Effect of restricted feeding schedule on seasonal shifting of daily demand-feeding pattern and food anticipatory activity in European sea bass (Dicentrarchus labrax L.)

The effect of restricted feeding schedule was investigated on the seasonal shifting of daily demand-feeding pattern and food anticipatory activity in European sea bass (Dicentrarchus labrax) held under natural environmental conditions in an outdoor laboratory. To that end, demand-feeding behavior was continuously monitored for approximately one year in four groups of 15 fish each exposed to natural fluctuations of water temperature (from 13.2 degrees C to 27.4 degrees C) and photophase (from 9.5 h to 14.5 h of light). When the animals were subjected to a time-restricted feeding schedule, the demand-feeding rhythm rapidly synchronized to the three periods of food availability: the first meal (FM) from 08:00 to 09:00 h, the second meal (SM) from 16:00 to 17:00 h, and the third meal (TM) from 00:00 to 01:00 h. The occurrence of demand-feeding activity into the three periods of food availability displayed a double seasonal shift: fish that self-fed mostly during the daytime periods of feeding availability (FM and SM) in summer and autumn changed to nocturnal feeding (TM) from December to April, returning to diurnal preferences in April. Food-demands appeared to be predominantly associated with feed availability, reaching its maximum levels during the hours of reward. In addition, feeding anticipatory activity (FAA) was observed. A relationship was detected between the duration of FAA and feeding-time, with shortest FAA (30-60 min) when mealtime occurred just after sunrise (FM) or sunset (TM). These findings demonstrate the ability of sea bass to self-feed under time-restricted schedules, and show a seasonal-phase inversion in demand-feeding activity in spite of the restrictions in their feeding availability. Sea bass can use external signals as reference to anticipate the time of feed availability. This information may be useful for designing new feeding strategies for European sea bass fish farming.     

Fattening responses to daily intervals of heat exposure in the Syrian hamster

Warm ambient temperature (38 degrees C) provided daily for one hr induced time-dependent changes in body weight and fat stores in the Syrian hamster. In animals held on 10-hr daily photoperiods and room temperature (23 degrees C), daily one-hour thermopulses at 8 and 20 hr after light onset stimulated increases in body weights and indices of body fat storage. Abdominal fat pad weights of these groups were twice those of untreated controls after 17 and 28 days of thermoperiodic treatments. On the other hand, daily thermopulses were completely ineffective at 0 and 16 hr after light onset. These results demonstrate that body fat stores may be influenced by a temporal interaction of environmental stimuli and implicate underlying circadian mechanisms in the regulation of body fat.     

Circadian locomotor activity rhythm under the influences of temperature cycle in the Djungarian hamster, Phodopus sungorus, entrained by 12 hour light-12 hour dark cycle

Effects of temperature cycle (25 degrees C during light and 10 degrees C during dark) on circadian locomotor activity rhythm entrained by 12 hr light-12 hr dark cycle were studied in the dark active Djungarian hamster. The amounts of activity per 24 hr were significantly greater under temperature cycle than under constant temperature of 25 degrees C. Phase angle difference between activity onset and light off was always more positive under temperature cycle than under constant temperature. These findings are discussed in terms of circadian physiology.     

Circannual rhythms of gonadal maturation in female rainbow trout (Oncorhynchus mykiss).

Individually identified, 2-year-old female rainbow trout were maintained for up to 51 months on a constant schedule of 6 hr light and 18 hr darkness (LD 6:18), constant temperature (8.5-9.0 degrees C), and constant feeding rate. The fish exhibited free-running circannual rhythms of gonadal maturation and ovulation, which were self-sustaining for up to three cycles. The periodicity of the rhythm showed variation between fish and in successive cycles for the same fish, ranging from approximately 11 to 15 months.     

Endocrine control of diurnal oocyte maturation in the kyusen wrasse,Halichoeres poecilopterus

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20beta-P and 20beta-S increased around the time of GVBD and ovulation, suggesting that 17,20beta-P and 20beta-S act as MIHs in the kyusen wrasse. Moreover, in vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.     

The growth of largemouth bass, Micropterus salmoides (Lacepede), under constant and fluctuating temperatures

Growth of largemouth bass, Micropterus salmoides , was tested under constant (15° C, 29° C) and cyclic (18 h at 15°C, 6 h at 29° C) temperature conditions. Six groups of 10 fish each were tested; one group under each constant condition, and four groups in the thermocycles. The four latter groups differed in the timing of their feeding; one group each was fed at the start and middle of the warm and cool periods, respectively. All groups were fed to near-satiation. The fish fed at the start of the warm cycle and start cool group grew most rapidly, followed by the constant warm, mid warm, mid cool, and constant cool groups. These data were evaluated in relation to vertical migrations of fish to test the physiological enhancements that may be important to vertical migrations. Apparently thermocycles allow more rapid consumption of food than constant cool conditions and lower metabolic rates than constant warm conditions, which allows some fish to grow more rapidly under thermocycles than under constant conditions at either extreme of the cycle. However, the timing of feeding in relation to temperature in the thermocycle is important in the degree of growth enhancement.     

Expression of warm temperature acclimation-related protein 65-kDa (Wap65) mRNA, and physiological changes with increasing water temperature in black porgy, Acanthopagrus schlegeli

We isolated the warm temperature acclimation-related protein 65-kDa (Wap65) cDNA from the liver of black porgy and investigated the expression by increasing water temperature in black porgy, Acanthopagrus schlegeli. Black porgy Wap65 full-length cDNA consists of 1,338 nucleotides, including an open reading frame, predicted to encode a protein of 425 amino acids and showed high homology to pufferfish (79%), Medaka (73%), carp (70%), and goldfish (68%) Wap65. Increase in water temperature (20 degrees C --> 30 degrees C; 1 degrees C/day) induced the rise of Wap65 mRNA expression in liver of black porgy. Also, the levels of cortisol and glucose in plasma were significantly higher at 30 degrees C than at 20 degrees C. To determine the high water temperature stressor specificity of the induction of Wap65, black porgy were transferred from seawater (SW) to freshwater (FW) for 24 hr. Wap65 expression was not detected when the fish were transferred from SW to FW (in fish transferred from SW to FW), although the levels of cortisol and glucose in plasma were increased. These results suggest that increase in Wap65 gene is related to high water temperature stress and play important roles in high water temperature environment of black porgy.     

Stressor-associated alterations in porcine plasma prolactin

Experiments were conducted to determine effects of restraint and thermal stressors on plasma prolactin (PRL) in castrated male pigs. A single 20-min restraining period in a restraining cage which prevented both movement and injury increased (P less than 0.05) plasma PRL when applied at either 0800 or 1600 hr. Exposure to 32 degrees C at 0800-1000 hr or at 1600-1800 hr produced more moderate increases (P less than 0.05). A combination of 20 min restraint and 2 hr at 32 degrees C produced a response similar to restraint alone. Twenty minutes after stressor application plasma PRL concentrations in pigs exposed to restraint or restraint +32 degrees C at 1600 h were greater (P less than 0.05) than concentrations measured in all other treatment groups at that time interval. However, there were no statistically significant differences in additional quantitative indices of the plasma PRL responses (maximal level, maximal change, or integrated response above basal levels) among restraint, 32 degrees C, or restraint +32 degrees C, nor between morning and afternoon applications of treatment. Such data do not provide, therefore, any strong evidence for stressor-dependent or circadian differences in plasma PRL response. A second study subjected castrated male pigs to 20 degrees C (controls), 20 +/- 12 degrees C (cyclic temperature, sine wave variation), 5 degrees C constant, and 5 +/- 12 degrees C cyclic for 20 days. After 6 days exposure to 5 degrees C constant or 5 +/- 12 degrees C cyclic there were decreases (P less than 0.05) of 59 and 67% respectively in plasma PRL when compared either with pretreatment levels or with levels in pigs at 20 or 20 +/- 12 degrees C. There were no differences in PRL responses between cyclic vs constant temperatures. These results are the first to indicate that plasma PRL in pigs is affected by acute restraint and thermal stressors.     

Variations in plasma melatonin levels of the rainbow trout (Oncorhynchus mykiss) under various light and temperature conditions

Daily variations in plasma melatonin levels in the rainbow trout Oncorhynchus mykiss were studied under various light and temperature conditions. Plasma melatonin levels were higher at mid-dark than those at mid-light under light-dark (LD) cycles. An acute exposure to darkness (2 hr) during the light phase significantly elevated the plasma melatonin to the level that is comparable with those at mid-dark, while an acute exposure to a light pulse (2 hr) during the dark phase significantly suppressed melatonin to the level that is comparable with those at mid-light. Plasma melatonin kept constantly high and low levels under constant darkness and constant light, respectively. No circadian rhythm was seen under both conditions. When the fish were subjected to simulative seasonal conditions (simulative (S)-spring: under LD 13.1:10.9 at 13 degrees C; S-summer: under LD 14.3:9.7 at 16.5 degrees C; S-autumn: under LD 11.3:12.7 at 13 degrees C; S-winter: under LD 10.1:13.9 at 9 degrees C), melatonin levels during the dark phase were significantly higher than those during the light phase irrespective of simulative seasons. The peak melatonin level in each simulative season significantly correlated with temperature but not with the length of the dark phase employed. In addition, the peak melatonin level in S-autumn was significantly higher than those in S-spring although water temperature was the same under these conditions. These results indicate that the melatonin rhythm in the trout plasma is not regulated by an endogenous circadian clock but by combination of photoperiod and water temperature.     

Testicular and some hormonal changes during the first four years of life in the mirror carp, Cyprinus carpio L.

Male carp bred in outside ponds in Poland were sampled monthly from 5 to 46 months old, to analyse changes in gonadosomatic index (GSI) and pituitary gonadotropin hormone (GTH) in blood serum and in pituitary, 17α20β8P and 11KT in blood and gonadotropin releasing hormone (GnRH) in pituitary and hypothalamus. First signs of puberty with significant testis development (a high GSI of 12% and strong mitotic activity of the type-B spermatogonia) were seen at 13 months and 4548° days. By 16 months the GSI had declined to 6%. At 25 months the GSI remained at 6%, active spermatogenesis was observed, with some accumulation of spermatozoa but no spermiation. During the years 4 and 5 the GSI increased regularly from 6 to 12% and spermiation was observed nearly all the time. Some GTH was found in the blood before gonad development occurred. Thereafter there was no major change in GTH (10–20 ng ml⁻¹ serum) except a peak of 200–130 ng ml⁻¹ at 38. 39 months: this peak was not related to any major biological event, except that all fish reached spermiation at that time. A progressive increase of the amount of GTH in the pituitary was observed during the sampling period. Opposite fluctuations in the GnRH content were observed in brain and pituitary. Circulating 11-ketotesterone (11-KT) increased to 20 30 ng ml⁻¹ serum in spring 22–25 months and at 36–37 months in parallel with the progressive rise of the water temperature, but independently of stage of testes development. These peaks of 11-KT were followed immediately (1984) or two months later (1983) by temporary major rises of 17αa-hydroxy-20β-dihydroprogesterone reaching 2–3 ng ml¹ serum; this was not related to the temperature nor to the spermatogenetic stage.     

Testosterone and estradiol potentiate the serum gonadotropin response to gonadotropin-releasing hormone in goldfish

The effects of gonadal steroids on gonadosomatic index (GSI; gonad wt/total body wt x 100), pituitary gonadotropin (GTH) content, and serum GTH response to [D-Ala6,Pro9-Net]-luteinizing hormone-releasing hormone (LHRH-A) were investigated throughout the seasonal reproductive cycle of the goldfish. Gonad-intact female fish were implanted i.p. for 5 days with silastic pellets containing no steroid (blank), testosterone (T; 100 micrograms/g), or estradiol (E2; 100 micrograms/g). The serum GTH response at 6 h following i.p. injection of saline or 0.1 microgram/g LHRH-A was assessed. In blank-implanted, saline-injected animals, seasonal variations in GSI, pituitary GTH content, and serum GTH levels were evident; maximal and minimal levels were noted in the spring and summer months, respectively. In blank-implanted fish, LHRH-A effectively stimulated GTH release in females undergoing gonadal recrudescence (late autumn and winter) and in sexually mature (spring) females, but not in sexually regressed (summer and early autumn) females. Implantation of T or E2 raised serum steroid levels to those found during ovulation in goldfish. Steroid treatments did not affect unstimulated serum GTH levels at any time of the year. Testosterone effectively potentiated the serum GTH response to LHRH-A during the entire reproductive cycle, whereas the positive effects of E2 were evident in sexually regressed and post-spawning females only. Both T and E2 potentiated the GTH response to LHRH-A in male fish. To examine the involvement of T aromatization in mediating its actions on induced GTH secretion, male and female fish were implanted with T or the nonaromatizable androgens 5 alpha-dihydroxytestosterone (DHT; 100 micrograms/g) and 11-keto-testosterone (11-KT; 250 micrograms/animal). Testosterone potentiated the GTH response to LHRH-A in both males and females whereas DHT and 11-KT were without effect. Furthermore, the positive action of T on induced GTH secretion was blocked by 2-day pretreatment with the aromatase inhibitor 1,4,6-androstatrien-3,17-dione (100 or 300 micrograms/g). Multiple i.p. injections of hCG (0.2 microgram/g every 3 days for 39 days), probably through stimulation of endogenous T secretion, resulted in potentiation of the GTH response to LHRH-A in mature male goldfish. These results clearly demonstrate that T, through aromatization to E2, can increase pituitary responsiveness to exogenous LHRH-A in gonad-intact male and female goldfish.     

Change in lipid composition in eastern oyster (Crassostrea virginica Gmelin) exposed to constant or fluctuating temperature regimes

A temperature decrease usually induces an ordering effect in membrane phospholipids that can lead to membrane dysfunction. Ectotherms typically counteract this temperature effect by remodeling membrane lipids as stipulated in the homeoviscous adaptation theory (HVA). Previous studies mostly focused on the remodeling of membrane lipids during long-term acclimatization or acclimation at constant temperature regimes, whereas in nature, many organisms experience variations in temperature on a daily basis and must react to this changing thermal environment. The objective of this study was to examine the composition of membrane lipids in oysters subjected to long-term acclimation at constant temperatures (12 or 25 degrees C) or to environmentally realistic daily fluctuations in temperature between 12 and 25 degrees C for 7 d. The lipid composition of gill in oysters subjected to long-term acclimation at a constant temperature or to daily temperature fluctuations varied in a way consistent with HVA: oysters adjusted their phospholipid to sterol ratio in response to long-term acclimation to a constant temperature but not to daily temperature fluctuations. In contrast, the unsaturation index of polar lipids in oysters varied in response to both long-term acclimation to a constant temperature and to daily temperature fluctuations, mainly due to changes in 22:6n-3 and 20:5n-3. The 20:4n-6 levels in oyster gills increased as temperature rose, suggesting an increasing availability of this fatty acid for eicosanoid biosynthesis during stress responses.     

Seasonal changes in serum thyroid hormone levels of the feral brown bullhead, Ictalurus nebulosus Lesueur

Seasonal changes in serum L-thyroxine (T4) and triiodo-L-thyronine (T3) concentrations were measured in a feral population of brown bullheads, Ictalurus nebulosus . Serum T4 was elevated in mid April concomitant with the onset of gonadal recrudescence and the increase in ambient temperatures from the winter level. Thereafter, serum T4 levels were constant throughout the late spring and summer (except for a significant lowering in levels associated with the spawning and early postspawning period), declining to a low overwintering level by mid December. Serum T3 levels increased in April, and apart from a significant decrease in July associated with the postspawning period, rose progressively until August, after which levels declined progressively to reach overwintering levels again by mid December. Between mid April and mid May serum T4/T3 ratios fell from the high values found in fish during the winter months. Apart from variations associated with the fluctuations of T4 and T3 concentrations in the spawning and early postspawning periods, serum T4/T3 ratios remained at a low level throughout the summer and early autumn months, and rose again in October to the overwintering levels. The circannual cycles are discussed in relation to the possible role of the thyroid hormone in gonadal maturation and metabolism in this species.     

Effects of photoperiod on pituitary gonadotropin levels in masu salmon

We examined the effects of photoperiod on pituitary levels of two types of gonadotropin (GTH), GTH I and GTH II, in masu salmon Oncorhynchus masou to study their mechanism of synthesis. In Experiment 1, the effects of long or short photoperiod combined with castration were examined using 8-month-old precocious males. Castration was carried out in early August and then the fish were reared under a short (8L16D) or long (16L8D) photoperiod for 60 days. In Experiment 2, the effects of photoperiod combined with testosterone treatment were examined using 12-month-old immature females. Silastic tubes containing testosterone (500 microg /fish) or vehicle were implanted intra-peritoneally in early October. Fish were reared under 16L8D for 60 days, and then half of the fish were transferred to 8L16D, while the remaining fish were kept under 16L8D until Day 90. In Experiment 1, GTH I contents were higher under 16L8D than under 8L16D in the castrated group on Day 30. Moreover, GTH I contents were higher in the castrated group than the control group under 16L8D on Day 30. GTH II contents increased with testicular maturation in the control groups, whereas they remained at low levels in the castrated groups regardless of photoperiodic treatment. In Experiment 2, GTH I contents did not change remarkably in all the groups, while GTH II contents were remarkably increased by testosterone treatment regardless of photoperiodic treatment. These results indicate that the synthesis of GTH I and GTH II are differently regulated by photoperiod and testosterone in masu salmon.     

Cold entrainment of the annual cycle of ovarian activity in the lizard Lacerta vivipara: thermoperiodic rhythm versus hibernation.

A constant warm thermoperiod maintains ovarian quiescence in the lizard Lacerta vivipara, whereas a 4-month artificial hibernation rapidly induces synchronized vitellogenesis after transfer to warmth. The present study examined the possibility of a thermoperiodic regulation of the ovarian cycle and the formal properties of an internal temporal program. These questions were addressed using 24-hr thermoperiodic conditions that combined a long or a short thermophase (6 or 2 hr of basking) with a warm (19-21 degrees C), a cool (5-15 degrees C), or a cold (3-7 degrees C) cryophase. Lizards were exposed to the natural photocycle or to LD 12:12. Occurrence and timing of vitellogenesis completion were monitored using immunodetection of plasma vitellogenin and laparotomies. Cold remained stimulatory when given intermittently with a 24-hr periodicity. However, under long-thermophase conditions, lizards responded poorly to cool cryophases but fully to cold ones (72.7-100% vitellogenesis). Thus a certain amount of cold must be provided during each 24-hr cycle in order to be effective through the succession of thermocycles. Reduction of the daily heat input from 6 to 2 hr modulated the stimulating effects of cold cryophases: The median date for the beginning of vitellogenesis occurred 1 month earlier, but the number of responding females decreased from 100% to 40%. The thermoperiodic regulation of the ovarian cycle also relies upon a precise heat-cold balance per nycthemeral unit. This ensures the entrainment of an internal rhythm, since the timing of reproductive responses varies with the date of transfer from the inhibitory warm thermoperiod to the inducing thermoperiod (long thermophase, cold cryophase). At least half the females started vitellogenesis within 1-2 months after a late transfer (winter solstice) instead of 6 months after an early one (autumn equinox), and the median date for onset differed by 1 month between the two groups. However, autumn transfer was the only one to induce a group response in close agreement with the natural timing.     

Acclimation to constant and variable temperatures in plethodontid salamanders--I. Rates of oxygen consumption

In preliminary experiments, salamanders of three species (Desmognathus ochrophaeus, Plethodon cinereus and Plethodon jordani) required 5-11 days to complete metabolic acclimation to a constant warm temperature; the rate of oxygen consumption (VO2) decreased 16-28% during acclimation. Unfed animals of each species underwent cyclic exposure to 5 and 21 degrees C at three different cycle periods (12 hr, 4-5 days, 51 days), or constant exposure to 14 degrees C for 102 days. The experimental treatments significantly affected the VO2 measured at 5, 14, 17.5 and 21 degrees C. The direction and magnitude of the acclimatory effects upon VO2 were inconsistent among species and among experimental temperatures, and resulted in little energy saving. The VO2 during exposure to cyclic temperatures averaged only 83% of that during preliminary experiments, perhaps as a response to starvation.     

Photoperiodic regulation of thyroid responsiveness to TSH in Fundulus heteroclitus

Treatment of the killifish, Fundulus heteroclitus, with ovine thyrotropin (oTSH) produced elevations in serum thyroxin (T4) that varied seasonally with a strong inverse correlation to environmental photoperiod (P less than 0.001). At 20 degrees C, oTSH (0.2 IU) increased serum T4 to levels ranging from 1.5 micrograms/100 ml in midsummer to 7.5 micrograms/100 ml in midwinter despite relatively stable resting levels throughout the two years of study. Similar rates of clearance of T4 in summer and winter suggest that an alteration in thyroid response to oTSH accounts for this change. The serum T4 response to oTSH is both photoperiod- and temperature-dependent. In laboratory studies, fish exposed to a short artificial photoperiod (LD 8:16) for one month responded to oTSH with T4 levels about twice as high as those in fish exposed to long days (LD 14:10). On the other hand the T4 response to oTSH was sharply reduced at 5 degrees C from that seen at 20 degrees C. This suggests that, in nature, seasonal changes in photoperiod and temperature have opposite effects on thyroidal responsiveness to TSH. Overall, the annual variation in the T4 response to oTSH appears to be driven by photoperiod, inasmuch as the alterations preceded major temperature changes in the wild and can be shown to occur at constant temperature in captivity. Photoperiodic induction of changes in thyroid sensitivity may aid in the maintenance of basal T4 levels under changing thermal conditions.     

诱导大鳍鱯和长吻鮠排卵过程中血清GTH水平的变化

繁殖期从嘉陵江收集性成熟的大鳍■ 和长吻 野生亲鱼,用Ｌｉｎｐｅ方法（即ＬＨＲＨ－Ａ加多巴胺Ｄ２受体拮抗剂地欧酮）或传统的ＬＨＲＨ—Ａ加脑垂体的方法进行催产,定时取血样,用放射免疫方法测定催产过程中血清ＧＴＨ水平的变化,进一步证实鲇形目鱼类ＧＴＨ的分泌受到下丘脑分泌的促性腺激素释放激素ＧｎＲＨ和多巴胺的双重调节;排卵和产卵也是以血清ＧＴＨ的急剧升高为先导的,而最终能否排卵还有赖于血清ＧＴＨ峰是否超过“排卵阈值”。尽管催产后的大鳍 和长吻 雄鱼血清ＧＴＨ水平也有一个高峰出现,但血清ＧＴＨ水平升高幅度都大大低于雌鱼,这种现象在硬骨鱼类可能具有普遍性。     

Glucose, Insulin, and Adrenaline Effects on Some Aspects of Fish Feeding Behavior

Effects of glucose, insulin, and adrenaline on the feeding latent time and the ration in 3 species of fresh-water teleost fish were studied. There has been shown a significant and uniform effect of glucose (an increase of the feeding latent period, a decrease of the ration) and of adrenaline (an increase of the feeding latent period) at initial stages of exotrophia in fish. The insulin effect on the fish feeding reaction rate is not uniform: apart from a statistically significant decrease of the feeding latent period, there were recorded opposite effects.     

Pathological changes caused by cold-water stress in Japanese eel Anguilla japonica

In the present study, histopathological changes in Japanese eel Anguilla japonica subjected to 'cold-water stress' were examined. Eels were exposed to cold water (13 to 15 degrees C) and warmer water (25 degrees C) as controls. Fish held in warm water did not show any apparent changes. Although none of the eels exposed to cold water died, they displayed nephrotic changes such as cloudy swelling and hyaline droplet degeneration of the renal tubular epithelia. Fish with nephroses had low levels of serum chloride (12 to 23 mg l-1 in fish with hyaline droplet degeneration, 71 to 81 mg l-1 in fish with cloudy swelling) compared with the control fish (87 to 109 mg l-1). In electron microscopy, affected tubular cells had variously damaged mitochondria and formations of secondary lysosomes of variable sizes. Electron microscopy also revealed mitochondrial degeneration in hepatocytes and degenerated granules of neutrophils in the hematopoietic tissue. 'Cold-water stress' was effective in damaging Japanese eels below 15 degrees C.