New haemoglobin genotypes in Atlantic cod, Gadus morhua: possible relation with growth

In a preliminary study, 121 individually tagged juvenile Atlantic cod (Gadus morhua) were classified according to their haemoglobin genotypes into four groups, i.e., two main haemoglobin genotypes [Hb-I(1/2), Hb-I(2/2)] and two sub-types [Hb-I(1/2b), Hb-I(2/2b)], and reared for 3 months at 10 degrees C, 13 degrees C and T-step (fish reared at 16 degrees C and then subsequently moved to 13 and later to 10 degrees C). Overall growth rates across temperatures were 10% and 19% higher in the Hb-I(2/2b), Hb-I(1/2b) sub-types compared to corresponding Hb-I(2/2) and Hb-I(1/2) main types, respectively. Individual growth rate trajectories varied between the genotypes at all temperatures studied. Our study indicates that under certain environmental conditions higher growth in the two sub-types compared to the main genotypes could be expected. This may indicate difference in other physiological characters not studied here, but seen in previous studies, i.e., oxygen affinity and competitive performance.     

Genetic variation and functional properties of Atlantic cod hemoglobins: introducing a modified tonometric method for studying fragile hemoglobins

Hemoglobin polymorphism in Atlantic cod has been investigated with respect to physiological performance at 10, 15 and 20 degrees C applying a modified tonometric method for O2 equilibrium analysis with full control of the equilibrating gas mixture. The results did not indicate any dissociation of the hemoglobins by a reduction in cooperativity and a parallel increase in affinity during the analytical procedure in contrast to the original tonometric method. With the applied preparation technique, we could store the hemolysate for 70 days at -25 degrees C without any significant changes in the O2 binding properties (P < 0.05) demonstrating the high quality of this procedure for analysing fragile fish hemoglobins. The present investigation demonstrates that the oxygen affinity of the hemoglobins varied between the genotypes. At all temperatures, except 20 degrees C and pH 8.0, Hb-I(2/2) had a higher O2 affinity than Hb-I(1/1). These results conform with previous results (16), suggesting Hb-I(2/2), the genotype which is the dominant allele in northern areas, to be the most efficient O2 carrier at low temperatures. The highest O2 affinity, however, was found for Hb-I(2/2b), supporting the results of Fyhn et al. (9), that this genotype is more restricted to coastal and warmer water and thus a better marker of the coastal population. Our results further suggest a correlation between genotype specific growth rates and oxygen affinities at all temperatures studied, with the highest growth rates observed in those genotypes having the highest O2 affinities. In conclusion, the hemoglobin polymorphism of cod seems to be correlated with physiological performance.     

Hypoxic avoidance behaviour in cod (Gadus morhua L.): The effect of temperature and haemoglobin genotype

Hypoxia can influence fish growth, survival and on larger scales, population structure. These effects may be influenced by water temperature, and may vary intra-specifically with genotype. In Atlantic cod (Gadus morhua L.), the two haemoglobin homozygotes (Hb-I?11 and Hb-I?22) vary in oxygen affinity at different temperatures, which is thought to correspond to variation in hypoxia tolerance. We therefore tested if hypoxic avoidance behaviour in cod 1) depends on ambient temperature and 2) is modified by haemoglobin genotype. In a laminar flow choice box, we subjected juvenile cod to an initial phase of non-escapable hypoxia, and a subsequent recovery phase, where one habitat was kept at 20% O₂ saturation while the other was raised in steps to full saturation. The experiment was performed at 5 and 15 °C with Hb-I?11 and Hb-I?22 cod. Cod responded to inescapable hypoxia by reducing their overall swimming speed and then, at the initial levels of the recovery phase, avoiding the most hypoxic habitat, irrespective of temperature or genotype. Fish recovered quickly as O₂ levels increased, as evidenced by increased swimming speed and time spent in the most hypoxic habitat. The avoidance response depended strongly on temperature: the relative reduction in speed and avoidance of the most hypoxic habitat was more pronounced at 15 than at 5 °C. During the recovery phase, stressed fish initially maintained a higher swimming speed in the most hypoxic habitat. However, as O₂ increased, swimming speed in both habitats converged. This point of convergence occurred at a lower O₂ saturation at 5 °C. Fish ventilation rate in inescapable hypoxia was also higher at 15 °C. Haemoglobin genotype did not influence either ventilation rates or the nature of the hypoxic avoidance response at either temperature, but Hb-I?11 cod swam faster than Hb-I?22 cod in normoxia at 15 °C. Our results indicate that increased temperature limits the ability of cod of both haemoglobin genotypes to exploit hypoxic habitats. This may have negative future consequences for coastal cod stocks in light of increasing global temperatures and eutrophication in coastal waters.     

Effects of temperature on growth and metabolism in juvenile turbot

The effects of constant temperatures on growth, food efficiency, and physiological status were studied in four different batches of juvenile turbot. The growth responses were studied in three experiments lasting 70–85 days under 8–20° C thermal conditions. There was a positive correlation between growth and temperature from 8 to 17° C and a plateau was observed from 17 to 20° C. In fish fed to satiety, specific growth rate was positively correlated to the food intake, which was double at 20° C, compared with 8° C. Minor changes were observed in food efficiency. Body fat deposition decreased as temperature increased (25% lower at 20° C, compared with 8° C). Apparent food conversion, PER (protein efficiency ratio) and PUC (protein utilization coefficient) ranges were 0.8–0.9, 2.1–2.3 and 33–38% respectively. In 70–300 g fish, routine MO₂ increased (2.5–6.5 μmol O₂ h^?1 g bw^?1) with temperature up to 20° C, while larger turbot (500–600 g) appeared relatively thermo-independent, with a lower oxygen consumption (1.5 ìmol h^?1 g^?1). The average daily total ammonia nitrogen (TAN) and urea-N excretion per fish biomass was positively related to temperature. TAN was 30% lower at 8° C, compared with 20° C. Ingested nitrogen was mainly excreted under the final form of TAN, urea-N representing 26% of the total amount. A post-prandial peak in TAN and a delayed peak in urea-N nitrogen were observed. The hydromineral status [osmolarity, sodium, chloride and potassium blood plasma, gill (Na⁺-K⁺)-ATPase activity] of turbot was not affected by progressive changes in temperature during the acclimation period. Juvenile turbots show remarkable homeostatic capacities and so they have a relatively thermo-independent physiology within the range of temperature studied.     

The interaction of temperature and fish size on growth of juvenile turbot

Growth rate of tagged juvenile turbot was significantly influenced by the interaction of temperature and fish size. The results suggest the optimum temperature for growth of juvenile turbot in the size range 25–75 g is between 16 and 19°C. Optimal temperature for growth decreased rapidly with increasing size, and is between 13 and 16°C for 100 g turbot. Although individual growth rates varied highly at all times within the temperature treatments, significant size rank correlations were maintained during the experimental period. The study confirms that turbot exhibit ontogenetic variation in temperature optimum, which might partly explain different spatial distribution of juvenile and adult turbot in ocean waters.     

SINGLE- AND MULTIPLE-LOCUS GENOTYPES AND LIFE-HISTORY RESPONSES OF GAMBUSIA HOLBROOKI REARED AT TWO TEMPERATURES

Eastern mosquitofish (Gambusia holbrooki) were reared from birth to 10 wk of age at 25°C and 32°C. Relationships of growth, time to maturity, and developmental stability to isozyme genotype were used to examine the hypothesis that more heterozygous individuals should exhibit superior performances, especially under thermally stressful (32°C) conditions. More heterozygous fish grew faster than homozygous individuals, especially at 32°C. Significant differences in time to maturity were detected among allozyme genotypes but not with heterozygosity. Multiple-locus heterozygosity was negatively related to fluctuating asymmetry. Thus, life-history traits were affected by both multiple-locus heterozygosity and single-locus genotype.     

Reverse temperature dependence of tuna hemoglobin oxygenation

At a pH around 7.5 with 0.05 M NaP_i, the shape of the oxygen equilibrium curve of hemoglobin from the bluefin tuna (Thunnus thynnus) is temperature dependent. The affinity at low saturation increases, and that at high saturation decreases on cooling from 20°C to 10°C. The equilibrium curves at the two temperatures therefore cross over. This behavior is physiologically advantageous to a warm-bodied fish. It may be explained in terms of the two-state model by supposing that the allosteric constant L increases markedly on cooling the solutions.     

GH–IGF system regulation of attenuated muscle growth and lipolysis in Atlantic salmon reared at elevated sea temperatures

Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 °C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 °C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at ≤17 °C, and dropped in fish reared at 19 °C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 °C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 °C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 °C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 °C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 °C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.     

Effect and interaction of temperature and photoperiod on growth and partitioning in three groundnut (Arachis hypogaea L.) genotypes1

Effect of temperature and photoperiod and their interaction on plant growth and partitioning of dry matter to pods was examined in three selected groundnut genotypes viz., TMV 2, NC Ac 17090 and VA 81B. The genotypes were grown in six walk-in growth chambers which were programmed to simulate three temperature regimes (22/18°C, 26/22°C and 30/26°C day/night) each under long (12 h) and short (9 h) photoperiods. The plant growth rates and partitioning of dry matter to pods were estimated on a thermal time basis. Plant growth rate (PLGR) was significantly influenced by temperature, photoperiod and genotype, whereas pod growth rate (PDGR) was influenced primarily by temperature and genotype. The interaction of genotype with photoperiod and with temperature was significant for both PLGR and PDGR. For example, at the 22/18°C temperature regime, VA 81B had a high PDGR, while NC Ac 17090 did not even initiate pod growth. The partitioning of dry matter to pods (Pf) was also significantly influenced by photoperiod, temperature and genotype, and significant interactions were found. Photoperiod did not significantly affect Pf under the low temperature regime, but at higher temperatures, partitioning to pods was significantly greater under short days. Pf of VA 81B was relatively insensitive to photoperiod compared with the other two genotypes. The study provided evidence of genotypic variability for photoperiod × temperature interactions which could influence adaptation of groundnut genotypes to new environments.     

The size and germinability of Scots pine pollen in different temperatures in vitro

The effect of genotype, the origin of genotype, and germination temperature on Scots pine pollen grain size, hydration rate, germinability, and tube growth was studied in vitro. The mean sizes of dry and germinated pollen grains varied among pollen genotypes in different ways, thus the hydration rate varied among genotypes. Pollen from Scots pine that originates in northern Finland hydrated more than pollen from a population in southern Finland. Germination temperature had no effect on the hydration rate. Germinability and tube growth rate of northern genotypes were higher at 20 °C than at 15 °C. Differences among southern genotypes were not significant. At 15 °C, the germinability and pollen tube growth rate of northern genotypes were lower than southern genotypes. At 20 °C, the differences were not significant. It appears that germination and growth of pollen from northern populations are enhanced at higher temperatures whereas pollen from southern populations is unaffected.     

A functional model for maximum growth of immature stone loach, Barbatula barbatula, from three populations in north-west England

1. The chief objective was to develop a functional model for growth of stone loach, Barbatula barbatula, using immature fish from three populations. The growth model had been developed previously for brown trout, Salmo trutta, but new estimates of the five parameters for the stone loach had to be obtained from laboratory experiments. 2. Fish from four size groups (initial arithmetic mean live weights 0.053 g, 0.231 g, 0.840 g, 1.612 g, with five fish per group) from Great Oaks Wood Beck were acclimatized to constant temperatures of either 3, 5, 10, 15, 20 or 25 °C. Each fish was kept in a separate tank and fed to satiation on freshwater shrimps. Weights and lengths of each fish were recorded at the start and finish of a growth period of 35 days. For each of the other populations (Black and Ford Wood Becks), there were only three temperatures (5, 10, 20 °C) with ten fish per temperature. 3. The growth model was an excellent fit (P < 0.001, R² > 0.99) for the 120 fish from Great Oaks Wood Beck. Growth rates were negative at 3 °C, close to zero at 5 and 25 °C, and positive at 10, 15 and 20 °C, with an optimum value of 19 °C. When growth rates were positive, they decreased markedly with increasing fish weight for small fish but decreased more slowly for larger fish. At the start of the experiments, weight–length relationships were similar for fish from all three populations and were well described by a power function. There was excellent agreement between growth rates estimated from the fitted growth model for fish from Great Oaks Wood Beck and values obtained for fish from Black and Ford Wood Becks. Data from all three populations were therefore pooled (n = 180) to obtain new estimates of the five parameters in the model. 4. Comparisons between parameter estimates for trout and stone loach showed that the latter grew better in warmer waters (e.g. optimum value for growth was 19.0 °C for stone loach and 13.1 °C for trout, with ranges for growth of 5.0–25.0 °C and 3.6–19.5 °C, respectively).     

Hemoglobin system of golden mullet (Liza aurata,Risso) at adaptation to conditions of outer hypoxia

Under conditions of experiment, effect of outer hypoxia on the hemoglobin heterogeneous system and respiratory blood characteristics were studied in the golden mullet Lisa aurata R. The control fish group was maintained at oxygen pressure of 158–162 gPa, the experimental group at 54–55 gPa. The exposure was 15 days, the water temperature—15°C. Under conditions of oxygen deficit in the mullet body there is noted development of a complex of compensatory reactions directed to maintenance of the oxygen blood capacity. Initially (the 1st–2nd day), the release of erythrocytes from spleen is noted, which increases the number of red blood cells and the hemoglobin concentration in blood (the emergency adaptation). Subsequently (the 10–15th day), there occur quantitative readjustments at the level of the hemoglobin system, which lead to an increase in the blood affinity to oxygen and to a decrease in its sensitivity to pH (the long-term adaptation). The revealed changes take place on the background of a decrease in volume of circulating erythrocytes, which reflects the character of change of erythrocytic parameters (MCV, MCHC) and seems to be a consequence of partial dehydration of blood plasma.     

A comparative study of blood oxygen transport in turbot and sea bass: effect of chronic hypoxia

A comparative study of blood oxygen binding and carrying capacities of turbot Scophthalmus maximus and sea bass Dicentrarchus labrax , two fish species differing in their demand for oxygen, was carried out under three levels of chronic hypoxia ( P o ₂ = 93, 65 and 40 mmHg) for 40 days. Blood O₂ affinity in normoxia was moderately high in both species ( P ₅₀ was c . 12–13 mmHg at pH 7·7). The Bohr factor was significantly lower in turbot (−0·52) than in sea bass (−0·85). In both species, blood O₂ affinity was not significantly affected by oxygen depletion whatever its level and duration. In turbot, however, P ₅₀ appeared to slightly decrease at the two more severe levels of hypoxia. In both species, blood O₂ carrying capacity was not affected by hypoxia and remained twice as high in sea bass than in turbot.     

Adaptive variation in energy acquisition and allocation among latitudinal populations of the Atlantic silverside

Understanding the evolution of growth rate requires knowledge of the physiology of growth. This study explored the physiological basis of countergradient variation (CnGV) in somatic growth across latitudinal populations of the Atlantic silverside, Menidia menidia. Energetics of northern (Nova Scotia, Canada) and southern (South Carolina, USA) genotypes were compared across resource levels, temperatures, and fish sizes to identify trade-offs to rapid growth. Offered unlimited resources, genotypes differed in both energy acquisition and allocation. Food consumption, growth, and efficiency of northern genotypes were consistently higher than in southern genotypes, across temperatures and body sizes. Feeding metabolism (specific dynamic action; SDA) was proportional to meal size, differing between genotypes to the extent that food consumption differed. Given limited resources, northern and southern genotypes displayed similar growth, efficiency, routine activity, and SDA across temperatures and fish sizes. Routine metabolism was equal at 17°C and 22°C, yet was significantly higher in northern fish at 28°C. Growth rates in M. menidia do not appear to trade off across environments or body sizes, i.e., at no temperature, ration, or size do southern fish outgrow northern conspecifics. Nor does submaximal growth result from increased costs of maintenance, tissue synthesis, or routine activity. Based on our findings, we propose that CnGV consumption and growth in M. menidia likely result from trade-offs with other energetic components, namely sustained and burst swimming. Received: 26 January 1999 / Accepted: 14 September 1999     

Effects of salinity on the ionic balance and growth of juvenile turbot

The effects of salinity changes (27, 19 and 10‰) on seawater-adapted juvenile turbot were studied on their plasma osmolarity and ion concentrations, on oxygen consumption, on gill Na⁺,K⁺-ATPase activity after 3 months and on growth parameters. All plasma concentrations (except chloride) were unchanged, suggesting that fish were well adapted to their environment. Oxygen consumption was significantly decreased in the 19 and 10‰ groups, where fish weighed significantly more 105 days after transfer than fish maintained in sea water. These results, and the fact that apparent food conversion rates were lower in a diluted environment, suggest that on a long term schedule growth conditions could be improved by adaptation to brackish waters (salinities between 10 and 19‰). The effects of transfer from sea water to 27, 19, 10 and 5‰ were also followed during the first 3 weeks. With salinity 10‰ a steady state was reached on day 21 with all plasma values within the same range. The significant differences observed in osmolarity, plasma ion concentrations and Na⁺,K⁺-ATPase activity 3 weeks after transfer of juveniles to 5‰ salinity, compared with transfers in higher salinities, suggest that there is a threshold of acclimation of turbot to a hypotonic environment.     

Long term t in vitro storage of lily: effects of temperature and concentration of nutrients and sucrose

Methods for long-term preservation of lily germplasm were examined. t In vitro regenerated bulblets of 10 lily (t Lilium L.) genotypes (Asiatic hybrids, Oriental hybrids, t L. longiflorum and t L. henryi) were stored for 28 months at -2 °C and 25 °C on four different media: 1/4 or full strength Murashige and Skoog nutrients with 9% (w/v) or 6% sucrose. Sprout growth, bulb growth, and viability were determined. The combination of 1/4 strength MS nutrients and 9% sucrose gave the highest reduction in sprout and bulb growth, the highest viability and the highest percentage of regrowth after 28 months of storage. At 25 °C, all lily genotypes survived 28 months of storage under these conditions. At -2 °C, Asiatic and Oriental hybrids survived 28 months of storage, whereas genotypes of t L. longiflorum and t L. henryi survived 6 months of storage, but died during prolonged storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of temperature on hardhead minnow (Mylopharodon conocephalus) blood-oxygen equilibria

Habitat perturbations, including dam construction with consequent temperature changes and the introduction of non-native species to California’s mid- to low-elevation streams, have negatively influenced some native fish populations’ historic distribution and abundance. Populations of hardhead, Mylopharodon conocephalus (Cyprinidae), have experienced such population declines, but environmental temperature effects on this large (to 60 cm SL), native species are poorly documented. We measured temperature effects on in vitro blood-oxygen affinity and equilibrium curve shape, key dynamics of the species’ oxygen-transport system, derived from blood collected from wild-caught hardhead. Over an 11–30 °C temperature range, the half-saturation value (P₅₀, an inverse measure of affinity) increased with the temperature from 0.51 to 1.80 kPa for low-PCO₂ (“arterial”) treatments and from 2.02 to 2.92 kPa for high-PCO₂ (“venous”) treatments. The apparent heat of oxygenation (temperature effect) was higher at temperatures > (absolute value) 19 °C. Therefore, hardhead’s blood has a decreased ability to bind oxygen at its gills at temperatures ≥25 °C, compared to that at temperatures ≤19 °C. The hardhead’s Bohr factors (Ф), non-bicarbonate buffer values (β), nucleoside triphosphate (NTP) concentrations, blood oxygen capacities (CBO₂), and mildly sigmoid-shaped oxygen equilibrium curves showed no relationship with temperature. Overall, their blood-oxygen equilibria suggest that hardhead can tolerate moderate hypoxia and temperature variations in its environment and that they have some capacity for sustained, high-aerobic activity.     

Localized temperature adaptation of cottonwoods from elevational ecoregions in the Rocky Mountains

To investigate temperature adaptation of cool-climate trees, we studied 14 cottonwood genotypes from riparian (streamside) zones in three ecoregions differing in elevation in the Rocky Mountains of Alberta, Canada. Black cottonwoods (Populus trichocarpa) were collected from the higher, cooler montane and intermediate (aspen) parkland, and narrowleaf cottonwoods (P. angustifolia) were collected from the lower, warmer fescue prairie. The genotypes were grown in growth chambers under three temperature regimes reflecting the ecoregion variation. Sapling growth (dry weight) varied significantly across temperatures and for the genotypes from within and particularly across the ecoregions. Significant temperature × genotype interactions further indicated differentiation of the temperature response. Growth of the montane clones increased by 209% between 15/10 and 20/15°C and was 37% lower at 25/20°C. In contrast, genotypes from the lower ecoregions grew more slowly at the cool and intermediate temperatures (59 and 58% of montane) and then demonstrated constant (−3% parkland) or slightly increased (+16% prairie) growth at 25/20°C. This suggests the existence of P. trichocarpa ecotypes, localized populations with different temperature responses. This differentiation may explain our previous observation of comparable growth across these ecoregions despite substantial temperature variation, and the existence of ecotypes would produce a range of responses to climate warming that should produce an upward shift of the mountain ecoregions.     

Temperature-independent genome-wide DNA methylation profile in turbot post-embryonic development

The morphological and biological characteristics of ectothermic vertebrates are known to be strongly influenced by environmental conditions, particularly temperature. Epigenetic mechanisms such as DNA methylation have been reported to contribute to the phenotypic plasticity observed in vertebrates in response to environmental changes. Additionally, DNA methylation is a dynamic process that occurs throughout vertebrate ontogeny and it has been associated with the activation and silencing of gene expression during post-embryonic development and metamorphosis. In this study, we investigated genome-wide DNA methylation profiles during turbot metamorphosis, as well as the epigenetic effects of temperature on turbot post-embryonic development. Fish growth and rates of development were greatly affected by rearing temperature. Thus, turbot raised at ambient temperature (18 °C) achieved greater body weights and progressed through development more quickly than those reared at a colder temperature (14 °C). Genome-wide DNA methylation dynamics analyzed via a methylation-sensitive amplified polymorphism (MSAP) technique were not significantly different between animals reared within the two different thermal environments. Furthermore, comparisons between phenotypically similar fish revealed that genome-wide DNA methylation profiles do not necessarily correlate with specific developmental stages in turbot.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.