Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution

The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids) have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR) binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional surfactant requirement is met by the leptin pulmonary surfactant production pathway which normally appears only to function in the mammalian foetus.

Rapid compensatory evolution promotes the survival of conjugative plasmids

Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome.

The role of glucocorticoids in naturally fasting grey seal (Halichoerus grypus) pups: dexamethasone stimulates mass loss and protein utilisation, but not departure from the colony

Seals must manage their energy reserves carefully while they fast on land to ensure that they go to sea with sufficient fuel to sustain them until they find food. Glucocorticoids (GCs) have been implicated in the control of fuel metabolism and termination of fasting in pinnipeds. Here we tested the hypothesis that dexamethasone, an artificial GC, increases fat and protein catabolism, and induces departure from the breeding colony in wild, fasting grey seal pups. A single intramuscular dose of dexamethasone completely suppressed cortisol production for 24–72 h, demonstrating activation of GC receptors. In experiment 1, we compared the effects of a single dose of dexamethasone or saline administered 10 days after weaning on fasting mass and body composition changes, cortisol, blood urea nitrogen (BUN) and glucose levels, and timing of departure from the colony. In experiment 2, we investigated the effects of dexamethasone on short-term (5 days) changes in mass loss, body composition and BUN levels. In experiment 1, dexamethasone induced a short-lived increase in mass loss, but there was no difference in timing of departure between dexamethasone- and saline-treated pups (N=10). In experiment 2, dexamethasone increased protein and water loss and prevented a decrease in BUN levels (N=11). Our data suggest changes in cortisol contribute to regulation of protein catabolism in fasting seal pups, irrespective of the sex of the animal, but do not terminate fasting. By affecting the rate of protein depletion, lasting changes in cortisol levels could influence the amount of time seal pups have to find food, and thus may have important consequences for their survival.

Effects of handling regime and sex on changes in cortisol, thyroid hormones and body mass in fasting grey seal pups

Survival of seal pups may be affected by their ability to respond appropriately to stress. Chronic stress can adversely affect secretion of cortisol and thyroid hormones, which contribute to the control of fuel utilisation. Repeated handling could disrupt the endocrine response to stress and/or negatively impact upon mass changes during fasting. Here we investigated the effects of handling regime on cortisol and thyroid hormone levels, and body mass changes, in fasting male and female grey seal pups (Halichoerus grypus). Females had higher thyroid hormone levels than males throughout fasting and showed a reduction in cortisol midway through the fast that was not seen in males. This may reflect sex-specific fuel allocation or development. Neither handling frequency nor cumulative contact time affected plasma cortisol or thyroid hormone levels, the rate of increase in cortisol over the first five minutes of physical contact or the pattern of mass loss during fasting in either sex. The endocrine response to stress and the control of energy balance in grey seal pups appear to be robust to repeated, short periods of handling. Our results suggest that routine handling should have no additional impact on these animals than general disturbance caused by researchers moving around the colony.

Effects of mass and body composition on fasting fuel utilisation in grey seal pups (Halichoerus grypus Fabricius): an experimental study using supplementary feeding

This study used supplementary feeding to test the hypothesis that fuel partitioning during the postweaning fast in grey seal pups is affected by size and composition of energy reserves at weaning, and by extra provisioning. Mass and body composition changes were measured during suckling and fasting to investigate the effect of natural differences in energy reserves at weaning on subsequent allocation of fat and protein to energy use. We fed seven pups for 5 days after weaning, to investigate the effect of increased fuel availability, and particularly protein, on fuel utilisation. After correcting for protein used during the moult, the proportional contribution of fat was 86–99% of total energy use. Pups with greater energy reserves, i.e. those that were heavier and fatter at weaning, had higher rates of fat and energy use. There was no significant relationship between adiposity at weaning and proportional contribution of fat to energy use, perhaps due to a limited sample size or range of body masses and adiposity. Supplemented individuals used energy, specifically fat, much faster and utilised proportionally less of their endogenous protein by departure than non-supplemented individuals. Fat metabolism contributed a similar percentage to daily energy use in both groups. These findings show that pups spare protein, even when energy use is dramatically increased. Pups that receive greater maternal provisioning and lay down more protein may have increased survival chances at sea. This study highlights the importance of protein reserves in first year survival of grey seal pups.

Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.

Factors influencing the radiative surface temperature of grey seal (Halichoerus grypus) pups during early and late lactation

The aim of this study was to examine the variation in body surface temperature of grey seal (Halichoerus grypus) pups throughout lactation in response to different environmental conditions. Radiative surface temperatures (T r, °C) of pups were measured on the Isle of May (56°11′N, 02°33′W), southeast Scotland from 29 October to 25 November 2003. Records were obtained from a total of 60 pups (32 female and 28 male) from three different pupping sites during early and late lactation. Pups were sheltered from high wind speeds but air temperature, humidity and solar radiation at pupping sites were similar to general meteorological conditions. The mean T r of all pups was 15.8°C (range 7.7–29.7°C) at an average air temperature of 10.2°C (range 6.5–13.8°C). There was no difference in the mean T r of pups between early and late lactation. However, the T r varied between different regions of the body with hind flippers on average 2–6°C warmer than all other areas measured. There was no difference in mean T r of male and female pups and pup body mass did not account for the variation in T r during early or late lactation. Throughout the day there was an increase in the T r of pups and this explained 20–28% of the variation in T r depending on stage of lactation. There was no difference in the mean T r of pups between pupping sites or associated with different substrate types. Wind speed and substrate temperature had no effect on the T r of pups. However, solar radiation, air temperature and relative humidity accounted for 48% of the variation in mean T r of pups during early lactation. During late lactation air temperature and solar radiation alone accounted for 43% of the variation in T r. These results indicate that environmental conditions explain only some of the variation in T r of grey seal pups in natural conditions. Differences in T r however indicate that the cost of thermoregulation for pups will vary throughout lactation. Further studies examining intrinsic factors such as blubber thickness and activity levels are necessary before developing reliable biophysical models for grey seals.

Shipping noise in a dynamic sea: a case study of grey seals in the Celtic Sea

Shipping noise is a threat to marine wildlife. Grey seals are benthic foragers, and thus experience acoustic noise throughout the water column, which makes them a good model species for a case study of the potential impacts of shipping noise. We used ship track data from the Celtic Sea, seal track data and a coupled ocean-acoustic modelling system to assess the noise exposure of grey seals along their tracks. It was found that the animals experience step changes in sound levels up to ~20dB at a frequency of 125Hz, and ~10dB on average over 10-1000Hz when they dive through the thermocline, particularly during summer. Our results showed large seasonal differences in the noise level experienced by the seals. These results reveal the actual noise exposure by the animals and could help in marine spatial planning.

Challenges in imaging and predictive modeling of rhizosphere processes

Background Plant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions. Scope In this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding. Conclusions We conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.