Keiron P. P. Fraser

Protein metabolism in marine animals: The underlying mechanism of growth

Growth is a fundamental process within all marine organisms. In soft tissues, growth is primarily achieved by the synthesis and retention of proteins as protein growth. The protein pool (all the protein within the organism) is highly dynamic, with proteins constantly entering the pool via protein synthesis or being removed from the pool via protein degradation. Any net change in the size of the protein pool, positive or negative, is termed protein growth. The three inter-related processes of protein synthesis, degradation and growth are together termed protein metabolism. Measurement of protein metabolism is vital in helping us understand how biotic and abiotic factors affect growth and growth efficiency in marine animals. Recently, the developing fields of transcriptomics and proteomics have started to offer us a means of greatly increasing our knowledge of the underlying molecular control of protein metabolism. Transcriptomics may also allow us to detect subtle changes in gene expression associated with protein synthesis and degradation, which cannot be detected using classical methods. A large literature exists on protein metabolism in animals; however, this chapter concentrates on what we know of marine ectotherms; data from non-marine ectotherms and endotherms are only discussed when the data are of particular relevance. We first consider the techniques available to measure protein metabolism, their problems and what validation is required. Protein metabolism in marine organisms is highly sensitive to a wide variety of factors, including temperature, pollution, seasonality, nutrition, developmental stage, genetics, sexual maturation and moulting. We examine how these abiotic and biotic factors affect protein metabolism at the level of whole-animal (adult and larval), tissue and cellular protein metabolism. Available gene expression data, which help us understand the underlying control of protein metabolism, are also discussed. As protein metabolism appears to comprise a significant proportion of overall metabolic costs in marine organisms, accurate estimates of the energetic cost per unit of synthesised protein are important. Measured costs of protein metabolism are reviewed, and the very high variability in reported costs highlighted. Two major determinants of protein synthesis rates are the tissue concentration of RNA, often expressed as the RNA to protein ratio, and the RNA activity (k(RNA)). The effects of temperature, nutrition and developmental stage on RNA concentration and activity are considered. This chapter highlights our complete lack of knowledge of protein metabolism in many groups of marine organisms, and the fact we currently have only limited data for animals held under a narrow range of experimental conditions. The potential assistance that genomic methods may provide in increasing our understanding of protein metabolism is described.

Hibernation in an Antarctic Fish: On Ice for Winter

Active metabolic suppression in anticipation of winter conditions has been demonstrated in species of mammals, birds, reptiles and amphibians, but not fish. This is because the reduction in metabolic rate in fish is directly proportional to the decrease in water temperature and they appear to be incapable of further suppressing their metabolic rate independently of temperature. However, the Antarctic fish (Notothenia coriiceps) is unusual because it undergoes winter metabolic suppression irrespective of water temperature. We assessed the seasonal ecological strategy by monitoring swimming activity, growth, feeding and heart rate (f H) in N. coriiceps as they free-ranged within sub-zero waters. The metabolic rate of wild fish was extrapolated from f H recordings, from oxygen consumption calibrations established in the laboratory prior to fish release. Throughout the summer months N. coriiceps spent a considerable proportion of its time foraging, resulting in a growth rate (Gw) of 0.18±0.2% day−1. In contrast, during winter much of the time was spent sedentary within a refuge and fish showed a net loss in Gw (−0.05±0.05% day−1). Whilst inactive during winter, N. coriiceps displayed a very low f H, reduced sensory and motor capabilities, and standard metabolic rate was one third lower than in summer. In a similar manner to other hibernating species, dormancy was interrupted with periodic arousals. These arousals, which lasted a few hours, occurred every 4–12 days. During arousal activity, f H and metabolism increased to summer levels. This endogenous suppression and activation of metabolic processes, independent of body temperature, demonstrates that N. coriiceps were effectively ‘putting themselves on ice’ during winter months until food resources improved. This study demonstrates that at least some fish species can enter a dormant state similar to hibernation that is not temperature driven and presumably provides seasonal energetic benefits.

Ice scour disturbance in Antarctic waters

The West Antarctic Peninsula is one of the fastest warming regions on Earth, and, as a consequence, most maritime glaciers and ice shelves in the region have significantly retreated over the past few decades. We collected a multiyear data set on ice scouring frequency from Antarctica by using unique experimental markers and scuba diving surveys. We show that the annual intensity of ice scouring is negatively correlated with the duration of the winter fast ice season. Because fast ice extent and duration is currently in decline in the region after recent rapid warming, it is likely that marine benthic communities are set for even more scouring in the near future.

Low heat shock thresholds in wild Antarctic inter-tidal limpets (Nacella concinna)

Heat shock proteins (HSPs) are a family of genes classically used to measure levels of organism stress. We have previously identified two HSP70 genes (HSP70A and HSP70B) in sub-tidal populations of the Antarctic limpet (Nacella concinna). These genes are up-regulated in response to increased seawater temperatures of 15°C or more during acute heat shock experiments, temperatures that have very little basis when considering the current Antarctic ecology of these animals. Therefore, the question was posed as to whether these animals could express HSP70 genes when subjected to more complex environmental conditions, such as those that occur in the inter-tidal. Inter-tidal limpets were collected on three occasions in different weather conditions at South Cove, Rothera Point, over a complete tidal cycle, and the expression levels of the HSP70 genes were measured. Both genes showed relative up-regulation of gene expression over the period of the tidal cycle. The average foot temperature of these animals was 3.3°C, far below that of the acute heat shock experiments. These experiments demonstrate that the temperature and expression levels of HSP production in wild animals cannot be accurately extrapolated from experimentally induced treatments, especially when considering the complexity of stressors in the natural environment. However, experimental manipulation can provide molecular markers for identifying stress in Antarctic molluscs, provided it is accompanied by environmental validation, as demonstrated here.

Growth in the slow lane: protein metabolism in the Antarctic limpet Nacella concinna (Strebel 1908)

SUMMARY Growth rates in Antarctic ectotherms are generally considered to be low in comparison to temperate and tropical species. Food consumption plays a major role in determining animal growth rates, but once food is ingested soft tissue growth rates are largely determined by the protein synthesis retention efficiency (PSRE), a measure of the efficiency with which proteins are synthesised and retained as protein growth. The effect of water temperatures on the PSRE of polar organisms has not previously been investigated, and it is possible that reduced PSRE at polar water temperatures may at least partially explain low growth rates in Antarctic organisms. We also currently lack any information on the potential effects of predicted increases in seawater temperatures on protein metabolism in Antarctic ectotherms. We have measured seasonal protein synthesis, degradation and growth rates in free-ranging Antarctic limpets (Nacella concinna), together with protein synthesis rates at temperatures ranging between –1.5°C and 6.0°C. PSRE were not significantly different in summer (15.69±4.41%) or winter (20.59±4.45%), but values were considerably lower than those previously reported in temperate and tropical species. A meta-analysis of published ectotherm PSRE suggested there was a positive relationship with temperature (y=449.9–114.9x, r2=28.8%, P<0.05). In turn, this suggests that temperature may be an important factor in determining ectotherm growth efficiency via an influence on PSRE. Maximal fractional and absolute protein synthesis rates occurred at ∼1°C in N. concinna, the approximate summer water temperature at the study site, and protein synthesis rates decreased above this temperature. In the absence of adaptation, predicted increases in Antarctic water temperatures would result in reduced, rather than increased, rates of protein synthesis and, in turn, possibly growth.

Protein Synthesis, RNA Concentrations, Nitrogen Excretion, and Metabolism Vary Seasonally in the Antarctic Holothurian Heterocucumis steineni (Ludwig 1898)

Seasonal changes in protein and nitrogen metabolism have not previously been reported in any Antarctic suspension‐feeding species that ceases feeding for extended periods in winter. To provide comparison with data reported on Nacella concinna, a species that continues to feed in winter, we have measured feeding activity; oxygen consumption; ammonia, urea, and fluorescamine‐positive substance (FPS) excretion; O:N ratios; body wall protein synthesis; RNA to protein ratios; and RNA activity at three times during the year in an Antarctic suspension‐feeding holothurian. Feeding activity ceased for 4 mo during winter, and oxygen consumption rates decreased from 8.79 ± 0.43 μmol h−1 to 4.48 ± 0.34 μmol h−1. Ammonia excretion also decreased during winter from 2,600 ± 177 nmol N h−1 to 974 ± 70 nmol N h−1, but urea excretion rates increased from 178 ± 36 nmol N h−1 to 281 ± 110 nmol N h−1, while FPS excretion rates remained unchanged throughout the year with a seasonal mean of 88 ± 13 nmol N h−1. Oxygen to nitrogen ratios ranged between 6 and 10, suggesting that proteins were used as the primary metabolic substrate. Body wall protein synthesis rates decreased from 0.35% ± 0.03% d−1 in summer to 0.23% d−1 in winter, while RNA to protein ratios decreased from 33.10 ± 1.0 μg RNA mg−1 protein in summer to 27.88 ± 1.3 μg RNA mg−1 protein in winter, and RNA activity was very low, ranging between 0.11 ± 0.01 mg protein mg−1 RNA d−1 in summer and 0.06 ± 0.01 mg protein mg−1 RNA d−1 in winter. Heterocucumis steineni shows a larger seasonal decrease in oxygen consumption and ammonia excretion between February (summer) and July (winter) than N. concinna, while the proportional decrease in protein synthesis rates is similar in both species.

Marine richness and gradients at Deception Island, Antarctica

Abstract Studies of the recovery of the fauna following the 1967–70 eruptions at Deception Island, South Shetland Islands, have made it one of the best-studied marine sites of the Southern Ocean for biodiversity. Using SCUBA we surveyed the mega- and macro-epifauna of its subtidal zones in the entrance (Neptunes Bellows), immediately inside the caldera (Whalers Bay) and well within the caldera (Fumarole Bay). Richness declined from 10 phyla, 13 classes and 35 species at Neptunes Bellows to three phyla, four classes and five species in Whalers Bay and just two phyla, classes and species at Fumarole Bay. Amongst the 35 species we found at Neptunes Bellows, 14 were previously unrecorded from Deception Island. Despite many ship visits and amongst the warmest sea temperatures in the Southern Ocean, the Non Indigenous Species (NIS) algae were not found in our survey. Deception Island has been recolonized considerably since the recent eruptions, but many taxa are still very poorly represented and the colonizers present are mainly those with planktotrophic larvae. Examination of the literature revealed that to date 163 named marine species have been found within the caldera as well as at least 50 more morphospecies, which are yet to be identified. Species accumulation has consistently increased across eight recent samples reported and the number of species reported there is likely to reach 300 when taxa such as the nematodes are identified to species level. This represents a first meaningful total species estimate for an Antarctic marine area and, as the site is comparatively impoverished, indicates how rich the surrounding Antarctic shelf must be.

Comparative phylogeography of three trematomid fishes reveals contrasting genetic structure patterns in benthic and pelagic species

Population genetics patterns of marine fish in general and of Southern Ocean fish in particular range from virtual panmixia over ocean-wide scale to deeply fragmented populations. However the causes underlying these different patterns are not properly understood. In this paper, we tested the hypotheses that population connectivity is positively related to a combination of life history traits, namely duration of pelagic larval period and the tendency towards pelagic life style in the adulthood. To do so, we analysed the variability of six microsatellite and one mitochondrial marker (cytochrome b) in three Southern Ocean fish species (Trematomus newnesi, Trematomus hansoni and Trematomus bernacchii). They share a recent common ancestor but notably differ in their duration of pelagic larval period as well as pelagic versus benthic lifestyle. We sampled over a range of more than 5000 km for all three species and used a number of population genetics tools to investigate past and contemporary levels of connectivity. All species experienced population fluctuations, but coalescent simulations suggested that contemporary populations are in migration-drift equilibrium. Although global F(ST) values were rather low, a significant population structure separated the High-Antarctic from the Peninsular regions in all species. The level of genetic differentiation was much lower in the pelagic versus benthic species. Present data suggest that past and present genetic structuring in the Southern Ocean are indeed related with the ecological traits of Antarctic fish, however the relative importance of individual factors remains unclear.

Heart rate and ventilation in Antarctic fishes are largely determined by ecotype

Extrinsic neural and humoral influences on heart rate (fH) and ventilation frequency (fV) were examined following varying periods of post-surgical recovery in eight related Antarctic fish species inhabiting an array of inshore niches. Resting fH after recovery from handling was lower than previous reports, and the novel measurement of routine fH in free-swimming Dissostichus mawsoni (6.14 beats min(-1), bpm) is the lowest recorded for any fish. The extent of cardio-depressive cholinergic (vagal) tonus explained the large range of fH among species and varied with behavioural repertoire, being lower in the more active species, apart from Notothenia coriiceps. Adrenergic tonus was low compared with cholinergic tonus, with the exception of Trematomus newnesi. Hence, high cardiac cholinergic tonus may be a genotypic trait of the notothenioids that diverged with ecotype. Power spectral analysis showed that the vagal influence produced comparable spectra among species of similar morphology and ecotype. Removal of autonomic tonus resulted in a remarkably similar intrinsic fH between species. Simultaneous measurements of cardio-respiratory variables and oxygen consumption (M(O(2))) were made in the benthic Trematomus bernacchii and cryopelagic Pagothenia borchgrevinki. The slopes of the relationship between fH and M(O(2)) were similar. Trematomus bernacchii, however, had a higher M(O(2)) for a given fH than P. borchgrevinki, and P. borchgrevinki required a two-fold larger range in fH to reach a similar maximum M(O(2)), suggesting that there is a difference in cardiovascular fitness between the two species. Overall, the data suggest that cardio-respiratory control in Antarctic nototheniids is largely determined by activity levels associated with a given ecotype.