Differentiation of swimming muscles and gills,and development of anaerobic power in the larvae of cyprinid fish (Pisces,Teleostei)

Summary Histological, histochemical, morphometric and electrophoretic methods were combined to study the differentiation of the swimming muscles and of the gills in the larvae and juveniles of cyprinids during the first 3 months after hatching. The bodies of recently hatched larvae are always surrounded by a single layer of muscle fibres (red layer) which possess strong cytochrome oxidase (COX) activity but whose myofibrils do not show the arrangement typical of the fibres of adult red muscle. The contribution of the red layer to total muscle mass decreases from about 12% on day 3 to 4% on day 40 post-hatching, after which the red layer becomes indistinguishable from the developing mass of adult red muscles. The adult fibres seem to originate through splitting from the larval fibres. The inner muscle masses of recently hatched larvae also display conspicuous COX activity which, however, disappears gradually. The development of the gill surface follows a time course which during the first 40 days is a mirror image of that of the larval red layer of muscle fibres, i.e. increasing at about the same rate as the latter decreases. The most cathodic of the isoenzymes of LDH (M₄), an indicator of the glycolytic capacity of the white muscle of adult fish, develops only slowly in the larvae. In whole body homogenates, the dominant isoenzyme after hatching is the aerobic H₄-form, and it is not until 2 weeks or so that the anaerobic M₄ is the strongest fraction in electropherograms of total body homogenates. In the bleak, Alburnus alburnus (Linné, 1758), which spends the first 8–10 days after hatching on the bottom of the aquarium, the M₄ isoenzyme takes much longer to develop than in Rutilus rutilus (Linné, 1758) or Chondrostoma nasus (Linné, 1758) which start swimming within 3 days after hatching. All these findings suggest that in the early larvae swimming is almost entirely aerobic, being powered by the deep layers of muscle fibres. During this developmental phase the superficial red layer perhaps is the main respiratory organ.     

Muscle development in larvae of a fast growing tropical freshwater fish, the curimatã-pacú

The distribution and ultrastructure of myotomal muscle fibres was studied in larvae and early juveniles of the curimatã-pacú Prochilodus marggravii , a tropical freshwater fish endemic to the São Francisco River system, Brazil. At 26°C, larvae hatched 15 h post-fertilization at a relatively early stage of development with the head still curved around the yolk-sac (head-trunk angle greater than 45°), and prior to pigmentation of the eyes and formation of the jaws, gut and pectoral fins. Although motile the swimming muscles of newly-hatched larvae were largely undifferentiated. The myotomes were made up of a single layer of superficial muscle fibres containing six to eight myofibrils and abundant mitochondria, surrounding an inner core of myoblasts, myotubes and immature muscle fibres. The volume densities of mitochondria and myofibrils in the immature inner muscle fibres of 1-day-old lavae were 14.5 and 6.4% respectively. The body axis straightened within 24 h of hatching and the yolk sac was completely absorbed by 72 h. Larval development was rapid with gill filaments, a muscular stomach, liver and swimbladder present after 7 days. The inner muscle fibres were well differentiated in 7-day-old larvae; the volume density of myofibrils had increased to 63.1% whereas the volume density of mitochondria had decreased to 3.5%. In 14-day-old juveniles the superficial muscle had thickened to a layer two to three fibres thick in the region of the lateral line nerve and capillaries were present in the inner muscle. Muscle growth until 14 days was largely due to the hypertrophy of the fibres present at hatching.     

Muscle growth in yolk-sac larvae of the Atlantic halibut as influenced by temperature in the egg and yolk-sac stage

Atlantic halibut eggs and yolk-sac larvae were incubated at 1, 5 and 8° C. Eggs incubated at 8° C gave slightly shorter larvae at hatching with a significantly smaller total cross-sectional area of white muscle fibres than eggs incubated at 5° C. Transport of eggs 2 days prior to hatching gave significantly longer larvae at hatching with a significantly larger red fibre cross-sectional area than when eggs were transported shortly after the blastopore closure. A higher survival until 230 degree days after hatching was also observed in the former group. All eggs incubated at 1° C died before hatching and all larvae incubated at 1° C died before 45 degree days after hatching. From hatching until 230 degree days the total white cross-sectional area increased threefold in all temperature groups. The increase in white cross-sectional area was entirely due to hypertrophy between hatching and 150 degree days (10 mm L _S). Recruitment of new white fibres increased in germinal zones at the dorsal, ventral and lateral borders of the myotome from 150 degree days onwards, but at 230 degree days (12–13 mm L _S) the recruitment fibre zone constituted <10% of the total white cross-sectional area. Larval incubation at 8° C gave slightly longer larvae with a significantly larger cross-sectional area of recruitment fibres at 230 degree days than incubation at 5° C. The larval group incubated at 8° C also had a significantly lower survival until 230 degree days than did the 5° C group. Incubation temperature regimes did not affect the volume density of myofibrils in the axial muscle fibres at 230 degree days. Thus hypertrophy is the predominant mechanism of axial white muscle growth in Atlantic halibut yolk-sac larvae and an increased rearing temperature during the yolk-sac stage increases white muscle fibre hyperplasia.     

Profiles of enzyme activity in larvae of two cyprinid species with contrasting life styles (Cyprinidae; Teleostei)

Growth and the development of gills, muscle fibres and 10 enzymes serving different metabolic functions were studied in larvae of Rutilus rutilus (L.) and Chalcalburnus chalcoides (Agassiz, 1832). R. rutilus starts swimming and feeding one to three days after hatching, whereas in C. chalcoides this process is delayed by about 10 days. This difference in behaviour is reflected in the time-course of growth, the differentiation of the red muscle fibres and the activity of the enzymes of aerobic energy metabolism. On the other hand, the activity of the enzymes of anaerobic energy metabolism increases steadily throughout the period of observation (up to 60 days post-hatch), this trend being more pronounced in C. chalcoides than in R. rutilus . A weight-independent and a weight-dependent phase of development can be distinguished in the enzymes of aerobic energy metabolism. It is suggested that, in accord with previous findings, the early phase of locomotor activity of cyprinid larvae is fuelled mainly by aerobic processes, and that the central muscle mass of the larvae is more aerobic than the white muscle fibres of the adults. The delayed development of aerobic enzyme activity in C. chalcoides is compensated by the accelerated development of anaerobic enzyme activity, particularly of creatine kinase and lactate dehydrogenase. This difference between the two species studied suggests differences in the metabolic basis of burst activity.     

Growth dynamics of white and red muscle fibres in fast- and slow-growing strains of rainbow trout

Compared with fish of a slow-growing strain, fast-growing rainbow trout exhibited significantly smaller white fibre diameters, throughout development from hatching to 24 cm body length, although possessing similar total number of fibres. In contrast, in red muscle, no differences were observed in fibre diameter between the two strains, but the fast growing fish showed a significantly higher number of red fibres. The differences in growth rate between the two strains were related to the mean white fibre diameter and were found to be matched by proportional adjustments in recruitment of new fibres to the growing muscle. Thus, the largest and fastestgrowing strain showed evidence of sustained higher recruitment of muscle fibres that endowed this strain with the potential to maintain rapid somatic growth for longer and accomplish greater muscle growth.     

Temperature and neuromuscular development in the tambaqui

The development of muscle innervation pattern was investigated in larvae of the Amazonian fish, the tambaqui Colossoma macropomum. The time to hatching decreased from 28–29 h at 23.5° C to 11–12 h at 31° C. The larvae hatched after the completion of somitogenesis (38-somite stage) at 23.5° C but only at the 33-somite stage at 28–31° C. Embryos were stained for acetylcholinesterase activity and with an acetylated tubulin antibody in order to visualize neural processes. All muscle fibre types were initially innervated at their myoseptal ends. The development of motor innervation to the trunk muscle was delayed with respect to hatching at higher temperatures. At hatching, muscle fibres were innervated only to somites 16–17 at 28–31° C and somite 23–26 at 23.5–25° C (counting from the head), although the larvae swam vigorously to avoid sinking. In contrast, in newly hatched larvae myofibrils were present right along the trunk at all temperatures in both the superficial and inner muscle fibres. At hatching numerous multi-layered membrane contacts with the ultrastructural characteristics of gap junctions, were found between muscle fibres and at the inter-somite junctions, suggesting the somites were initially electrically coupled. These structures disappeared concomitant with the development of muscle endplates right down the trunk. The larvae started feeding 5 days post-hatch at 28° C. First feeding was associated with a dramatic decrease in the volume density of mitochondria and an increase in the volume density of myofibrils in the inner muscle fibres. The polyneuronal and multi-terminal pattern of innervation characteristic of adult slow-muscle fibres also developed around the time of first feeding.     

Early ontogeny of Ancherythroculter nigrocauda and effects of delayed first feeding on larvae

Development of embryos and larvae in Ancherythroculter nigrocauda Yih et Woo (1964) and effects of delayed first feeding on larvae were observed after artificial fertilization. The fertilized eggs were incubated at an average temperature of 26.5°C (range: 25.7–27) and the larvae reared at temperatures ranging from 21.8 to 28°C. First cleavage was at 50 min, epiboly began at 7 h 5 min, heartbeat reached 72 per min at 24 h 40 min and hatching occurred at 43 h 15 min after insemination. Mean total length of newly hatched larvae was 4.04 ± 0.03 mm (n = 15). A one‐chambered gas bladder was observed at 70 h 50 min, two chambers occurred at 15 days, and scales appeared approximately 30 days after hatching. Larvae began to feed exogenously at day 4 post‐hatch at an average temperature of 24°C. Food deprivation resulted in a progressive atrophy of skeletal muscle fibres, deterioration of the larval digestive system and cessation of organ differentiation. Larval growth under food deprivation was significantly affected by the time of first exogenous feeding. Starved larvae began to shrink, with negative growth from day 6 post‐hatch. The point of no return (PNR) was reached at day 11 after hatching. Mortality of starved larvae increased sharply from day 12 after hatching.     

Cellular localization of insulin-like growth factor-II protein in the sea bass (Dicentrarchus labrax) from hatching to adult

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.     

Minute tubercles on the skin surface of larvae in the Korean endemic bitterling, Rhodeus pseudosericeus (Pisces, Cyprinidae)

The morphology and distribution of the minute tubercles on the skin surface of larvae in Korean bitterling, Rhodeus pseudosericeus, were observed during larval development. Just after hatching, the epidermis of the larvae consists of a thin single cell layer having smaller basophilic flat or round‐flattened basal cells. As the larvae grow, the epidermis contains more small flat cells and large epidermal cells that are round or hemisphere‐shaped. These large unicellular epidermal cells, called minute tubercles, consist of more or less homogeneous cytoplasm that is PAS (Periodic acid‐Schiff method) positive. They are more densely distributed in the wing‐like yolk sac projection. Vestigial minute tubercles occur in the body region and the caudal fin‐fold region. These minute tubercles grow in number and height from 6 to 8 days after hatching onward. However, they become reduced in height and number as the larvae develop. At day 31 after hatching (i.e. free‐swimming stage), minute tubercles no longer exist on the larval skin. The sequence of occurrence and gradual disappearance of these cell structures are described and histologically documented for comparative purposes of beta, taxnomomic and environmental studies.     

Expression of titin isoforms in red and white muscle fibres of carp (Cyprinus carpio L.) exposed to different sarcomere strains during swimming

Titin (also known as connectin) is a striated-muscle-specific protein that spans the distance between the Z- and M-lines of the sarcomere. The elastic segment of the titin molecule in the I-band is thought to be responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscle types express isoforms of titin that differ in their molecular mass. To help to elucidate the relation between the occurrence of titin isoforms and the functional properties of different fibre types, we investigated the presence of different titin isoforms in red and white fibres of the axial muscles of carp. Gel electrophoresis of single fibres revealed that the molecular mass of titin was larger in red than in white fibres. Fibres from anterior and posterior axial muscles were also compared. For both white and red fibres the molecular mass of titin in posterior muscle fibres was larger than in anterior muscle fibres. Thus, the same fibre type can express different titin isoforms depending on its location along the body axis. The contribution of titin to passive tension and stiffness of red anterior and posterior fibres was also determined. Single fibres were skinned and the sarcomere length dependencies of passive tension and passive stiffness were determined. Measurements were made before and after extracting thin and thick filaments using relaxing solutions with 0.6 mol · l⁻¹ KCl and 1 mol · l⁻¹ KI. Tension and stiffness measured before extraction were assumed to result from both titin and intermediate filaments, and tension after extraction from only intermediate filaments. Compared to mammalian skeletal muscle, intermediate filaments developed high levels of tension and stiffness in both posterior and anterior fibres. The passive tension-sarcomere length curve of titin increased more steeply in red anterior fibres than in red posterior fibres and the curve reached a plateau at a shorter sarcomere length. Thus, the smaller titin isoform of anterior fibres results in more passive tension and stiffness for a given sarcomere strain. During continuous swimming, red fibres are exposed to larger changes in sarcomere strain than white fibres, and posterior fibres to larger changes in strain than anterior fibres. We propose that sarcomere strain is one of the functional parameters that modulates the expression of different titin isoforms in axial muscle fibres of carp. Accepted: 7 May 1997     

Morphological and biochemical variations in the gills of 12 aquatic air-breathing anabantoid fish

All fish species in the Anabantoidei suborder are aquatic air-breathing fish. These species have an accessory air-breathing organ, called the labyrinth organ, in the branchial cavity and can engulf air at the surface of the water to assist in gas exchange. It is therefore necessary to examine the extent of gill modification among anabantoid fish species and the potential trade-offs in their function. The experimental hypothesis that we aimed to test is whether anabantoid fishes have both morphological and functional variations in the gills among different species. We examined the gills of 12 species from three families and nine genera of Anabantoidei. Though the sizes of the fourth gill arch in three species of Trichogaster were reduced significantly, not all anabantoid species had morphological and functional variations in the gills. In these three species, the specific enzyme activity and relative protein abundance of Na(+)/K(+)-ATPase were significantly higher in the anterior gills as compared with the posterior gills and the labyrinth organ. The relative abundance of cytosolic carbonic anhydrase, an indicator of gas exchange, was found to be highest in the labyrinth organ. The phylogenetic distribution of the fourth gill's morphological differentiation suggests that these variations are lineage specific, which may imply a phylogenetic influence on gill morphology in anabantoid species.     

Histomorphology of the early yolk-sac larvae of the Atlantic halibut (Hippoglossus hippoglossus L.)—an indication of the timing of functionality

Halibut larvae hatch at a very immature stage, and the duration of the yolk-sac period is very long (up to 50 days). This paper describes the histomorphological development of organs of the yolk-sac larvae (6° C) by use of light and electron microscopy. Rudimentary branchial cavities were open from 2 days after hatching. Kidneys seemed functional 16 days after hatching and onwards, and primitive lamellae on the gill arches were beginning to form at this age. Pancreatic zymogen granules were first observed 20daysafter hatching. The liver was segmented into lobes between 20 and 23 days after hatching, and the gall bladder seemed functional from day 23. The hindgut became extensively folded from day 26, and branchial capillaries were first observed at this stage. The larvae were able to catch food particles 24 days after hatching. Judging from ultrastructural observations, it seemed that halibut larvae were able to digest food particles between day 24 and 26 after hatching (around 150 daydegrees and 50% yolk absorption).     

Histochemical and immunohistochemical profile of pink muscle fibres in some teleosts

Summary The pink muscle of several Teleosts was examined immunohistochemically using antisera specific for the myosins of red and white muscle, and histochemically using various methods for demonstrating myosin ATPase (in ATPase) activity.In the catfish the pink muscle consists of 2 different layers of fibres. The superficial layer has a low mATPase activity after both acid and alkali pre-incubation, whereas the deeper layer has a high mATPase activity after acid and alkali pre-incubation, being more resistent to these conditions even than is the white muscle.In the trout the pink muscle is composed of fibres with the same mATPase activity as in the superficial pink muscle of the catfish, whereas in the rock goby, goldfish, mullet and guppy the pink muscle is like the deep pink layer of the catfish.Immunohistochemically the fibres of the pink muscle behave like the white muscle fibres except in the guppy and rock goby in which at the level of the lateral line there occurs a transition zone between red and pink fibres. The fibres of this region react with both anti-fast and (to a lesser extent) anti-slow myosin antisera, and have a mATPase activity which, going from the superficial to the deeper fibres, gradually loses the red muscle characteristics to acquire those of the main pink muscle layer.     

Ultrastructure aspects of Brycon gouldingi (Teleostei,Characidae) related to swimming ability and feeding during larval development

The larval ultrastructure of Brycon gouldingi related to swimming and feeding from hatching to total yolk absorption is described from scanning electron micrographs. Newly hatched larvae (time zero) had no mouth opening, undefined optic vesicles, an olfactory plate visible as a shallow depression, rudimentary gill arches, neural groove, embryonic fin and a primary neuromast in the dorsal region of the head. At the time of yolk absorption, 55 h post hatching, the larvae presented an optic vesicle comprising an optic cup and crystalline lens; a mouth with tongue, tapered teeth and taste buds; a ciliated olfactory cavity; branched gill arches; filled neural groove signalling central nervous system development; caudal, pectoral, dorsal and anal fins; and neuromasts distributed throughout the head and body. These characters are related to prey capture and swimming ability, key aspects of survival during the larval stage. The results of this study provide important information for exploitation and aquaculture of B. gouldingi.     

Histochemical and immunohistochemical profile of pink muscle fibres in some teleosts

The pink muscle of several Teleosts was examined immunohistochemically using antisera specific for the myosins of red and white muscle, and histochemically using various methods for demonstrating myosin ATPase (mATPase) activity. In the catfish the pink muscle consists of 2 different layers of fibres. The superficial layer has a low mATPase activity after both acid and alkali pre-incubation, whereas the deeper layer has a high mATPase activity after acid and alkali pre-incubation, being more resistent to these conditions even than is the white muscle. In the trout the pink muscle is composed of fibres with the same mATPase activity as in the superficial pink muscle of the catfish, whereas in the rock goby, goldfish, mullet and guppy the pink muscle is like the deep pink layer of the catfish. Immunohistochemically the fibres of the pink muscle behave like the white muscle fibres except in the guppy and rock goby in which at the level of the lateral line there occurs a transition zone between red and pink fibres. The fibres of this region react with both anti-fast and (to a lesser extent) anti-slow myosin antisera, and have a mATPase activity which, going from the superficial to the deeper fibres, gradually loses the red muscle characteristics to acquire those of the main pink muscle layer.     

Growth of swimming muscles and its metabolic cost in larvae of whitefish at different temperatures

The temperature relationship of routine metabolic rate (R_r) of non-feeding, non-growing Coregonus lavaretus larvae between 2 and 15°C is characterized by Q₁₀-values ranging from l.8-2.45. The rate of growth, based on weight determinations, of first-feeding larvae amounted to 3.5, 7.6 and 9.4% day-¹ at 5, 10 and 12°C respectively, from which Q₁₀-values between 4.0 and 4.8 can be calculated. The rate of increase of muscle mass between 5 and 10°C, based on the determination of the cross-sectional area of inner muscle fibres, resulted in a Q₁₀-value of 4.5. Water temperature influenced the pattern of growth of the inner muscle fibres. At hatching, after 360 day degrees, total muscle mass of larvae reared at 4 and 8°C was independent of temperature, but at 4°C the rate of mass increase owed more to hyperplasia (increase in fibre number) than to hypertrophy (increase in fibre mass), whereas at 8°C the opposite was the case. The calculation of power budgets (including the metabolic cost of growth) of first-feeding larvae yielded net conversion efficiencies (K₂) increasing with temperature from 46.3% at 5°C to 54.7% at 12°C. Comparing our data with literature data two general conclusions can be drawn. (1) In first-feeding larvae the net, but not the gross, conversion efficiency of food energy increases with temperature. This is due to net energy input being characterized by a much higher Q₁₀-value than energy expenditures. (2) In embryos of freshwater fish so far investigated hyperplasia plays a greater role in the increase of fibre mass than hypertrophy at the lower temperature, whereas in embryos of marine fish hyperplasia prevails at the higher temperature. It is suggested that this discrepancy correlates with the high concentration of free amino acids in the eggs of marine species which provide an additional, easily available, source of metabolic energy absent in freshwater species.     

Respiratory system of arachnids II: morphology of the tracheal system of Leiobunum rotundum and Nemastoma lugubre (Arachnida, Opiliones)

The tracheal system of two species of harvestmen with different life styles was investigated: the long-legged, Leiobunum rotundum (Phalangioidea, Phalangiidae) and the short-legged Nemastoma lugubre (Troguloidea, Nemastomatidae). The morphology of the tracheae is very similar in both species: The branching pattern is basically asymmetric and dichotomous, and the tracheae taper between branching points. The tracheal diameters range from 160 to 0.3 mum in L. rotundum (mean body mass 25.3 mg), and from 70 to 0.5 mum in N. lugubre (mean body mass 3.8 mg). Ultrastructurally, the tracheal walls are similar to those of insects, consisting of an outer hypodermal layer and an inner cuticular layer with taenidial structures. Tracheae of all diameters have close contact with organs and muscles, indicating that diffusive gas exchange may take place through the walls of all tracheae. The finest tracheae end freely in the hemolymph or at the surface of organs and muscles. Exceptions are tracheal penetration of the central nerve mass in the prosoma in both species, and of the muscles in L. rotundum. The latter may correlate with the active perambulatory life style of L. rotundum.     

Amphibian hatching gland cells: pattern and distribution in anurans

The hatching gland (HG) is a transient organ, found in most anuran embryos and early larvae, and located on the dorsal side of the head. The enzymes secreted by hatching gland cells (HGCs) aid the embryos to escape from their enveloping coats. Analysis of HG morphology and distribution in 20 anuran species from six families using scanning electron microscopy revealed small differences in the shape and pattern of the gland particularly in the length and width of the posterior mid-dorsal extension of the gland. The four species of foam-nest making leptodactylids examined had HGs of a somewhat different shape to the others, but otherwise, there was little sign of a relationship between HG shape and taxonomic position. In the single Eleutherodactylus species examined, cells with the appearance and location of HGCs were transiently present long before the active stage of hatching. No sign of HGCs was seen on the head surface of one species, Phyllomedusa trinitatis. It seems possible that in this species, hatching is achieved by a mechanical rather than an enzymatic mechanism. The microvilli characteristic of the surfaces of HGCs were quite variable in density and length from species to species, and at different stages. HGCs remained at the surface of the embryo for some time after hatching and the possibility of a post-hatching function is briefly discussed.     

版纳鱼螈胚胎和幼体鳃的退化观察

为了解版纳鱼螈(Ichthyophis bannanicus)胚胎和幼体鳃的退化特征,同窝卵中8枚在胚胎成熟阶段早期被随机取出剖产,随后余下的9枚卵在胚胎成熟阶段晚期时孵出或剖产,观察幼体或胚胎鳃的变化。成熟阶段早期的胚胎,初时其鳃色鲜红,鳃枝充盈,鳃丝与鳃轴近乎垂直;随后鳃色发白,鳃枝疲软,鳃丝卷曲,鳃丝与鳃轴夹角变小。成熟阶段晚期的胚胎和刚孵出的幼体,初时其鳃色纯白,鳃枝疲软,鳃丝卷曲,鳃丝与鳃轴夹角较小;随后鳃逐渐脱落。观察发现,版纳鱼螈胚胎或幼体鳃的退化模式不是重吸收,也不是重吸收加脱落,而仅为脱落。鳃退化过程经历外鳃供血减少-供血停止-鳃枝组织死亡和脱落3个阶段,每个阶段都有明显的外部形态特征。胚胎进入成熟阶段后,越早孵化,鳃枝留存越多,反之,鳃枝留存越少。发白后的鳃枝随机性脱落。