Synopsis

1.1. Assessing Standard Indices of Condition

Despite its importance as a good predictor of fitness, there is currently no consensus on the ‘best’ way to estimate the overall body condition of cetaceans. Using data from stranded animals, I investigated morphometric indices of body condition across three cetacean families. These indices are commonly used in terrestrial ecology, and I identified the most appropriate indices as mass/length2 _{and girth/length (where mass data were unavailable). Blubber thickness, the most} commonly used index in cetaceans, was a consistently poor indicator of condition across all three families. Interestingly, beaked whales showed very little variability in their apparent energy stores. I used these morphometric indices to assess other potential markers of condition where a ‘ground truthing’ approach was required to evaluate novel biomarker methods.

Blubber lipid content was investigated as another commonly used condition index in cetaceans. Again, the variation in blubber lipid content was assessed across three cetacean families. Blubber thickness was not correlated with lipid content. This is likely because the blubber must maintain a certain thickness to fulfil its other roles, separate from energy storage, in thermoregulation, streamlining and buoyancy, for example. Therefore, the extent to which blubber can be reduced in thickness is limited. Together, the results from Chapters 1 and 2 demonstrate that using blubber thickness as a standard measure of condition should be avoided.

The lipid content analysis showed that blubber lipid content is not a suitable biomarker of body condition in these species for two reasons. Firstly, layering through the tissue means the outer layer accessible for biopsy sampling is not representative of the lipid available for mobilisation through the full blubber depth. Secondly, blubber lipid content was not correlated with morphometric indices of body condition. In keeping with the results of Chapter 1, there was little variation in lipid content both within and between the ziphiid samples, further suggesting little variation in energy stores. There may be trade-offs occurring between the different functions of blubber (energy storage, maintaining hydrodynamic shape, controlling buoyancy, contributing to water balance and preserving thermoregulatory properties) in these deep-diving species. The idea that the main role of the blubber is to act as an energy storage depot across all cetacean species is therefore challenged in Chapters 1 and 2. Its other roles may be of equal or even greater importance in some species depending on their reliance on endogenous energy stores during parts of their life cycle.

1.2. Investigating Novel Biomarkers of Condition

Cortisol is the main glucocorticoid hormone known to be important for the regulation of lipolysis and lipogenesis in mammals. As it has been previously suggested to be involved in the control of fasting metabolism in pinnipeds, it appeared to be a good candidate as a condition marker. Cortisol

was extracted and concentrations were measured in the blubber of stranded harbour porpoises and balaenopterids. Blubber cortisol concentrations showed a negative relationship with morphometric indices of body condition. It is suggested in Chapters 3 and 4, that concentrations of this hormone in the tissue are not a marker of an acute stress response, but are an integrated marker of physiological state. A long-term dataset of biopsy samples from female humpback whales on their feeding grounds in the North Atlantic showed significant inter-annual variation in cortisol concentrations.

Pregnancy rates were estimated for this population based on blubber progesterone concentrations. There was some evidence that inter-annual variation in pregnancy rates were negatively correlated with cortisol concentrations. Specifically, years with lower blubber cortisol concentrations corresponded to higher overall pregnancy rates. I hypothesise that this could be as a result of changing environmental conditions between years. The individual variability was probably a result of the multifunctional nature of cortisol that was not explained by differences in reproductive state, season, and a morphometric index of body condition obtained from aerial photogrammetry image analysis. Cortisol concentrations likely provide a ‘snap shot’ of the metabolic state of the tissue at the time of sampling.

The use of cortisol concentrations as a single marker to measure a ‘snap-shot’ in time highlights a general question of the applicability of using a single condition index to assess and compare animals across a range of life history stages or even different populations and ecotypes. Limiting the use of certain measures to compare within age classes, life-history stages or different environments may be more informative. In terms of life-history stage variability, using a single index with the same numerical value to compare the overall body condition of an individual at the end of a breeding season, to another individual the end of a feeding season, is meaningless unless the context in which the animal is sampled is considered. Specifically, animals should have accumulated significant energy reserves by the end of the feeding season, so two animals sampled at these different life-history stages with the same condition index value, does not mean that they are equally healthy. In terms of population-level variability, humpback whales tagged with suction cup tags in the Gulf of St Lawrence had higher overall body density values (indicative of lower total body fat stores), suggesting that they were in poorer body condition, compared to whales instrumented with the same tags in Northern Norway (Aoki, pers. comm.). However, differences in the water salinity, and therefore the density of these two aquatic environments, could have led the whales in these separate populations to adapt to an ‘optimum’ body density, close to neutral buoyancy, in order for locomotion, and therefore foraging, to be as efficient as possible in each environment. Thus, the use of a single body condition index, with a ‘one size fits all’ approach, does not apply in many cases as there are a number of other factors to consider when interpreting these estimates. For these reasons, there should be a movement towards multiple measures that, when integrated together, can provide composite indicators of body condition and overall health in order to add more detailed and robust information to condition index estimates.

Multiple other blubber biomarkers of interest that, together, reflect overall health and condition were investigated. The proteome of cetacean blubber tissue was explored for the first time. In Chapters 5 and 6, I developed a method for total protein extraction and applied it to samples from stranded animals and biopsies from live animals. Over 400 different proteins were identified and classed into ten functional groups based on their involvement in different physiological processes in terrestrial species. The most abundant proteins were involved in cell function and metabolism, immune response and inflammation, and lipid metabolism. The identified proteins are thought to have been secreted from the various cell types within the tissue, and to have accumulated from the circulation. These proteins capture the range of cellular and physiological processes experienced by individuals at the time of sampling. Protein band profiles generated through 1D SDS-PAGE across individuals of different ages, sexes, reproductive states, cause of death classes, morphometric body condition and species were compared. Analysis did not reveal any protein bands, or patterns of protein bands that explained the individual variation in protein profiles. There was some evidence that the presence and absence of a number of protein bands were correlated with blubber cortisol concentrations in biopsy samples from humpback whales. These bands showed relative increases and decreases in lipid metabolism and amino acid metabolism compared to the full dataset of identified proteins. Using multiple analytical approaches, endocrine markers, like cortisol concentrations, can potentially be linked to downstream changes in protein abundance. A finer scale approach, using 2D PAGE for example, is necessary to identify individual proteins or classes of proteins that vary in relative abundance as a function of physiological state. The importance of furthering this proteomic approach is two-fold: Firstly, it could improve our understanding of tissue function, and its involvement in animal physiology by assigning novel roles to marine mammal blubber similar to those identified in the adipose tissue of other mammals. These include the regulation of appetite and energy balance, immune system and inflammatory responses and nutrient transport for example. Secondly, it could lead to the development of a suite of biomarkers that can be used as tools to better assess the health of live individuals though remote blubber biopsy sampling.