on day 4. By day 8 the quadratomandibular joint has formed and the tectum posterius has appeared. Cartilage reduction first affects the trabecular bars, then, markedly, the visceral arches. By day 18 the braincase floor has almost disappeared.

Parvalbumin isotypes PA II, PA III, PA IVa, and PA IVb were isolated by chromatography from trunk white muscle of barbel and physicochemically characterized. Electrospray ionization mass spectroscopy revealed that PA II has a lower molecular weight than the other isotypes and that PA IVa and PA IVb each consist of two subforms. Isotype distribution was studied by polyacrylamide gel electrophoresis. In adult fish, the total parvalbumin titre decreased and the isotype distribution varied from the anterior to the posterior myotomes. In the course of barbel development, the total parvalbumin titre increased rapidly as fish standard length increased from 1·3 to 5 cm; then sloped down gently as the length increased to 60 cm. At least six parvalbumin isotypes were identified, three of which are different forms (a, b, and c) of PA II. These three forms were present together at the larval stage, but PA IIc and chiefly PA IIb appeared as early isotypes, contrary to PA IIa which was present until the adult period. Later PA IVb accounted for up to 90% of the total parvalbumin content; PA III and PA IVa are minor adult isotypes. Temporal and spatial variations in the total parvalbumin titre and in the differential expression of barbel parvalbumin isotypes very likely reflected the functional requirements of the fish axial musculature according to fish size and myotome location. Physiologically, the larval isotypes could promote faster relaxation of fast fibres than the adult isotypes, and hence favour shorter contraction times.

Polyacrylamide gel electrophoresis was used to analyse the distribution of parvalbumin, myosin light chain, and troponin I isoforms in white muscles of larval, juvenile, and adult Chrysichthys auratus (catfish, siluriforms) and to study the kinetics of their synthesis. Parvalbumin isoform PA II was first detected from day 5 post-hatching and was the main "larval" isoform in this species. PA III appeared at the beginning of the juvenile stage but always remained the minor isoform, even in adult fish. Young mature specimens (approximately 12 cm long) displayed the highest total parvalbumin content. Adult-type myosin light chains were detected from day 8. Densitometric analysis confirmed the light-chain distribution typical of fish muscles, with a relatively high amount of LC3 and a low amount of LC1. We evidenced a "larval" form of troponin-I and its progressive replacement by an "adult" form.

At hatching, Heterobranchus longifilis does not display any primordia of the cephalic skeleton. The latter appears 12 h post–hatching and develops in three stages up to day 16. The first stage (12 h to 2 days) involves almost exclusively the development of the chondrocranium. During the second period (days 3–8), dermal elements of the splanchnocranium appear. The final stage is marked by resorption of the cartilages, progressively replaced by ossifications (days 10–16). At their appearance the elements of the splanchnocranium are fused together, as are the first neurocranial elements. Later, the splanchnocranium splits up. By the time the yolk sac is completely resorbed, the buccal and pharyngeal jaws are present, the suspensoria and hyoid bars are partially developed, and the parasphenoid partially closes the hypophyseal fenestra. These structures delimit a buccal cavity that is probably functional, i.e. capable of participating in the intake of exogenous food. Next to continue its development is principally the splanchnocranium, completing the walls of the buccal cavity. Cartilage resorption parallels the appearance of endochondral ossifications (except for the trabecular bars). Braincase closure begins to accelerate once the buccal system is complete.

Five goat latissimus dorsi muscles (LDM) were submitted to a progressive chronic electrostimulation program to reach an integrated understanding of the fast-to-slow transformation process in large mammals. LDM were regularly sampled and followed during a period of 8 months. Each sample was simultaneously assessed for histoenzymological study, myosin and LDH isoforms and bioenergetic capacities [NADH dehydrogenase cytochrome c oxidoreductase (NADH Cyt c OR), succinate dehydrogenase cytochrome c oxidoreductase (Succ Cyt c OR), cytochrome c oxidase (Cyt c Ox) and LDH]. Such muscles were also tested with and without completion of II to I transformation for their mechanical properties in isometric and isotonic strain gauge testing. The conversion of fast-to-slow myosin monitored by heavy chain (HC I) and light chain slow component (LC2S) began a few days after stimulation and was almost 100% after 100 days. The H-LDH isoforms evolved similarly but did not reach 100% conversion after 200 days. The activity of respiratory chain oxidases increased within 36 h but to a variable extent and peaked after 32 days, corresponding to a 75% transformation of myosin compared to initial levels. NADH Cyt c OR, Succ Cyt c OR, and Cyt c Ox, respectively increased 10-, 5- and 5-fold. These activities then significantly decreased before the completion of the myofibrillar transformation and reached a plateau with stable activities that remained 2- to 3-fold higher than the unstimulated LDM. LDH activity sharply decreased until day 62 (5-fold) and then plateaued. Functionally, muscle showed a reduced speed of contraction and moderate reduction in power output but had become fatigue-resistant. This study documents the transformation process in large mammals and suggests the dynamic relation between workload, aerobic-anaerobic metabolism and the contractile myofibrillar system.

The inception, and development of the cephalic skeleton of Barbus barbus from hatching to 24 days passes through periods of fast and slow growth; these rates are not the same in different parts of the skull. Trabeculae, parachordal plates, Meckelian cartilages and hyposymplectics are present at hatching. Then the cartilaginous floor of the neurocranium develops, the pars quadrata, the hyoid bars and branchial arches elements appear shortly before the first movable dermal bones, the dentaries, maxillae and opercles. The first bone of the braincase to appear is the parasphenoid; other bones develop subsequently and at the same time: the angular, quadrate, interopercle and fifth ceratobranchial. Later the splanchnocranium continues to develop at a relatively fast rate while the neurocranium shows little growth. The braincase does not begin to close before the 24th day, nor do the first bones of the skull roof appear, while the bucco‐pharyngeal apparatus is complete, having the adult shape. The early constitution of the latter structures seems to be linked with the mechanical demands of biological functions such as breathing and feeding.

Muscle proteins were investigated in two large European barbels, Barbus barbus and B. meridionalis , and in four small tropical barbels native to SE Asia: B. conchonius , B. tetrazona , B. sachsi and B. titteya . Polyacrylamide gel electrophoresis was used to analyse myosin heavy and light chains and parvalbumin isotypes from white trunk muscle. Each species could be biochemically identified. The myosin subunit and parvalbumin isotype patterns obtained for the two European barbels were similar. The Asian barbels, on the other hand, not only differed from the European species but displayed a greater diversity within their group. These biochemical results are largely in agreement with morphological and genetic data, but fail to substantiate suggested close relationships between Asian barbel species.

1. Actomyosin extracts of trunk, heart, and head muscles from barbel (Barbus barbus L.) were analyzed by SDS-polyacrylamide gel electrophoresis to study their myosin heavy chain composition. 2. Four heavy chain isoforms were found: trunk white, trunk red, and ventricle muscles yielded one heavy chain typical of the muscle type; head muscles devoid of red fibers displayed two heavy chain isoforms, the slow migrating one corresponding to the trunk white muscle type. 3. The electrophoretic mobility of red and ventricle myosin heavy chains related to that of white isoforms appeared highly modified by the glycerol content of the gels.