Metallomics approach for the identification of the iron transport protein transferrin in the blood of harbour seals (Phoca vitulina)

The health status of marine mammals such as harbour seals (Phoca vitulina) represents an indirect but powerful way for the assessment of environmental changes. The present work illustrates the first investigation and characterisation of Tf isolated from blood samples of North Sea harbour seals with a view to using changes in Tf isoform patterns as an additional parameter in extended studies of their health status. Therefore, an HPLC-ICP-MS approach has been developed which allows the highly resolved separation and fractionation of up to eight different Tf isoforms, as well as their sensitive and specific detection on the basis of their characteristic iron content. Molecule-specific detection techniques such as nanoLC-ESI-QTRAP-MS or MALDI-TOF-MS were used as complementary techniques to unambiguously identify the isolated proteins as Tf via cross species protein identification and to further characterise the molecular weight as well as the sialic acid content, which is responsible for the elution behaviour of the different isoforms during their ion exchange separation. A molecular mass above 80 kDa has been measured for the different seal Tf isoforms, which is in good agreement with the known molecular mass in other mammalian species, while the estimated pI of the different isoforms indicates some differences in comparison to other species. A number of homologies to known Tf sequences have been observed, which finally allows the cross species protein identification. The combined metallomics orientated analytical approach, which includes the complementary application of element and molecule-specific detection techniques, opens up interesting possibilities for the fast and targeted isolation and identification of a diagnostically relevant metal containing protein from an un-sequenced mammalian species prior to its utilisation in extended studies.     

An assessment of population size and distribution of harbour seals in the Republic of Ireland during the moult season in August 2003

The status of Ireland's harbour seal Phoca vitulina vitulina population is poorly understood. The most recent national population estimate dates back to the breeding season in 1978 and did not cover the entire coastline. Reliable up-to-date information on the abundance and distribution of harbour seals in Ireland is necessary to assess the conservation status of the species and for the effective identification, management and monitoring of special areas of conservation required for harbour seals under the EU Habitats Directive. To provide comprehensive current information on Ireland's harbour seal population, a geographically extensive survey was conducted along the coastline of the Republic of Ireland during the species' annual moult in August 2003. This complemented a similar survey of Northern Ireland, which was conducted in 2002. Using thermal imaging technology, haul-out groups of harbour seals and grey seals Halichoerus grypus were identified from the air, aerial-counts were obtained and compared with simultaneous ground-count data from selected sites. Harbour seal distribution recorded during the 2003 moult season was concentrated in the south-west, west and north-west of the country. This national survey yielded a minimum population estimate for the Republic of Ireland of 2905 harbour seals, delivering an effective baseline for current and future population research.     

Trophic niche overlap between sympatric harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) at the southern limit of their European range (Eastern English Channel)

Sympatric harbour (Phoca vitulina) and grey seals (Halichoerus grypus) are increasingly considered potential competitors, especially since recent local declines in harbour seal numbers while grey seal numbers remained stable or increased at their European core distributions. A better understanding of the interactions between these species is critical for conservation efforts. This study aimed to identify the trophic niche overlap between harbour and grey seals at the southern limit of their European range, in the Baie de Somme (BDS, Eastern English Channel, France), where numbers of resident harbour seals and visiting grey seals are increasing exponentially. Dietary overlap was identified from scat contents using hierarchical clustering. Isotopic niche overlap was quantified using δ¹³C and δ¹⁵N isotopic values from whiskers of 18 individuals, by estimating isotopic standard ellipses with a novel hierarchical model developed in a Bayesian framework to consider both intraindividual variability and interindividual variability. Foraging areas of these individuals were identified from telemetry data. The three independent approaches provided converging results, revealing a high trophic niche overlap due to consumption of benthic flatfish. Two diet clusters were dominated by either small or large benthic flatfish; these comprised 85.5% [CI95%: 80.3%–90.2%] of harbour seal scats and 46.8% [35.1%–58.4%] of grey seal scats. The narrower isotopic niche of harbour seals was nested within that of grey seals (58.2% [22.7%–100%] overlap). Grey seals with isotopic values similar to harbour seals foraged in coastal waters close to the BDS alike harbour seals did, suggesting the niche overlap may be due to individual grey seal strategies. Our findings therefore provide the basis for potential competition between both species (foraging on benthic flatfish close to the BDS). We suggest that a continued increase in seal numbers and/or a decrease in flatfish supply in this area could cause/amplify competitive interactions and have deleterious effects on harbour seal colonies.     

The 1988 and 2002 phocine distemper virus epidemics in European harbour seals

We present new and revised data for the phocine distemper virus (PDV) epidemics that resulted in the deaths of more than 23 000 harbour seals Phoca vitulina in 1988 and 30,000 in 2002. On both occasions the epidemics started at the Danish island of Anholt in central Kattegat, and subsequently spread to adjacent colonies in a stepwise fashion. However, this pattern was not maintained throughout the epidemics and new centres of infection appeared far from infected populations on some occasions: in 1988 early positive cases were observed in the Irish Sea, and in 2002 the epidemic appeared in the Dutch Wadden Sea, 6 wk after the initiation of the outbreak at Anholt Island. Since the harbour seal is a rather sedentary species, such 'jumps' in the spread among colonies suggest that another vector species could have been involved. We discussed the role of sympatric species as disease vectors, and suggested that grey seal populations could act as reservoirs for PDV if infection rates in sympatric species are lower than in harbour seals. Alternatively, grey seals could act as subclinical infected carriers of the virus between Arctic and North Sea seal populations. Mixed colonies of grey and harbour seal colonies are found at all locations where the jumps occurred. It seems likely that grey seals, which show long-distance movements, contributed to the spread among regions. The harbour seal populations along the Norwegian coast and in the Baltic escaped both epidemics, which could be due either to genetic differences among harbour seal populations or to immunity. Catastrophic events such as repeated epidemics should be accounted for in future models and management strategies of wildlife populations.     

Acute phase protein haptoglobin in blood plasma samples of harbour seals (Phoca vitulina) of the Wadden Sea and of the isle Helgoland

Haptoglobin (Hp) which is synthesized in response to infection, inflammation, trauma or toxicological damage is known as a major acute phase protein in numerous species. Quantification of the circulating concentration of this protein can provide an objective measure of the health status, but there is a lack of investigations on harbour seals. We investigated the Hp concentration in samples of 123 seals (Phoca vitulina) from the German and Danish Wadden Sea to study physiological ranges of Hp levels. Hp levels between 2002, the end of the phocine distemper virus epidemic (PDV), and 2007 were considered, and Hp concentrations between animals of different sex, ages as well as living areas were compared. Furthermore, as a case study, six animals from the open sea isle Helgoland were investigated in 2006. Influences on the health status of the seal population e.g. the PDV epidemic were reflected by increased Hp levels in North Sea seals in 2002. The results of the Wadden Sea seals showed no significant age-, sex-, or geographical area-related differences. Interestingly, for the seals of the open sea isle Helgoland higher Hp values were measured compared to the Wadden Sea seals. The present study demonstrates that Hp can be used as a diagnostic tool to monitor the health status of harbour seals.     

Cytochrome P450 1A expression and organochlorine contaminants in harbour seals (Phoca vitulina): evaluating a biopsy approach

We previously reported in vivo induction of cytochrome P450 1A (CYP1A) by beta-naphthoflavone in skin and liver biopsies of captive harbour seals (Phoca vitulina richardsi). The present study evaluated CYP1A expression (immunoblot analysis and ethoxyresorufin O-deethylase activity-EROD) in harbour seals using two study designs: i) skin and liver biopsies from 20 harbour seal pups captured from coastal British Columbia (BC, Canada) and temporarily housed in captivity; and ii) skin biopsies from 42 free-ranging harbour seals captured and sampled on-site in multiple locations in BC and Washington State (USA). Toxic Equivalency Quotients (TEQs) were calculated for polychlorinated biphenyl, polychlorinated dibenzo-p-dioxin, and polychlorinated dibenzofuran residues measured in blubber from a subset of study animals (n=30). CYP1A data from the seal pups held temporarily in captivity show that CYP1A protein levels were greater in liver than skin and that CYP1A protein and EROD activity were correlated in skin and liver. However, analysis of free-ranging seals from different sites revealed that blubber organochlorine TEQ values did not correlate with skin CYP1A levels. CYP1A protein levels and EROD activities in skin of seal pups from the BC locations and from Puget Sound were relatively low, possibly reflecting contaminant levels that were not high enough to elicit a response, a small sample size, or methodological limitations. Our results show that CYP1A measurements in skin show promise as a biomarker of contaminant exposure, but that refinements to techniques and a larger sample size are needed.     

Maximum likelihood population size estimation of harbour seals in the Dutch Wadden Sea based on a mark–recapture experiment

1. The harbour seal population Phoca vitulina in the entire Wadden Sea was severely depleted due to a virus-epizootic during 1988. A comprehensive study on the population biology and activity patterns was subsequently initiated to design a management and conservation plan. The main objective of this study was to estimate harbour seal abundance in the different regions of the Wadden Sea.
2. We investigated the potential of a mark–recapture experiment using VHF radio-tags in combination with repeated aerial surveys to estimate the number of harbour seals in the Dutch part of the Wadden Sea. The number of harbour seals hauled-out and the presence of any radio-tagged seals was monitored during seven aerial surveys of all known haul-out sites in the Dutch Wadden Sea over the 1994 breeding season.
3. A maximum likelihood (ML) estimator was developed to infer the rate of tag-loss and the size of the local prepupping population.
4. The ML estimate of the number of harbour seals in the Dutch Wadden Sea was 1536 (95% confidence limits were 1225 and 2200). The corresponding maximum proportion of seals hauled-out was 68%.
5. The use of VHF radio-tags which can be monitored from the air provides a way of correcting aerial survey counts for the proportion of harbour seals hauled-out during the surveys. Since haul-out behaviour may be influenced by local conditions, such as exposure time of sand banks, we recommend this technique be repeated in other areas of the Wadden Sea rather than using the estimates from the current study in other areas.     

Increasing numbers of harbour seals and grey seals in the Solent

Harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) both occur within the UK, but display regional contrasting population trends. While grey seals are typically increasing in number, harbour seals have shown varying trends in recent decades following repeated pandemics. There is a need for monitoring of regional and local populations to understand overall trends. This study utilized a 20‐year dataset of seal counts from two neighboring harbours in the Solent region of south England. Generalized additive models showed a significant increase in the numbers of harbour (mean 5.3–30.5) and grey (mean 0–12.0) seals utilizing Chichester Harbour. Conversely, in Langstone Harbour there has been a slight decrease in the number of harbour seals (mean 5.3–4.0). Accompanying photographic data from 2016 to 18 supports the increase in seal numbers within Chichester Harbour, with a total of 68 harbour and 8 grey seals identified. These data also show evidence of site fidelity of harbour seals in this area, with almost a quarter of animals resighted within the past three years. Overall, this long‐term study indicates an increasing number of both harbour and grey seals within the Solent. However, more research is required to identify the drivers of this trend.     

Separation and characterization of metallothionein in two species of seals by high performance liquid chromatography-atomic absorption spectrophotometry

Metallothionein (MT) was separated from the livers of two species of seals, ribbon seal (Histriophoca fasciata) and harbour seal (Phoca vitulina), by high performance liquid chromatography--atomic absorption spectrophotometry (HPLC-AAS). Both gel permeation and anion-exchange HPLC demonstrated that MT was present as a mixture of at least two isoforms in the livers of both species of seals.     

Rookery through rehabilitation: Microbial community assembly in newborn harbour seals after maternal separation

Microbial community assembly remains largely unexplored in marine mammals, despite its potential importance for conservation and management. Here, neonatal microbiota assembly was studied in harbour seals (Phoca vitulina richardii) at a rehabilitation facility soon after maternal separation, through weaning, to the time of release back to their native environment. We found that the gingival and rectal communities of rehabilitated harbour seals were distinct from the microbiotas of formula and pool water, and became increasingly diverse and dissimilar over time, ultimately resembling the gingival and rectal communities of local wild harbour seals. Harbour seal microbiota assembly was compared to that of human infants, revealing the rapid emergence of host specificity and evidence of phylosymbiosis even though these harbour seals had been raised by humans. Early life prophylactic antibiotics were associated with changes in the composition of the harbour seal gingival and rectal communities and surprisingly, with transient increases in alpha diversity, perhaps because of microbiota sharing during close cohabitation with other harbour seals. Antibiotic-associated effects dissipated over time. These results suggest that while early life maternal contact may provide seeding for microbial assembly, co-housing of conspecifics during rehabilitation may help neonatal mammals achieve a healthy host-specific microbiota with features of resilience.     

Diet differences among age classes of Arctic seals: evidence from stable isotope and mercury biomarkers

A basic understanding of current food web dynamics and baseline data from which to measure future change is necessary to understand species re-distribution and altered competition for food with climate change. We use mercury (Hg) and carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios as biomarkers to understand species diet differences and age class differences among ringed (Phoca hispida), bearded (Erignathus barbatus), and harbour (P. vitulina) seals in a subarctic marine ecosystem. Adult bearded seals had significantly lower δ¹⁵N and muscle Hg than bearded seal pups, whereas the opposite was observed in ringed seals where pups had lower δ¹⁵N than adults, suggesting age specific foraging differences in trophic food level for both species. For harbour seals, δ¹⁵N did not differ significantly among ages while Hg and δ¹³C did. The δ¹³C in muscle supports that bearded seals in this study are benthic feeders and are part of a separate food web from ringed seals and harbour seals. Harbour seals had the highest levels of mercury and δ¹⁵N, indicating they feed at a higher trophic level relative to the other two seal species. Carbon and nitrogen isotopic ratios and Hg levels illustrate how resources are partitioned among three seal species and offer evidence for separation based on life stages within species.     

Historical population size of the harbour seal,Phoca vitulina,in the Delta area,SW Netherlands

Seals in the Dutch Delta area have been subject to hunting pressure for centuries, promoted by a bounty system which generated a sort of hunting statistics. Hunting mortality is used to estimate historical population size. Based on ranges for most likely net recruitment rates, corresponding population trajectories are back calculated from an assessed population size of 350 seals in 1960. It is concluded that the size of the harbour seal population in the Delta area in 1900 will have been close to 11 500 animals. Significant loss of habitat has occurred due to closing off parts of the larger estuaries and the enlargement of the entrance to the harbour of Rotterdam in the 1960s and early 1970s. It is estimated that about 4000 harbour seals could inhabit the remaining Delta area under tidal influence. This outcome, based on retrospective population analysis, will be an important reference in defining management objectives for the recovery of the harbour seal population in the Delta area, which amounted to 18 seals in 1992.     

Apparent decline in the harbour seal Phoca vitulina population near Hvaler, Norway, following an epizootic

From 1986-90 we tagged 98 harbour seal pups at Hvaler, Norway Fifteen were recovered later the same year of which 10 were recovered in 1988, when the seal population was infected by a morbillivirus The epizootic in 1988 resulted in a population decline of c 75% Predicted birth weight did not vary significantly before and after the epizootic Newborn harbour seals have antibodies against the morbillivirus two years after the outbreak of the disease     

Transmission of lungworms of harbour porpoises and harbour seals: Molecular tools determine potential vertebrate intermediate hosts

Harbour porpoises (Phocoena phocoena) and harbour seals (Phoca vitulina) from German waters are infected by six species of lungworms (Metastrongyloidea). These nematodes parasitise the respiratory tract, are pathogenic and often cause secondary bacterial infections. In spite of their clinical and epidemiological significance, the life cycle and biology of lungworms in the marine environment is still largely unknown. Regions of ribosomal DNA (ITS-2) of all lungworms parasitising harbour porpoises and harbour seals in German waters were sequenced to characterise and compare the different species. The phylogenetic relationship among the lungworm species was analysed by means of their ITS-2 nucleotide sequences and the species-specific traits of the ITS-2 were used to screen wild fish as possible intermediate hosts for larval lungworms. Molecular markers were developed to identify larval nematodes via in-situ hybridisation of tissues of harbour porpoise and harbour seal prey fish. Potential wild intermediate fish hosts from the North Sea were dissected and found to harbour larval nematodes. Histological examination and in-situ hybridisation of tissue samples from these fish showed lungworm larvae within the intestinal wall. Based on larval ITS-2 nucleotide sequences, larval nematodes were identified as Pseudalius inflexus and Parafilaroides gymnurus. Turbot (Psetta maxima) bred and raised in captivity were experimentally infected with live L1s of Otostrongylus circumlitus and ensheathed larvae were recovered from the gastrointestinal tract of turbot and identified using molecular tools. Our results show that fish intermediate hosts play a role in the transmission of metastrongyloid nematodes of harbour porpoises and harbour seals.     

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.     

Using sparse survey data to investigate the declining abundance of British harbour seals

This study presents an analysis of changes in the regional abundance of harbour seals Phoca vitulina based on repeated aerial surveys of haulouts, and demonstrates the use of sparse data to deliver advice about population status and management. Generalized linear models with negative binomial errors were used to represent these overdispersed data. The shape parameter of the negative binomial distribution was directly estimated from the data where this was possible. Information from time-series of counts where there were few gaps in the data was used to improve the estimation of this parameter in areas where fewer surveys had been carried out. The results show that the number of harbour seals in eastern England has not increased since the end of the 2002 phocine distemper epidemic. There is also evidence of a general decline in most of the large harbour seal colonies around Britain. The populations in the Inner Hebrides were an exception, with numbers appearing to be stable or increasing. Between 2001 and 2006, the population in Orkney and Shetland declined by 40% (95% confidence interval: 30–50%), indicating harbour seals in these areas experienced substantially increased mortality or very low recruitment over this period. The widespread declines, ranging from Shetland to The Wash, suggest that the causes may have been present over a large part of the North Sea.     

Assessment of errors associated with harbour seal (Phoca vitulina) faecal sampling

Six harbour seals, ages 4–8 years, were held as pairs in a 10 times 20 times 2 m tank filled with sea water, and on 60 occasions were fed a meal of a specific species of fish or cephalopod of known size. The tank was drained periodically, and harbour seal faeces were collected on a 0.5 mm sieve. Number and size of otoliths and beaks found in faeces were determined. Fifty-eight percent of 670 fish and 37% of 36 cephalopods fed to harbour seals were represented by their otoliths or beaks in faeces. Estimated number of prey consumed was determined from the greatest number of left or right otoliths or upper or lower beaks collected in faeces. Estimated length ofprey was determined from measurements of otoliths and beaks recovered in the tank and relationships of otolith and beak measurements to prey length. Estimated number of fish eaten was not significantly different among pairs of harbour seals, but was different among species of fishes. Only 24–35% of fish species with small otoliths were represented in faeces, whereas more robust otoliths from other species were less apt to be completely dissolved. Estimated length of fishes was significantly less than lengths of fishes fed to harbour seals in 39 (76.5%) of 51 trials. Cephalopod beaks were not affected by passage through the harbour seal digestive tract. Amount of otolith dissolution was not related to species of fish; estimated fish length was underestimated by an average 27.5%. Although some (7.4%) of the otoliths were collected within 100 h after the fish were ingested, more than 90% were recovered within 24 h after the fish was eaten. Correction factors were developed which will allow researchers to estimate more reliably number and size of fish and cephalopod prey eaten by harbour seals.     

Evaluating the function of the male harbour seal, Phoca vitulina, roar through playback experiments

The aim of this study was to evaluate the role of vocalizations produced during the breeding season by the male harbour seal, an aquatically breeding pinniped. During the spring of 1999, playback experiments were conducted at eight locations in Elkhorn Slough, California, U.S.A. Through an underwater speaker, we presented male harbour seals with three acoustic stimuli: a long-duration, low-frequency roar (LL), a short-duration, high-frequency roar (SH) and amplified water noise (control). Male responses to the playback boat were characterized by increased approach rates and aggressive flipper slapping during 62.5% of SH sessions (N=8), 25% of LL sessions (N=8) and 0% of control sessions (N=8). No more than one identifiable seal responded during each playback location. We observed no responses by female harbour seals to playbacks. We conclude from these experiments that territorial male harbour seals use roars given by intruders to locate and challenge intruders.     

Assessing the potential impact of salmon fisheries management on the conservation status of harbour seals (Phoca vitulina) in north-east Scotland

Conservation efforts are often constrained by uncertainty over the factors driving declines in marine mammal populations. In Scotland, there is concern over the potential impact of unrecorded shooting of seals, particularly where this occurs near Special Areas of Conservation. Here, we show that the abundance of harbour seals Phoca vitulina in the Moray Firth, north-east Scotland, declined by 2–5% per annum between 1993 and 2004. Records from local salmon fisheries and aquaculture sites indicated that 66–327 harbour seals were shot each year between 1994 and 2002. Matrix models and estimates of potential biological removal indicate that this level of shooting is sufficient to explain observed declines. Nevertheless, uncertainty over the number and identity of seals shot means that other factors such as changes in food availability may be contributing. Recent conservation measures markedly reduced the recorded levels of shooting in 2003 and 2004. In 2005, a coordinated management plan was developed to protect salmon fisheries interests while minimizing impacts on local seal populations. Comprehensive monitoring of future population trends and improved regulation of culls are now required to provide more robust assessments of the impact of human persecution on harbour seal populations in the Moray Firth and in other parts of the UK.     

Serum chemistry and antibodies against pathogens in antarctic fur seals, Weddell seals, crabeater seals, and Ross seals

Information on health parameters, such as antibody prevalences and serum chemistry that can reveal exposure to pathogens, disease, and abnormal physiologic conditions, is scarce for Antarctic seal species. Serum samples from Antarctic fur seals (Arctocephalus gazella, n=88) from Bouvet?ya (2000-2001 and 2001-2002), and from Weddell seals (Leptonychotes weddellii, n=20), Ross seals (Ommatophoca rossii, n=20), and crabeater seals (Lobodon carcinophagus, n=9) from the pack-ice off Queen Maud Land, Antarctica (2001) were analyzed for enzyme activity, and concentrations of protein, metabolites, minerals, and cortisol. Adult Antarctic fur seal males had elevated levels of total protein (range 64-99 g/l) compared to adult females and pups (range 52-79 g/l). Antarctic fur seals had higher enzyme activities of creatine kinase, lactate dehydrogenase, and amylase, compared to Weddell, Ross, and crabeater seals. Antibodies against Brucella spp. were detected in Weddell seals (37%), Ross seals (5%), and crabeater seals (11%), but not in Antarctic fur seals. Antibodies against phocine herpesvirus 1 were detected in all species examined (Antarctic fur seals, 58%; Weddell seals, 100%; Ross seals, 15%; and crabeater seals, 44%). No antibodies against Trichinella spp., Toxoplasma, or phocine distemper virus (PDV) were detected (Antarctic fur seals were not tested for PDV antibodies). Antarctic seals are challenged by reduced sea ice and increasing temperatures due to climate change, and increased anthropogenic activity can introduce new pathogens to these vulnerable ecosystems and represent a threat for these animals. Our data provide a baseline for future monitoring of health parameters of these Antarctic seal species, for tracking the impact of environmental, climatic, and anthropogenic changes in Antarctica over time.