Estimating the risks of exposure to harmful algal toxins among Scottish harbour seals.

Harmful algal bloom (HAB) toxins consumed by marine predators through fish prey can be lethal but studies on the resulting population consequences are lacking. Over the past approximately 20 years there have been large regional declines in some harbour seal populations around Scotland. Analyses of excreta (faeces and urine from live and dead seals and faecal samples from seal haulout sites) suggest widespread exposure to toxins through the ingestion of contaminated prey. A risk assessment model, incorporating concentrations of the two major HAB toxins found in seal prey around Scotland (domoic acid (DA), and saxitoxins (STX)), the seasonal persistence of the toxins in the fish and the foraging patterns of harbour seals were used to estimate the proportion of adults and juveniles likely to have ingested doses above various estimated toxicity thresholds. The results were highly dependent on toxin type, persistence, and foraging regime as well as age class, all of which affected the proportion of exposed animals exceeding toxicity thresholds. In this preliminary model STX exposure was unlikely to result in mortalities. Modelled DA exposure resulted in doses above an estimated lethal threshold of 1900 µg/kg body mass affecting up to 3.8 % of exposed juveniles and 5.3 % of exposed adults. Given the uncertainty in the model parameters and the limitations of the data these conclusions should be treated with caution, but they indicate that DA remains a potential factor involved in the regional declines of harbour seals. Similar risks may be experienced by other top predators, including small cetaceans and seabirds that feed on similar prey in Scottish waters.

Toxins from harmful algae in fish from Scottish coastal waters.

Harmful algal bloom events are increasing in a number of water bodies around the world with significant economic impacts on the aquaculture, fishing and tourism industries. As well as their potential impacts on human health, toxin exposure from harmful algal blooms (HABs) has resulted in widespread morbidity and mortality in marine life, including top marine predators. There is therefore a need for an improved understanding of the trophic transfer, and persistence of toxins in marine food webs. For the first time, the concentrations of two toxin groups of commercial and environmental importance, domoic acid (DA) and saxitoxin (including Paralytic Shellfish Toxin (PST) analogues), were measured in the viscera of 40 different fish species caught in Scotland between February and November, 2012 to 2019. Overall, fish had higher concentrations of DA compared to PSTs, with a peak in the summer / autumn months. Whole fish concentrations were highest in pelagic species including Atlantic mackerel and herring, key forage fish for marine predators including seals, cetaceans and seabirds. The highest DA concentrations were measured along the east coast of Scotland and in Orkney. PSTs showed highest concentrations in early summer, consistent with phytoplankton bloom timings. The detection of multiple toxins in such a range of demersal, pelagic and benthic fish prey species suggests that both the fish, and by extension, piscivorous marine predators, experience multiple routes of toxin exposure. Risk assessment models to understand the impacts of exposure to HAB toxins on marine predators therefore need to consider how chronic, low-dose exposure to multiple toxins, as well as acute exposure during a bloom, could lead to potential long-term health effects ultimately contributing to mortalities. The potential synergistic, neurotoxic and physiological effects of long-term exposure to multiple toxins require investigation in order to appropriately assess the risks of HAB toxins to fish as well as their predators.

Declining reproductive success in the Gulf of St. Lawrence's humpback whales (Megaptera novaeangliae) reflects ecosystem shifts on their feeding grounds.

Climate change has resulted in physical and biological changes in the world's oceans. How the effects of these changes are buffered by top predator populations, and therefore how much plasticity lies at the highest trophic levels, are largely unknown. Here endocrine profiling, longitudinal observations of known individuals over 15 years between 2004 and 2018, and environmental data are combined to examine how the reproductive success of a top marine predator is being affected by ecosystem change. The Gulf of St. Lawrence, Canada, is a major summer feeding ground for humpback whales (Megaptera novaeangliae) in the North Atlantic. Blubber biopsy samples (n = 185) of female humpback whales were used to investigate variation in pregnancy rates through the quantification of progesterone. Annual pregnancy rates showed considerable variability, with no overall change detected over the study. However, a total of 457 photo-identified adult female sightings records with/without calves were collated, and showed that annual calving rates declined significantly. The probability of observing cow-calf pairs was related to favourable environmental conditions in the previous year; measured by herring spawning stock biomass, Calanus spp. abundance, overall copepod abundance and phytoplankton bloom magnitude. Approximately 39% of identified pregnancies were unsuccessful over the 15 years, and the average annual pregnancy rate was higher than the average annual calving rate at ~37% and ~23% respectively. Together, these data suggest that the declines in reproductive success could be, at least in part, the result of females being unable to accumulate the energy reserves necessary to maintain pregnancy and/or meet the energetic demands of lactation in years of poorer prey availability rather than solely an inability to become pregnant. The decline in calving rates over a period of major environmental variability may suggest that this population has limited resilience to such ecosystem change.

Mercury in cetaceans: Exposure, bioaccumulation and toxicity.

The fate and transportation of mercury in the marine environment are driven by a combination of anthropogenic atmospheric and aquatic sources, as well as natural geological inputs. Mercury biomagnifies up the food chain, resulting in the bioaccumulation of toxic concentrations in higher trophic organisms even when concentrations in their habitat remain below the threshold level for direct toxicity. As a result, mercury exposure has been recognised as a health concern for both humans and top marine predators, including cetaceans. There appears to be no overall trend in the global measured concentrations reported in cetaceans between 1975 and 2010, although differences between areas show that the highest concentrations in recent decades have been measured in the tissues of Mediterranean odontocetes. There is increasing concern for the impacts of mercury on the Arctic marine ecosystem with changes in water temperatures, ocean currents, and prey availability, all predicted to affect exposure. The accumulation of mercury in various tissues has been linked to renal and hepatic damage as well as reported neurotoxic, genotoxic, and immunotoxic effects. These effects have been documented through studies on stranded and by-caught cetaceans as well as in vitro cell culture experiments. Demethylation of methylmercury and protection by selenium have been suggested as possible mercury detoxification mechanisms in cetaceans that may explain the very high concentrations measured in tissues of some species with no apparent acute toxicity. Thus, the ratio of selenium to mercury is of importance when aiming to determine the impact of the contaminant load at an individual level. The long-term population level effects of mercury exposure are unknown, and continued monitoring of odontocete populations in particular is advised in order to predict the consequences of mercury uptake on marine food chains in the future.

Not just fat: investigating the proteome of cetacean blubber tissue.

Mammalian adipose tissue is increasingly being recognized as an endocrine organ involved in the regulation of a number of metabolic processes and pathways. It responds to signals from different hormone systems and the central nervous system, and expresses a variety of protein factors with important paracrine and endocrine functions. This study presents a first step towards the systematic analysis of the protein content of cetacean adipose tissue, the blubber, in order to investigate the kinds of proteins present and their relative abundance. Full depth blubber subsamples were collected from dead-stranded harbour porpoises (Phocoena phocoena) (n = 21). Three total protein extraction methods were trialled, and the highest total protein yields with the lowest extraction variability were achieved using a RIPA cell lysis and extraction buffer based protocol. Extracted proteins were separated using 1D Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry (nLC-ESI-MS/MS). A range of proteins were identified (n = 295) and classed into eight functional groups, the most abundant of which were involved in cell function and metabolism (45%), immune response and inflammation (15%) and lipid metabolism (11%). These proteins likely originate both from the various cell types within the blubber tissue itself, and from the circulation. They therefore have the potential to capture information on the cellular and physiological stresses experienced by individuals at the time of sampling. The importance of this proteomic approach is two-fold: Firstly, it could help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue in other mammals. Secondly, it could lead to the development of a suite of biomarkers to better monitor the physiological state and health of live individuals though remote blubber biopsy sampling.

Exposure of harbour seals Phoca vitulina to Brucella in declining populations across Scotland.

Since 2000 there has been a major decline in the abundance of Scottish harbour seals Phoca vitulina. The causes of the decline remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n = 150). Serum samples collected from live capture-released seals (n = 343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). In total, 16% of seals cultured had Brucella isolated from one or more tissues, but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results, showing that overall 25.4% of seals were seropositive, with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.

Evaluating morphometric and metabolic markers of body condition in a small cetacean, the harbor porpoise (Phocoena phocoena).

Mammalian body condition is an important individual fitness metric as it affects both survival and reproductive success. The ability to accurately measure condition has key implications for predicting individual and population health, and therefore monitoring the population-level effects of changing environments. No consensus currently exists on the best measure to quantitatively estimate body condition in many species, including cetaceans. Here, two measures of body condition were investigated in the harbor porpoise (Phocoena phocoena). First, the most informative morphometric body condition index was identified. The mass/length2 ratio was the most appropriate morphometric index of 10 indices tested, explaining 50% of the variation in condition in stranded, male porpoises with different causes of death and across age classes (n = 291). Mass/length2 was then used to evaluate a second measure, blubber cortisol concentration, as a metabolic condition marker. Cortisol is the main glucocorticoid hormone involved in the regulation of lipolysis and overall energy balance in mammals, and concentrations could provide information on physiological state. Blubber cortisol concentrations did not significantly vary around the girth (n = 20), but there was significant vertical stratification through the blubber depth with highest concentrations in the innermost layer. Concentrations in the dorsal, outermost layer were representative of concentrations through the full blubber depth, showed variation by sex and age class, and were negatively correlated with mass/length2. Using this species as a model for live cetaceans from which standard morphometric measurements cannot be taken, but from which blubber biopsy samples are routinely collected, cortisol concentrations in the dorsal, outermost blubber layer could potentially be used as a biomarker of condition in free-ranging animals.

Seasonal variation in harbour seal (Phoca vitulina) blubber cortisol - A novel indicator of physiological state?

Cortisol is one of the main glucocorticoid hormones involved in both the mammalian stress response, and in fat metabolism and energy regulation, making it of increasing interest as a biomarker for stress, health and overall physiological state. However, transient stress responses to animal handling and sampling may be important sources of measurement artefact when investigating circulating concentrations of this hormone in wildlife. Here, cortisol concentrations were measured in the plasma and, for the first time, in the blubber of live captured adult harbour seals (Phoca vitulina). Plasma cortisol concentrations were positively correlated with capture time, suggesting that they were largely driven by a stress response to the capture event. In contrast, blubber cortisol concentrations were shown not to be significantly affected by capture time and varied significantly by sex and by season, with higher concentrations during natural fasting periods of their life cycle, particularly during the moult. These results suggest that cortisol may play a key role in increased fat metabolism during highly energetically demanding periods, and that blubber concentrations have the potential to be used as physiological state indicators in phocid seals.