Can the impacts of cold-water pollution on fish be mitigated by thermal plasticity?

Increasingly, cold-water pollution (CWP) is being recognised as a significant threat to aquatic communities downstream of large, bottom-release dams. Cold water releases typically occur during summer when storage dams release unseasonably cold and anoxic hypolimnetic waters, which can decrease the temperature of downstream waters by up to 16°C. Depending on the release duration, these hypothermic conditions can persist for many months. The capacity of ectothermic species to tolerate or rapidly adjust to acute temperature changes may determine the nature and magnitude of the impact of CWP on affected species. This study assessed the impacts of an acute reduction in water temperature on the physiological function and locomotor performance of juvenile silver perch (Bidyanus bidyanus) and examined their capacity to thermally compensate for the depressive effects of low temperatures via phenotypic plasticity. Locomotor performance (Ucrit and Usprint) and energetic costs (routine and maximum metabolic rate) were measured at multiple points over a 10-week period following an abrupt 10°C drop in water temperature. We also measured the thermal sensitivity of metabolic enzymes from muscle samples taken from fish following the exposure period. Cold exposure had significant depressive effects on physiological traits, resulting in decreases in performance between 10% and 55%. Although there was partial acclimation of Ucrit (~35% increase in performance) and complete compensation of metabolic rate, this occurred late in the exposure period, meaning silver perch were unable to rapidly compensate for the depressive effects of thermal pollution. The results of this study have substantial implications for the management of cold water releases from large-scale dams and the conservation of native freshwater fish species, as this form of thermal pollution can act as a barrier to fish movement, cause reduced recruitment, ecological community shifts and disruptions to timing and success of reproduction.

Measuring Ucrit and endurance: equipment choice influences estimates of fish swimming performance.

This study compared the critical swimming speed (Ucrit ) and endurance performance of three Australian freshwater fish species in different swim-test apparatus. Estimates of Ucrit measured in a large recirculating flume were greater for all species compared with estimates from a smaller model of the same recirculating flume. Large differences were also observed for estimates of endurance swimming performance between these recirculating flumes and a free-surface swim tunnel. Differences in estimates of performance may be attributable to variation in flow conditions within different types of swim chambers. Variation in estimates of swimming performance between different types of flumes complicates the application of laboratory-based measures to the design of fish passage infrastructure.

Osmoregulation by juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, in hypo- and hyper-saline waters.

While there is a considerable body of work describing osmoregulation by elasmobranchs in brackish and saltwater, far fewer studies have investigated osmoregulation in hypersaline waters. We examined osmo- and ionoregulatory function and plasticity in juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, exposed to three experimental salinities (25, 34 and 40‰) for two weeks. C. punctatum inhabits sheltered coastal areas and bays which can naturally become hypersaline as a consequence of evaporation of water but can also become hyposaline during flood events. We hypothesised that C. punctatum would demonstrate a phenotypically plastic osmoregulatory physiology. Plasma osmolality, urea, Na(+) and Cl(-) levels increased significantly with increasing environmental salinity. Rectal gland and branchial sodium-potassium ATPase (NKA) activities were unaffected by salinity. Using immunohistochemistry and Western Blotting we found evidence for the presence of the key ion-regulatory proteins vacuolar H(+)-ATPase (VHA), pendrin (Cl(-)/HCO3(-) co-transporter) and the Na(+)-H(+) exchanger isoform 3 (NHE3) in discrete cells within the branchial epithelia. These results indicate that C. punctatum is a partially euryhaline elasmobranch able to maintain osmo- and ionoregulatory function between environmental salinities of 25‰ and 40‰. As suggested for other elasmobranchs, the gills of C. punctatum likely play a limited role in maintaining Na(+) homeostasis over the salinity range studied, but may play an important role in acid-base balance.

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectotherm Cyclorana alboguttata.

Animals that undergo prolonged dormancy experience minimal muscle disuse atrophy (MDA) compared to animals subjected to artificial immobilisation over shorter timeframes. An association between oxidative stress and MDA suggests that metabolic depression presumably affords dormant animals some protection against muscle disuse. Because aerobic metabolism is temperature sensitive, we proposed that MDA in dormant (aestivating) ectotherms would be enhanced at elevated temperatures. In the green-striped burrowing frog, Cyclorana alboguttata, the thermal sensitivity of skeletal muscle metabolic rate is muscle-specific. We proposed that the degree of atrophy experienced during aestivation would correlate with the thermal sensitivity of muscle metabolic rate such that muscles with a relatively high metabolic rate at high temperatures would experience more disuse atrophy. To test this hypothesis, we examined the effect of temperature and aestivation on the extent of MDA in two functionally different muscles: the M. gastrocnemius (jumping muscle) and M. iliofibularis (non-jumping muscle), in C. alboguttata aestivating at 24 or 30 °C for 6 months. We compared a range of morphological parameters from muscle cross-sections stained with succinic dehydrogenase to show that muscle-specific patterns of disuse atrophy were consistent with the relative rates of oxygen consumption of those muscle types. However, despite muscle-specific differences in thermal sensitivity of metabolic rate, aestivation temperature did not influence the extent of atrophy in either muscle. Our results suggest that the muscles of frogs aestivating at high temperatures are defended against additional atrophy ensuring protection of muscle function during long periods of immobilisation.

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs.

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na(+)K(+)ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.

Effect of incubation temperature on muscle growth of barramundi Lates calcarifer at hatch and post-exogenous feeding.

Muscle morphology was investigated in newly hatched barramundi Lates calcarifer larvae incubated at set temperatures (26, 29 and 31 degrees C) prior to hatching. Three days after hatching (the start of exogenous feeding), larvae from the 26 and 31 degrees C treatments were each divided into two groups and reared at that temperature or transferred over the period of several hours to 29 degrees C (control temperature). Incubation temperature significantly affected muscle cellularity in the developing embryo, with larvae incubated at 26 degrees C (mean +/-s.e. 223.3 +/- 7.9) having on average 14.4% more inner muscle fibres than those incubated at 31 degrees C (195.2 +/- 8.8) and 4.8% more than those incubated at 29 degrees C (213.5 +/- 4.7). Conversely, inner muscle fibre cross-sectional area significantly increased at the warm incubation temperature in L. calcarifer, so that the total cross-sectional muscle area was not different between treatment groups. The total cross-sectional area of superficial muscle fibres and the proportion of superficial to total fibre cross-sectional area in just hatched L. calcarifer were also affected by incubation temperature, with incubation at the cool temperature (26 degrees C) increasing both the total cross-sectional area and proportion of superficial muscle fibres. By 9 days post-hatch, the aforementioned differences were no longer significant. Similarly, there was no difference in total superficial fibre cross-sectional area between any treatment groups of L. calcarifer, whereas incubation temperature still significantly affected the proportion of superficial to total muscle fibre cross-sectional area. Larvae hatched and grown at 31 degrees C had a significantly reduced percentage of superficial muscle cross-sectional area (mean +/-s.e. 5.11 +/- 0.66%) compared with those incubated and grown at 29 degrees C (8.04 +/- 0.77%) and 26 degrees C (9.32 +/- 0.56%) and those incubated at 26 degrees C and transferred to 29 degrees C (7.52 +/- 0.53%), and incubated at 31 degrees C and transferred to 29 degrees C (6.28 +/- 0.69%). These results indicate that changes in muscle cellularity induced by raising or lowering the incubation temperature of L. calcarifer display varying degrees of persistence over developmental time. The significance of these findings to the culture of L. calcarifer is discussed.

Characterization of an inducible nitrilase from a thermophilic bacillus.

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M(r), 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying kcat/KM values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced kcat, while orthosubstitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65 degrees C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50 degrees C, 2.5 h at 60 degrees C, 13 min at 70 degrees C, and less than 3 min at 80 degrees C.

Molecular characterisation of a novel thermophilic nitrile hydratase.

The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases.

Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes.

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom. Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known. Nitrile hydratases are metalloenzymes, incorporating FeIII or CoII ions in thiolate ligand networks where they function as Lewis acids. In comparison, nitrilases are thiol-enzymes and the two enzyme groups have little or no apparent sequence or structural homology. The hydratases typically exist as alpha beta dimers or tetramers in which the alpha- and beta-subunits are similar in size but otherwise unrelated. Nitrilases however, are usually found as homomultimers with as many as 16 subunits. Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions. Purified mesophilic nitrile-degrading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50 degrees C without rapid inactivation. However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells. Low molecular stability has frequently been cited as a reason for the limited industrial application of "nitrilases"; such statements notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to the smaller-scale production of nicotinic acid, R-(-)-mandelic acid and S-(+)-ibuprofen. There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage.

Isolation and structure determination of two novel phenazines from a Streptomyces with inhibitory activity against metallo-enzymes, including metallo-beta-lactamase.

Two novel metabolites, SB 212021 and SB 212305, have been isolated from a Streptomyces and shown to have molecular formulae of C15H10N2O5 and C20H17N3O8S, respectively. The structures were deduced by a combination of NMR techniques and mass spectral fragmentation patterns and shown to be novel members of the phenazine group of antibiotics. In the absence of added zinc, both compounds had IC50's of 1-75 microM for the Bacteroides fragilis 262 CfiA and Xanthomonas maltophilia L-1 metallo-beta-lactamases. The compounds also inhibited ACE with IC50's of 55 and 68 microM, respectively. Mode of action studies illustrate that the compounds inhibit some metalloenzymes by chelation of the active site metal ion. They exhibit poor antibacterial activity.

Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid).

The effects of altered endogenous indole-3-acetic (IAA) levels on elongation in garden pea (Pisum sativum L.) plants were investigated. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) were applied to elongating internodes of wild-type and mutant lkb plants. The lkb mutant was included because elongating lkb internodes contained 2- to 3-fold less free IAA than those of the wild type. In the wild type, TIBA reduced both the IAA level and internode elongation below the site of application. Both TIBA and HFCA strongly promoted the elongation of lkb internodes and also raised IAA levels above the application site. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) also markedly increased internode elongation in lkb plants and virtually restored petioles and tendrils to their wild-type length. In contrast, treatment of wild-type plants with TIBA, HFCA, or 2,4-D caused little or no increase in elongation above the application site. The ethylene synthesis inhibitor aminoethoxyvinylglycine also increased stem elongation in lkb plants, and combined application of HFCA and aminoethoxy-vinylglycine restored lkb internodes to the wild-type length. It is concluded that the level of IAA in wild-type internodes is necessary for normal elongation, and that the reduced stature of lkb plants is at least partially attributable to a reduction in free IAA level in this mutant.

Rapid identification of metallo- and serine beta-lactamases.

Simple methods to detect, identify, and differentiate metallo- and serine beta-lactamases were developed and used to differentiate enzymes produced by 17 clinical isolates of Xanthomonas maltophilia. All isolates exhibited beta-lactamase activity, and in 16 strains this was induced by imipenem. All but one isolate hydrolyzed imipenem (and meropenem), and in all cases this activity was inhibited by 1 mM EDTA. The metallo- and serine beta-lactamases in the cell extracts were distinguished on isoelectric focusing (IEF) gels by using the following procedures. (i) Cell lysates were preincubated with 83 mM EDTA prior to IEF and subsequent visualization with nitrocefin, and (ii) after IEF, the gels were overlaid with either 1 mM zinc sulfate or 100 microM BRL 42715 before staining with nitrocefin. Bands of beta-lactamase activity which were removed by BRL 42715 but unaffected by EDTA or zinc sulfate were categorized as serine beta-lactamases. Bands which were unaffected by BRL 42715 but inhibited by EDTA or enhanced by zinc sulfate were classified as metallo-beta-lactamases. By using this approach, seven metallo-beta-lactamases were differentiated with pI values of 4.8 (two strains), 5.5 (four strains), 5.7 (one strain), 6.0 (one strain), 6.4 (four strains), 6.6 (one strain), and 6.8 (three strains). The metallo-beta-lactamase band with a pI of 6.4 aligned with the recently characterized metallo-beta-lactamase from X. maltophilia 511. Heterogeneity was also observed for the serine beta-lactamases: 14 isolates elaborated serine beta-lactamase activity which focused with major bands with at least eight different pIs. The remaining three strains produced serine beta-lactamases which focused with five distinct bands with pIs of 6.4, 6.2, 5.7, 5.5, and 5.2. We conclude that X. maltophilia produces many types of metallo- and serine beta-lactamases distinguishable by these new methods and that the previously reported L-1 and L-2 enzymes are not solely representative of the beta-lactamases produced by this species.

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases.

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.