Comparison of whole animal costs of protein synthesis among polar and temperate populations of the same species of gammarid amphipod.

Protein synthesis can account for a substantial proportion of metabolic rate. Energetic costs of protein synthesis, should in theory, be the same in marine invertebrates from a range of thermal habitats, and yet direct measurements using inhibitors produce widely differing values, especially in the cold. The present study aimed to remove any potential confounding interspecific effects by determining costs of protein synthesis in two latitudinally separated populations of the same species (amphipod, Gammarus oceanicus) living in two different thermal regimes; polar vs cold-temperate. Costs of protein synthesis were determined in summer acclimatised G. oceanicus from Svalbard (79°N) at 5°C and from Scotland (58°N) at 13°C. Amphipods were injected with the protein synthesis inhibitor, cycloheximide (CHX), at 9mmoll-1 in crab saline to give a tissue concentration of 0.05mgCHXg-1FW and left for 60min before the injection of [3H] phenylalanine. After incubation for 120min (180min in total from initial injection), both whole-animal rates of oxygen uptake and absolute rates of protein synthesis were significantly reduced in CHX-treated amphipods vs controls injected with saline. Both populations exhibited similar costs of protein synthesis of ~7µmolO2mg-1protein which is close to the estimated theoretical minimum for peptide bond formation, and similar to the values obtained in cell-free systems. The study demonstrates that in G. oceanicus, costs of protein synthesis rates were not elevated in the cold but were fixed among polar and cold-temperate populations.

Congeneric amphipods show differing abilities to maintain metabolic rates with latitude.

Metabolic variability across latitudinal populations of gammarid amphipods was examined in the summer by determining whole-animal rates of oxygen uptake (M(o)2) in four species with overlapping distribution patterns in the northeast Atlantic and Arctic oceans. Comparisons were made between an arctic/boreal species, Gammarus setosus, a subarctic/boreal species, Gammarus oceanicus, a boreal/temperate species, Gammarus duebeni duebeni, and a temperate species, Gammarus locusta. Measurements included acclimatized M(o)2 in all four species and M(o)2 after acclimation to 10°C in two populations of G. oceanicus and G. locusta. In G. oceanicus, acclimatized M(o)2 declined with latitude (13° to 5°C) so that metabolic rates were lower in subarctic (79°N) relative to temperate (58°N) populations and similar to the values in G. setosus at 79°N. Consequently, there was no evidence for metabolic rate compensation in the colder-water, high-latitude populations in the summer. Further examination of the specific effects of temperature revealed similarities in M(o)2 between populations of G. oceanicus acclimated at 10°C and similarities in thermal sensitivity (Q(10)) and activation energies (E(a)) on exposure to acute temperature change. In sharp contrast, there was no variation in summer acclimatized M(o)2 with latitude in either G. d. duebeni between 48° and 70°N or G. locusta between 38° and 53°N. Instead, the two species maintained relatively high metabolic rates across latitudes, which were associated in G. locusta with differences in M(o)2 and with Q(10) and E(a) values in amphipods acclimated at 10°C. The ability to compensate metabolic rate with latitude in the summer suggests greater metabolic flexibility, which predicts a greater capacity for survival during climate change of the temperate/boreal over the subarctic and arctic gammarid species.

Characterisation of myosin heavy chain gene variants in the fast and slow muscle fibres of gammarid amphipods.

Recent molecular work has revealed a large diversity of myosin heavy chain (MyHC) gene variants in the abdominal musculature of gammarid amphipods. An unusual truncated MyHC transcript from the loop 1 region (Variant A(3)) was consistently observed in multiple species and populations. The current study aimed to determine whether this MyHC variant is specific to a particular muscle fibre type, as a change in net charge to the loop 1 region of Variant A(3) could be functionally significant. The localisation of different fibre types within the abdominal musculature of several gammarid species revealed that the deep flexor and extensor muscles are fast-twitch muscle fibres. The dorsal superficial muscles were identified as slow fibres and the muscles extrinsic to the pleopods were identified as intermediate fibres. Amplification of loop 1 region mRNA from isolated superficial extensor and deep flexor muscles, and subsequent liquid chromatography and sequence analysis revealed that Variant A(3) was the primary MyHC variant in slow muscles, and the conserved A(1) sequence was the primary variant in fast muscles. The specific role of Variant A(3) in the slow muscles remains to be investigated.

Linking functional molecular variation with environmental gradients: myosin gene diversity in a crustacean broadly distributed across variable thermal environments.

To investigate the molecular basis of temperature adaptation in natural populations we used the candidate gene approach, targeting the myosin heavy chain (MyHC) gene. The functional effects of genetic variation in MyHC have been well characterised, and changes in the flexibility of the surface loops 1 and 2, caused by modulations in length, amino acid composition and charge can play an important role in thermal acclimation in fish. However, the extent that MyHC diversity is influenced by natural thermal gradients is largely unknown. Sequence variation in MyHC cDNA was examined in 7 species of gammarid amphipod with broad latitudinal distributions and differing intertidal thermal habitats in the NE Atlantic and Arctic Oceans. A high degree of diversity was detected in the loop 1 nucleotide sequences, although not all are likely to be functional transcripts, and their deduced amino acid sequences indicated no differences in the length and charge of loop 1 and associated binding kinetics. Four isoforms for loop 2 were detected which differed in sequence length and charge distribution, suggesting functional differences in sliding velocities and ATPase activities. While all species, and indeed most individuals, expressed multiple loop 2 isoforms, analysis of the two species with the greatest number of sequenced clones revealed that G. duebeni, a high-shore species with the highest thermal tolerance, expressed a greater diversity of forms than G. oceanicus, a low intertidal species more sensitive to temperature change. Latitude further influenced MyHC loop 2 diversity in G. duebeni, as the number of isoforms increased in the northern populations. Species-specific variations in MyHC diversity were observed, irrespective of phylogenetic associations revealed by analysis of the mitochondrial cytochrome oxidase 1 (CO1) gene. Overall, it appears that the temporal temperature variations associated with higher intertidal habitat may be a greater selective agent for MyHC isoform diversity in gammarid muscles than broad spatial changes with latitude.

Effects of salinity on rates of protein synthesis and oxygen uptake in the post-larvae and juveniles of the tropical prawn Macrobrachium rosenbergii (de Man).

Protein synthesis is a major determinant of growth and yet little is known about the environmental factors that influence protein synthesis rates in farmed freshwater prawns. To this end, post-larvae and juveniles of Macrobrachium rosenbergii were exposed to various salinities (0, 14, 30 per thousand) to determine whole-animal rates of fractional protein synthesis (k(s)) and oxygen uptake. In the post-larvae that migrate upstream from brackish to freshwater areas, whole-animal k(s) was unaffected by salinity, but rates of oxygen uptake were significantly lower at 14 per thousand. In the freshwater juveniles, a different response was observed, as mean k(s) was significantly higher at 14 per thousand compared with 0 per thousand, but rates of oxygen uptake remained unchanged. Such differences are thought to be related to the energetic costs of osmoregulation and to the ability to maintain osmotic gradients in freshwater. In an additional experiment, acclimation temperature (20, 26, 30 degrees C) had a direct effect on k(s) in juveniles held at 0 per thousand. In all cases, changes in k(s) resulted from alterations in RNA activity at constant RNA capacity. In juveniles at least, whole-animal rates of protein synthesis were highest at 14 per thousand and 30 degrees C which corresponds to the optimal salinity and temperature recommended for the growth and culture of M. rosenbergii.

Phylogeography and environmental diversification of a highly adaptable marine amphipod, Gammarus duebeni.

Genetic diversity and phylogeographic population structure in the gammarid amphipod, Gammarus duebeni, were investigated across its broad latitudinal distribution in the NE and NW Atlantic by analysis of mitochondrial DNA sequence. Gammarus duebeni has exceptional tolerance of salinity change and inhabits environments ranging from marine to freshwater. The longstanding debate on whether there are distinct marine and freshwater subspecies was assessed by sampling populations from sites characterized by different salinities. Our sequence data demonstrates that there are two major lineages, with little internal geographic structuring. Evidence is provided to suggest a pre-glacial divergence of these two clades, involving segregation between a region historically associated with the freshwater form and the majority of the marine localities on both sides of the Atlantic. A modern contact zone between the marine and freshwater forms is proposed in western Britain.

Polar cod, Boreogadus saida (Gadidae), show an intermediate stress response between Antarctic and temperate fishes.

To examine whether the attenuated stress response observed in Antarctic notothenioid fishes is a specialism for life in sub-zero waters, the polar cod, Boreogadus saida, and the temperate shorthorned sculpin, Myoxocephalus scorpius, were subjected to various stress treatments. Activity stress in both species had no effect on plasma catecholamine and cortisol levels, splenic mass, and on the haematological variables in B. saida. In contrast, heat stress caused a significant rise in circulating noradrenaline and adrenaline levels in B. saida, accompanied by a significant increase in haematocrit and haemoglobin concentrations, at constant plasma cortisol levels, red blood cell count and splenic mass. A concomitant rise in blood lactate concentrations indicated that heat-stressed B. saida were hypoxaemic. The capacity to synthesise catecholamines in B. saida was 38% of the value in M. scorpius, but similar to the values for Antarctic notothenioids. The lack of any adrenergic response to activity stress suggests that dominance of cholinergic control of the cardiovascular system may not be restricted to Antarctic notothenioids. Rather, the stress response in B. saida appears to be intermediate between Antarctic and temperate teleosts, in keeping with their relatively recent occupation of cold Arctic waters.

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation.

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.

Comparison of the variable loop regions of myosin heavy chain genes from Antarctic and temperate isopods.

The evolutionary adaptations of functional genes to life at low temperatures are not well characterised in marine and fresh water invertebrates. Temperature has been shown to affect the functional characteristics of fish muscles, with changes in the velocity of shortening and ATPase activity being associated with myosin heavy chain (MyHC) isoform composition and the structure of the surface loop regions. Two PCR products spanning loops 1 and 2 of a MyHC gene from an Antarctic isopod (Glyptonotus antarcticus) were sequenced and compared with those of a temperate isopod (Idotea resecata), slow and fast fibres from lobster (Homarus gammarus) and a cold water amphipod (Eulimnogammarus verrucosus), revealing specific differences between the species, possibly related to fibre type and habitat temperature. The loop 2 region from G. antarcticus myosin was cloned and used for Northern analysis of total RNA from the other species. The cloned myosin cDNA hybridised specifically to a 6.6-kb transcript, in G. antarcticus muscle. In contrast, cDNA probes for lobster slow myosin and actin hybridised to muscle RNA from all species, demonstrating that a distinct MyHC isoform is expressed in the Antarctic isopod, as opposed to the temperate species. The inter- and intra-specific sequence differences in loop 2 region suggest that this may be a site for muscle adaptation to enable function at the low temperatures found in the Southern Ocean.

Protein synthesis and specific dynamic action in crustaceans: effects of temperature.

Temperature influences the specific dynamic action (SDA), or rise in oxygen uptake rate after feeding, in eurythermal and stenothermal crustaceans by changing the timing and the magnitude of the response. Intra-specific studies on the eurythermal crab, Carcinus maenas, show that a reduction in acclimation temperature is associated with a decrease in SDA magnitude, resulting from an increase in SDA duration but a decrease in peak factorial scope (the factorial rise in peak SDA over prefeeding values). Inter-specific feeding studies on stenothermal polar isopods revealed marked differences in SDA response between the Antarctic species, Glyptonotus antarcticus and the Arctic species, Saduria entomon. Compared to S. entomon held at 4 and 13 degrees C, the SDA response in G. antarcticus held at 1 degrees C was characterised by a lower absolute oxygen uptake rate at peak SDA and an extended SDA duration. At peak SDA, whole animal rates of protein synthesis increased in proportion to the postprandial increase in oxygen uptake rate in the Antarctic and the Arctic species. Rates of oxygen uptake plotted against whole animal rates of protein synthesis gave similar relationships in both isopod species, indicating similar costs of protein synthesis after a meal, despite their differences in SDA response and thermal habitat.

Effects of water salinity on acid-base balance in decapod crustaceans.

Extracellular acid-base balance in decapod crustaceans is influenced by water salinity, although the nature of this relationship is unclear. In euryhaline crabs, a decrease in salinity results in a metabolic alkalosis in the haemolymph and an increase in salinity results in a metabolic acidosis. Alterations in acid-base status by external changes in salinity are thought to be secondary to the adjustments required for ionic and osmotic regulation. In the present study, acid-base adjustments in the haemolymph of Eriocheir sinensis after transfer to 30 % sea water accompanied alterations in muscle pH and [HCO(3)(-)], as an initial acidosis coincided with an alkalosis in the leg muscle. By 48 h transfer, haemolymph pH increased as muscle pH and HCO(3)(-) declined. Haemolymph [Cl(-)] decreased significantly 3 h after transfer to a new steady state but haemolymph [Na(+)] and muscle [Na(+)] and [Cl(-)] remained unchanged. Muscle free amino acid concentration increased twofold 6 h after transfer, followed by a 2.5-fold increase in the haemolymph after 24 h. In contrast, 30 % sea water had no effect on haemolymph acid-base adjustments in the osmoconforming crab, Necora puber, which lacks ion and osmo-regulatory mechansims. Collectively these observations support the view that salinity-induced alterations in acid-base status are caused by adjustments consistent with cell volume regulation.

Antarctic fishes have a limited capacity for catecholamine synthesis.

To determine whether an attenuated stress response is a general feature of Antarctic fish or is dependent on ecotype, the capacity for catecholamine synthesis within the head kidney and plasma levels of the primary stress hormones (catecholamines and cortisol) were determined in species with a range of activity patterns. Tyrosine hydroxylase (TH) activities were similar in both sluggish (Gobionotothen gibberifons, 153+/-22 nmol g(-)(1 )h(-)(1), mean +/- s.e.m.) and active (Notothenia rossii, 185+/-39 nmol g(-)(1 )h(-)(1), Dissostichus mawsoni, 128+/-31 nmol g(-)(1 )h(-)(1)) pelagic nototheniids, but only 30 % of those in Atlantic cod (Gadus morhua, 393+/-88 nmol g(-)(1 )h(-)(1)) at the same temperature. TH activities were even lower in white-blooded channichthyids (Chaenocephalus aceratus, 74+/-16 nmol g(-)(1 )h(-)(1) and Champsocephalus gunnari, 53+/-17 nmol g(-)(1 )h(-)(1)), although values in Chionodraco rastrospinosus were similar to red-blooded species (178+/-45 nmol g(-)(1 )h(-)(1)). Circulating catecholamine levels were extremely high in all species after fishing stress, with adrenaline levels 3-4 times higher than noradrenaline levels. Cortisol levels remained low, ranging from 1.33+/-0.58 ng ml(-)(1) in Champsocephalus gunnari to 44.9+/-25.0 ng ml(-)(1 )in Dissostichus mawsoni. These data suggest that depressed catecholamine synthesis is typical of Antarctic fish regardless of life style, although they are able to release extensive stores from the chromaffin tissue under conditions of extreme trauma. Cortisol does not appear to be an important primary stress hormone in these species.

A comparison of the metabolic cost of protein synthesis in stenothermal and eurythermal isopod crustaceans.

To examine the presence of metabolic cold adaptation in Antarctic isopods, whole animal rates of oxygen uptake (MVo2) and protein synthesis were measured in Glyptonotus antarcticus at 0 degree C and compared with the temperature isopod Idotea rescata at 4 and 14 degrees C. The specific relationship between rates of metabolism and protein synthesis was investigated by injecting animals with cycloheximide, a protein synthesis inhibitor. In G. antarcticus, routine MVo2 was 11.10 +/- 0.89 mumol.kg-1.min-1 (n = 19 animals), and ks was 0.24 +/- 0.04% protein synthesized/day (n = 8 animals). Comparison with I. rescata showed that standardized whole animal MVo2 decreased with temperature (temperature quotient = 1.99), but whole animal ks was considerably lower in the Antarctic isopod; 66 and 22% of total MVo2 was attributable to protein synthesis in G. antarcticus at 0 degree C and I. rescata at 4 degrees C, respectively. The energetic cost of protein synthesis was four times higher in G. antarcticus at 885 +/- 141 mmol ATP/g protein (n = 5 animals) compared with 237 +/- 76 mmol ATP/g protein (n = 6) in I. rescata. G. antarcticus does not show metabolic rate compensation and maintains extremely low ks levels because of the relatively high energetic cost of protein synthesis.

Regulation of muscle gene expression in Crustacea over the moult cycle.

Muscle growth in Crustacea may occur during specific stages of the moult cycle, focused around ecdysis when the old cuticle is shed and the new cuticle expands. In order to determine the moult stages in which sarcomeric proteins are synthesized and the regulatory factors involved, actin mRNA levels have been measured in the muscles of two crustaceans. These levels have been followed throughout the moult cycle and in response to passive stretch of walking leg muscle in vivo and to exogenous ecydsteroids applied to muscle preparations in vitro. Actin mRNA levels in both claw and leg muscles were elevated during the pre and postmoult stages of the moult cycle. However, varying patterns of expression are found in claw and leg muscle at specific stages of pre and postmoult. There was no increase in actin mRNA expression in extensor leg muscles in vitro after 6 hours exposure to elevated premoult levels of ecdysteroids. Immobilization of intemoult walking legs to maintain the extensor muscle in continuous passive stretch did not result in increased levels of actin mRNA after 5 days. These results are discussed in relation to the regulation of muscle growth over the moult cycle and to the molecular processes which may be responsible for controlling muscle protein synthesis.

Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus.

Robustoxin, the lethal neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus, is of unique structural type and physiological mode of action. The primary structure of this 42-residue peptide was determined to be H2N-Cys-Ala-Lys-Lys-Arg-Asn-Trp-Cys-Gly-Lys-Asn-Glu-Asp-Cys-Cys-Cys-Pro- Met-Lys-Cys-Ile-Tyr-Ala-Trp-Tyr-Asn-Gln-Gln-Gly-Ser-Cys-Gln-Thr-Thr-Ile- Thr-Gly-Leu-Phe-Lys-Lys-Cys-H. The disposition of disulphide-bridged cysteine residues at both the amino- and carboxy-termini and as a triplet at residues 14-16 appears to have no precedent amongst neurotoxins.

Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells.

Attachment, ""tight binding'' and eclipse of radioactive poliovirus 2 (P2) and human rhinovirus 2 (HRV 2) were investigated. The activation energy for attachment of both HRV2 and P2 was about 13 kcal/mol. HRV2 differed from P2 in two respects: the Arrhenius plot for attachment of HRV2 showed a break at 15 to 19 degrees C when the cells were first treated several hours at 0 degrees C, and attachment of HRV2 was inhibited by treatment of cells with metabolic poisons able to reduce cellular ATP by more than 90%. Tight binding was determined by isolation of a specific P2-membrane complex or by loss of EDTA dissociability of HRV2. Tight binding of both viruses was slowed by 0.01 M iodoacetamide but not by 0.02 M F-; F- plus 0.002 M CN- slowed tight binding of HRV2 but not of P2. Eclipse, the irreversible alteration of parental virions, was detected by isolation of cell-associated subviral particles or by loss of cell-associated infectious virus. Eclipse of both viruses is slowed by iodoacetamide or F-. It seems likely that the early steps of infection with picornaviruses may be sensitive to alterations in the cell membrane produced by metabolic inhibitors or by treatment at low temperature.