Temperature acclimation in crucian carp, Carassius carassius L., morphometric analyses of muscle fibre ultrastructure

Carp show a partial compensation in metabolic rate and activity following temperature acclimation. In the present study crucian carp, Carassius carassius , were acclimated for eight weeks to either 2deg; C or 28deg; C. The effects of temperature acclimation on muscle fibre ultrastructure has been investigated. The fractional volume (%) of each fibre type occupied by mitochondria and myofibrils was determined using a point counting morphometric method. Mitochondrial density was found to be higher in the muscles of cold (red fibres 25%; pink fibres 20% and white fibres 4%) than in those of warm acclimated fish (red fibres 14%, pink fibres 11%, white fibres 1%). The proportion of subsarcolemmal to intra-myofibrillar mitochondria was significantly lower in the red fibres of cold acclimated fish. Metabolic compensation to low temperatures are therefore associated with an increase in the number of mitochondria per cell. In contrast, the fractional volume occupied by myofibrils actually decreased following cold acclimation. Evidence is reviewed that temperature compensation of contractile activity results from qualitative rather than quantitative changes in myofibrillar proteins.     

Plasticity of myosin heavy chain expression with temperature acclimation is gradually acquired during ontogeny in the common carp (Cyprinus carpio L.)

Common carp (Cyprinus carpio L.) were reared from hatching until 61 mm total length (TL) at 21 degrees C. At 14 weeks and 20 weeks post-hatch, corresponding to initial lengths of 30 mm and 44 mm respectively, fish were acclimated to 10 degrees C using a rate of cooling of 1 degrees C per day. A statistical model was used to compare the time course in the change of white muscle myofibrillar ATPase activity with temperature acclimation. The myosin heavy chain (MHC) composition of white muscle myofibrils was investigated using peptide mapping. A significant increase in myofibrillar ATPase activity was observed after 2-3 weeks in the 44 mm group, but not until 4-5 weeks in the 30 mm group. when they had reached 37 mm TL. The MHC banding pattern of 120 mm TL fish acclimated to 10 degrees C or 21 degrees C for a minimum of 6 weeks were distinct from each other. The MHC peptide map characteristic of 10-degrees C-acclimated fish was not observed in individuals less than 37 mm length. We therefore conclude that the capacity to alter the composition and properties of myofibrils with cold acclimation is acquired in juvenile carp at around 37 mm TL.     

Homeostatic regulation of acetazolamide-sensitive esterase activity in the bluegill sunfish, Lepomis macrochirus, following acute cold shock

Acetazolamide-sensitive esterase activity was elevated in branchial homogenates of control juvenile bluegill sunfish, Lepomis macrochirus , acclimated at 20° C but decreased rapidly within 9 h following an acute hypothermal shock to 8° C. After 2 weeks at 8° C, shocked-fish enzyme activity was similar to control fish. At 20° C acclimation temperature, specific activity of bluegills was similar in swimbladder, liver, kidney, gill, spleen, and gonad homogenates and was significantly higher (α=0.05 level) in whole blood homogenates. The pH optima for enzymes extracted from fish acclimated at 20° and 8° C were 7.29 and 8.00, respectively. Polyacrylamide gel electrophoresis (PAGE) demonstrated two distinct forms of acetazolamide-sensitive esterase activity present in both 20° and 8° C acclimated fish. Specific activity for homogenates from both 20° and 8° C acclimated fish differed significantly when assayed at 20° C, suggesting both qualitative and quantitative changes in acetazolamide-sensitive esterase. It is postulated that relatively rapid alterations in esterase activity promote survival in bluegill following acute cold shock through the central role of enzymes in the regulation of plasma ion concentrations and acid/base equilibria.     

The effects of temperature acclimation on organ/tissue mass and cytochrome c oxidase activity in juvenile cod (Gadus morhua)

Cod were acclimated to 5 and 15° C (cold and warm acclimation, respectively) for at least 43 days after which tissue-somatic indices, tissue protein, DNA content, and cytochrome c oxidase (CCO) activity were measured. Liver, stomach, intestine, total heart and ventricle-somatic indices were all increased significantly in the cold acclimated animals compared with their warm acclimated counterparts. There were no differences in gill or white muscle-somatic indices between the acclimation temperatures. Tissue protein concentration (mg protein g tissue⁻¹) was generally unaffected by temperature acclimation. Cold acclimation resulted in higher white muscle and lower ventricle CCO specific activities(μmol cytochrome c oxidized min⁻¹· g tissue⁻¹) compared with the respective warm acclimated tissues. No significant differences in CCO specific activity were observed in the remaining tissues (when measured at an intermediate temperature of 10° C). Total tissue CCO activity (measured at an intermediate temperature of 10° C) did not differ significantly between the cold and warm acclimated fish.     

Energy reserves during food deprivation and compensatory growth in juvenile roach: the importance of season and temperature

The effect of 21 days of starvation, followed by a period of compensatory growth during refeeding, was studied in juvenile roach Rutilus rutilus during winter and summer, at 4, 20 and 27° C acclimation temperature and at a constant photoperiod (12L : 12D). Although light conditions were the same during summer and winter experiments and fish were acclimated to the same temperatures, there were significant differences in a range of variables between summer and winter. Generally winter fish were better prepared to face starvation than summer fish, especially when acclimated at a realistic cold season water temperature of 4° C. In winter, the cold acclimated fish had a two to three‐fold larger relative liver size with an approximately double fractional lipid content, in comparison to summer animals at the same temperature. Their white muscle protein and glycogen concentration, but not their lipid content, were significantly higher. Season, independent of photoperiod or reproductive cycle, was therefore an important factor that determined the physiological status of the animal, and should generally be taken into account when fish are acclimated to different temperature regimes. There were no significant differences between seasons with respect to growth. Juvenile roach showed compensatory growth at all three acclimation temperatures with maximal rates of compensatory growth at 27° C. The replenishment of body energy stores, which were utilized during the starvation period, was responsible for the observed mass gain at 4° C. The contribution of the different energy resources (protein, glycogen and lipid) was dependent on acclimation temperature. In 20 and 27° C acclimated roach, the energetic needs during food deprivation were met by metabolizing white muscle energy stores. While the concentration of white muscle glycogen had decreased after the fasting period, the concentrations of white muscle lipid and protein remained more or less constant. The mobilization of protein and fat was revealed by the reduced size of the muscle after fasting, which was reflected in a decrease in condition factor. At 20° C, liver lipids and glycogen were mobilized, which caused a decrease both in the relative liver size and in the concentration of these substrates. Liver size was also decreased after fasting in the 4° C acclimated fish, but the substrate concentrations remained stable. This experimental group additionally utilized white muscle glycogen during food deprivation. Almost all measured variables were back at the control level within 7 days of refeeding.     

Consequences of thermal change on the myofibrillar ATPase of five freshwater teleosts

Myofibrillar ATPase activity was measured in the epaxial musculature of five freshwater species of fish acclimated to extremes of temperature within their tolerance ranges. Changes in the enzyme activity were apparent in carp, tench and roach, cold acclimated fish (10°C) having higher enzyme activity levels than hot acclimated fish (28°C). Such changes were not apparent in eels or brook trout. Alteration of the enzyme activity took less than 4 weeks, and was totally reversible. This suggests that seasonal adaptation to environmental temperatures is possible, thus maintaining locomotory efficiency.     

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.     

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.     

Post-larval growth in the lateral white muscle of the eel, Anguilla anguilla

The post-larval growth of lateral white muscle was studied in eels at different stages of post-larval development (glass, yellow and silver eels) by means of histochemical methods for myosin-ATPase (mATPase) activity, immunohistochemistry (for myosin isoforms) and electron microscopy.
Morphological, histo- and immunohistochemical data reveal a uniform appearance of white muscle in glass eels, whereas in following stages the typical mosaic appearance is present. Small-diameter fibres show a more acid-labile mATPase activity than large fibres and react with anti-F, anti-FHC and anti-S sera, but not with anti-SHC serum. In the silver stage, the small fibres tend to decrease in number as the size of the eels increases.
Electron microscopy reveals the presence of satellite cells at every stage: in glass eels there are also 'activated' elements showing scarce myofilaments in their cytoplasm; in yellow eels very small fibres are present, enveloped within the basal lamina of well-differentiated muscle fibres; in silver eels there are no fibres showing signs of immaturity.
Presumably the post-larval development of white muscle involves in juvenile eels a substantial recruitment of fibres from the satellite cell population; later the hyperplasia decreases or ceases and hypertrophy remains the only mechanism for muscle growth.     

A study of the swimming performance of the Crucian carp Carassius carassius (L.) in relation to the effects of exercise and recovery on biochemical changes in the myotomal muscles and liver

A study has been made of the maximum sustained swimming speed of Crucian carp Carassius carassius (L.) using a fixed velocity technique. The data obtained from swimming tests on 214 carp have been analysed using the method of probit analysis. The 50% fatigue level for 13–16 cm fish acclimated to 9.5±0.6°C has been estimated to be 3.35 lengths/sec. Biochemical measurements have been made on the red and white myotomal muscles and liver of fish subjected to both varying intensities of sustained swimming and short periods of vigorous swimming. Free creatine was found to increase only during high speed swimming in the white muscle. Elevated lactate concentrations occurred at both low and high sustained swimming speeds in the red superficial muscle but not during short periods of strenuous exercise. Glycogen depletion from the red musculature also only took place at the sustained swimming speeds investigated. The reverse situation was operative in the white muscle, significant glycogen depletion occurring only at the highest swimming speed studied. Lactate levels were only significantly different from non-exercised fish in the fish swimming at the higher velocities. The effects of periods of recovery following 200 min of sustained swimming were also investigated. White muscle lactate was at a higher level than non-exercise fish 5 h post-exercise, while both red muscle glycogen and lactate rapidly returned to pre-exercise concentrations. Biochemical measurements on the myotomal muscle types have been discussed in relation to the swimming performance of the fish and the division of labour between red and white fibres.     

Muscle development in Atlantic salmon (Salmo salar) embryos and the effect of temperature on muscle cellularity

Salmon eggs were incubated at 5, 8 or 11° C from fertilization to hatching. At Gorodilov stages 25, 27, 29, 31 and 33 transverse sections of whole embryos (at somite level 10–15) were prepared for histochemistry and electron microscopy. At every stage up to hatching, cross–sectional areas of the embryos were not different between temperatures, and from stage 27 onwards there was also no difference in the ratio of white to red muscle. However, there were more muscle fibres but of smaller average diameter in both the red and white muscle for the colder temperature embryos. At hatching there were also more nuclei (per cross–section) in the colder embryos but more nuclei per muscle fibre in the warmer embryos. In all cases the 8° C embryos were intermediate between 5 and 11° C embryos in their muscle parameters. Fast and slow muscle fibres could only be distinguished in the embryos by alkali–stable ATPase reactions. Succinic dehydrogenase activity was low in embryonic fish. No differences between the temperature groups were detected in the histochemical reactions for either ATPase or succinic dehydrogenase activities.     

Temperature acclimation and the expression of contractile protein isoforms in the skeletal muscles of the common carp (Cyprinus carpio L.)

Summary Common carp (Cyprinus carpio L.) were acclimated to either 2, 5, 8, 11, 15, 20, or 23°C for 12 weeks (12 h light: 12 h dark). Fish did not feed after 6 weeks at temperatures below 8°C. Skinned fibres were prepared from fast myotomal muscle by freeze-drying. Measured at 0°C unloaded contraction velocity (Vmax) and maximum isometric tension generation (Po) were 2–3 times higher in the 11°C-than 23°C-acclimated groups, and had intermediate values in 15 °C-acclimated fish. Po and Vmax at 0°C were not significantly different for carp maintained at 2, 5, 8, or 11°C. Measured at the acclimation temperature of each group Vmax and Po were 51% and 71% lower for fibres from 2°C- than 23°C-acclimated fish. The results indicate a partial capacity adaptation of muscle power output in fish acclimated between 11°C and 23°C. At 8°C the ATPase activity of myofibrils was 2 times higher in fish acclimated to 8°C than to 20°C. The effects of temperature acclimation on the protein composition of myofibrils was investigated using one- and two-dimensional electrophoresis. Peptide maps of purified myosin heavy chains and actin prepared by proteolytic digestion with either Staphylococcus aureus V8 protease or chymotrypsin were similar for both acclimation groups. The molecular weights and isoelectric points of the major isoforms of tropomyosin, troponin C, troponin I, troponin T, and myosin light chains (MLC1, MLC2 and MLC3) were also similar in 8°C- and 20°C-acclimated carp. A 20 kDa molecular weight protein with a pI intermediate between that for MLC2 and MLC3 was found in myofibrils and single fibres from carp acclimated to 8°C but was not present in carp acclimated to 20°C. It is suggested that this band corresponds to a myosin light chain isoform unique to cold-acclimated fish. Evidence was also obtained that myofibrils from warm-acclimated fish contained a second minor isoform of troponin I.     

The effects of temperature acclimation on the oxygen consumption and enzyme activity of red and white muscle fibres isolated from the tropical freshwater fish Oreochromis niloticus

The standard oxygen consumption rate and the activities of muscle citrate synthase, creatine phosphokinase and lactate dehydrogenase in the tropical fish Oreochromis niloticus acclimated to either 20.5 ± 0.3° C or 26.5 ± 0 ± 5 ± C for at least 3 months were investigated. The standard oxygen consumption rate of individual fish from the two acclimation temperatures was determined at 20, 25 and 30 ± C. At all experimental temperatures, the standard oxygen consumption rate of fish acclimated to 20.5 ± 0.3° C was significantly higher than that of fish kept at 26.5 ± 0.5 ± C. In both groups smaller individuals had a higher oxygen consumption rate than large ones.
Analyses of the activity levels of citrate synthase (CS), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) in both red and white muscles isolated from fish kept under the two temperature regimes were performed at 26 ± C. The activity of CS in both red and white muscles isolated from the 20.5 ± 0.3° C acclimated fish was significantly higher than that of muscles isolated from the 26.5 ± 0.5 ± C acclimation group. Similarly, the CPK activity in white muscles isolated from fish acclimated to 20.5 ± 0.3 ± C was higher than that of muscles obtained from the 26.5 ± 0.5 ± C acclimation group. However, the CPK activity in red muscles isolated from the two fish groups was not significantly different. The opposite results were obtained for LDH activity. For example, the LDH activity of white muscles isolated from fish acclimated to 26.5 ± 0.5 ± C was significantly higher than that of the same muscles but from the 20.5 ± 0.3 ± C acclimated fish. No differences were observed in the LDH activity of red muscles isolated from the two fish groups.     

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.     

Enhanced contractile activity of goldfish skeletal muscle related to cold acclimation

Dorsal muscles were isolated from goldfish acclimated to 8 degrees C and 25 degrees C, and stimulated electrically at 5-35 degrees C. Contractile activity of isolated muscle from the 8 degrees C fish was highest at 15 degrees C, whilst that of the 25 degrees C fish was independent of experimental temperature. The activity was significantly increased with cold acclimation. Pyruvate and lactate contents of red and white muscles increased with work. Addition of iodoacetic acid into the incubation medium caused a decrease of lactate production and contractile activity in muscle from the 8 degrees C fish, suggesting that the increase of the activity was partly associated with an increased activity of anaerobic glycolysis of the tissues.     

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0·0%; T2 = 1·1%; T3 = 1·7%; T4 = 4·0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH‐TR reaction and strong acid‐stable mATPase activity, and a deep white one, most of the muscle mass, formed by fibres with weak NADH‐TR reaction and strong alkali‐stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid‐stable or acid‐labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1–T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters ≤8 μm was higher in the lysine treatments, and with diameters between 16 and 24 μm, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 μm and in T1–T4 were between 8 and 16 μm. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy.     

Muscle development in the tambaqui, an important Amazonian food fish

Eggs of the tambaqui Colossoma macropomum were incubated at 28 and 31) C. Somitogenesis started shortly after the formation of the neural plate and notochord. New somites were added at the rate of one every 13 min at 28) C and one every 11 min at 31) C. Myogenesis started in the most rostral myotomes at the 9-somite stage and proceeded in a caudal direction. Mononuclear myotubes with the morphological characteristic of muscle pioneer cells were observed lateral to the notochord. The majority of myotubes were formed from the fusion of 3–6 spindle-shaped myoblasts. Myofibril synthesis started soon after cell fusion at the periphery of myotubes. Close membrane contacts and 'gap'-type junctions were observed between myotubes, immature muscle fibres and at the inter-somite boundary, suggesting that the cells were electrically coupled. Embryos exhibited rhythmic movements at the 20-somite stage, and hatched at the 29–30-somite stage 15–18 h post-fertilisation (PFT) at 28° C and 11 h PFT at 31° C. Larvae hatched at a comparatively early stage of development prior to the completion of somitogenesis and the formation of eye pigment, pectoral fins and jaws. The myotomes comprised a single superficial layer of well-differentiated muscle fibres which contained abundant mitochondria, overlying an inner core of myotubes (presumptive white muscle layer). Differentiation and growth during the larval stages was extremely rapid, and the juvenile stage was reached after little more than 6 days at 28° C.     

Alterations to the swimming performance of carp, Cyprinus carpio, as a result of temperature acclimation

Groups of common carp were acclimated to either 10°C or 28°C for 6 weeks. Fish were then exercised at 10°C or 20°C, and the critical swimming speed (fatigue velocity) was measured. At 10°C, cold-acclimated carp were capable of significantly higher swimming speeds. When exercised at 20°C. however, the situation was reversed, and warm-acclimated carp exhibited improved swimming ability. These results provide direct evidence that acclimation of the contractile proteins is beneficial across a wide temperature range. Following acclimation to low environmental temperatures the critical swimming speed exhibited a Q₁₀ of only 1.1 for the temperature range 10–20°C. compared to a value of 2.9 for fish acclimated to the higher temperature.     

Temperature acclimation of gross cardiovascular morphology in common carp (Cyprinus carpio)

Temperature is of fundamental importance in the functioning of the cardiovascular system of ectothermic fish, with cold-induced ventricular hypertrophy and increased red muscle mass being reported in a number of fish species upon cold acclimation. This study demonstrates a non-linear cold-induced ventricular hypertrophy in common carp (relative ventricular mass (RVM)=0.086±0.003%, 0.074±0.005% and 0.074±0.004% at 5, 15 and 25 °C, respectively), but a cold-induced atrophy of the lateral red muscle mass (RMM) with respect to total muscle mass (2.504±0.554%, 3.982±0.818% and 4.490±0.256% at 5, 15 and 25 °C, respectively). The ventricular hypertrophy in carp acclimated to 5 °C reflects a compensation against the increased workload of pumping viscous blood at low temperatures and the negative inotropic effects of the cold, whereas atrophy of the red muscle is likely due to disuse resulting from reduced locomotory activity in the cold.     

The effect of reduced temperature and salinity on the blood physiology of juvenile Atlantic cod

The osmolality and ionic composition of the blood of juvenile Atlantic cod Gadus morhua and their response to conditions of reduced temperature and salinity in summer‐ and winter‐acclimated individuals was investigated. Haematocrit percentage was relatively stable throughout the experimental procedures. Summer‐acclimated juvenile Atlantic cod had higher plasma osmolality than winter‐acclimated fish in ambient conditions. Plasma Na⁺ levels were, however, higher in winter conditions, while Cl⁻ did not vary between seasons. Temperature reduction (12, 9 and 6° C in summer and to 6 and 4° C in winter) induced a significant response in plasma osmolality and Na⁺ levels in summer, but only in Na⁺ levels in winter‐acclimated fish. A pronounced effect was seen in the summer 6° C treatment. Salinity treatments (24, 16 and 8) had a significant effect on almost all the variables in both summer and winter and resulted generally in dilution of ionic and osmotic concentrations of the plasma. This effect was pronounced in the lowest temperature treatments, with the greatest reduction observed in the summer 6° C treatment. This could suggest that winter‐acclimated fish are physiologically adapted to cope with lower seawater temperatures as opposed to summer‐acclimated fish.