Characterization of xenobiotic-metabolizing cytochrome P450 (CYP) forms in ringed and grey seals from the Baltic Sea and reference sites

Earlier studies have shown that members of the cytochrome P4501 (CYP1) enzyme family are constitutively expressed, and are elevated in the livers of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) living in the heavily polluted Baltic Sea. In this study, we compared the expression profiles of several additional CYP enzymes in the liver and extrahepatic tissues of Baltic ringed and grey seals with the corresponding CYP expression in seals from relatively unpolluted waters. We used marker enzyme activity levels, diagnostic inhibitors and immunoblot analysis to assess members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A sub-families. Coumarin 7-hydroxylation (COH), a marker of CYP2A activity, was high in the liver and the lungs of all the studied seal populations. The presence of a putative CYP2A form in these seals was further supported by the strong inhibition of COH activity by a chemical inhibitor and by an anti-CYP2A5 antibody. However, antibodies to human and rodent CYP2B, CYP2C and CYP2E forms did not recognize any proteins in these seal species. Dextromethorphan O-demethylation (marker for CYP2D activity) and chlorzoxazone 6-hydroxylation (marker for CYP2E activity) were measurable in the livers of all the seals we studied. Both activities were elevated in the Baltic seal populations, showed a strong positive correlation with CYP1A activity and were at least partly inhibited by a typical CYP1A inhibitor, alpha-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6beta-hydroxylation, a CYP3A marker, showed a relatively high level of activity in the livers of both seal species and was potently inhibited by ketoconazole, a CYP3A-selective inhibitor. The putative CYP3A activity showed an opposing geographical trend to that of CYP2D and CYP2E, since it was elevated in the control area. CYP3A protein levels, revealed by immunoblotting, showed a positive correlation with testosterone 6beta-hydroxylation. We conclude tentatively that CYP2A- and CYP3A-like enzymes are expressed in ringed and grey seals, but that CYP2B- and CYP2C-like ones are not. Further information on the individual contaminant profile is needed before any conclusions can be drawn on a possible connection between the varying CYP expressions and the contaminant load.     

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.     

Hematology and serum chemistry of harp (Phoca groenlandica) and hooded seals (Cystophora cristata) during the breeding season, in the Gulf of St. Lawrence, Canada

Standard hematologic and serum chemistry parameters were determined from 28 harp seals (Phoca groenlandica) and 20 hooded seals (Cystophora cristata) sampled from 6 March 2001 to 13 March 2001 during the breeding season. Whole blood was collected immediately postmortem from harp seal mother-pup pairs and from six hooded seal pups, and from live-captured adult hooded seals and three hooded seal pups; blood was analyzed within 24 hr at a local human hospital. A certified veterinary laboratory validated subsamples of whole blood and analyzed all serum chemistry parameters. Significant interlaboratory differences in mean values of packed cell volume (PCV) and mean cell volume (MCV) were found. Significant differences were found between samples from the five seal groups (adult male hooded seals, lactating female hooded seals, unweaned hooded seal pups; lactating female harp seals, and unweaned harp seal pups) for hematology and most serum chemistry parameters. In general, age-class influenced mean values of PCV, hemoglobin (HB), red blood cell (RBC) counts, MCV, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and nucleated red blood cell (NRBC) counts per 100 leucocytes, but most age-related variations were species specific. Harp seal pups had significantly lower mean values of HB, PCV, MCH, and MCHC than did other seal groups, and significantly lower mean RBC counts than did hooded seal pups. Mean NRBC counts per 100 leukocytes were more than three times higher in harp seal pups than in hooded seal pups, but this difference was not statistically significant. Mean MCV were significantly lower in harp and hooded seal pups compared to those of adult harp and hooded seals. Differences in hemograms between pup species were likely because of the precocious development of hooded seal pups, which are weaned within 4 days, compared to 12 days for harp seal pups. Among adult seal groups, male hooded seals had significantly higher mean values of PCV and HB than did female harp and hooded seals, and significantly higher mean RBC counts than did adult female hooded seals. Among adult females, mean values of MCH and MCHC were statistically higher in hooded seals than in harp seals. Adult female harp and hooded seals did not differ significantly in other RBC parameters and mean leukocyte counts. Mean values of glucose, blood urea nitrogen, total bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, and albumin showed species-specific variations between adults and pups. Except for ALP, few significant differences in mean enzyme activities of aspartate aminotransferase (AST), ALT, creatine kinase and gamma-glutamyltransferase were found between seal groups. Mean concentrations of electrolytes (calcium, phosphorus, sodium, potassium, chloride, magnesium, and total carbon dioxide) varied with age class, but variations in potassium and magnesium were species specific. Harp seal pups had significantly higher mean phosphorus and potassium levels compared to other seal groups.     

Histological,histochemical, and ultrastructural investigations on the gastrointestinal system of antarctic seals: Weddell seal (Leptonychotes weddellii) and crabeater seal (Lobodon carcinophagus)

The morphology of the principal sections of the gastrointestinal system of two Antarctic seals with different dietary habits, namely, the Weddell seal (Leptonychotes weddellii) and the crabeater seal (Lobodon carcinophagus), has been investigated. Histologically examined by light microscopy, the tissue layers of the gastrointestinal tract of both seals are almost identical to those observed in most other mammals and no major differences in principle organization could be found between the two seal species. The ultrastructure of the gastric and intestinal epithelial cells has been examined and is also closely comparable to that of these cells in other mammals; however, Paneth cells have not been found in our material. In general, therefore, adaptations of the gastrointestinal tract to the aquatic environment or the diet are not obvious at the morphological levels of organization studied. Histochemical differences are found between the two closely related species; mucins of the surface epithelium in the stomach of Weddell seals are highly sulfated, while those in the crabeater seal are not. Mucous neck cells in Weddell seals contain acid mucosubstances, while those of crabeater seals contain neutral ones. Goblet cells in the small and large intestine in Weddell seals contain both neutral and acid mucosubstances. Both mucin types are detected in the crabeater seal; however, the mucins of the colon in the crabeater seal are more highly sulfated than those in the Weddell seal. The ratio of globet cells to enterocytes in the large intestine of crabeater seals is higher than that in Weddell seals. © 1995 Wiley-Liss, Inc.     

A Novel Tool to Mitigate By-Catch Mortality of Baltic Seals in Coastal Fyke Net Fishery

Developing methods to reduce the incidental catch of non-target species is important, as by-catch mortality poses threats especially to large aquatic predators. We examined the effectiveness of a novel device, a “seal sock”, in mitigating the by-catch mortality of seals in coastal fyke net fisheries in the Baltic Sea. The seal sock developed and tested in this study was a cylindrical net attached to the fyke net, allowing the seals access to the surface to breathe while trapped inside fishing gear. The number of dead and live seals caught in fyke nets without a seal sock (years 2008–2010) and with a sock (years 2011–2013) was recorded. The seals caught in fyke nets were mainly juveniles. Of ringed seals (Phoca hispida botnica) both sexes were equally represented, while of grey seals (Halichoerus grypus) the ratio was biased (71%) towards males. All the by-caught seals were dead in the fyke nets without a seal sock, whereas 70% of ringed seals and 11% of grey seals survived when the seal sock was used. The seal sock proved to be effective in reducing the by-catch mortality of ringed seals, but did not perform as well with grey seals.     

Identification of hairs found in leopard seal (<Emphasis Type="Italic">Hydrurga leptonyx</Emphasis>) scats

The leopard seal is a top-order predator in the Southern Ocean ecosystem and preys on a wide variety of vertebrate species including seals and penguins. We assessed the use of hairs found in leopard seal scats to identify the species of pinniped consumed. A reference collection of hairs was obtained from four potential leopard seal prey species including crabeater, Weddell, Ross, and Southern elephant seals. Discrimination techniques applied to terrestrial mammals did not allow for identification of the seal hairs. Instead, a 2-dimensional (2-D) and 6-dimensional (6-D) analysis technique utilising Mahalanobis distances (D ²) was used. The smallest Mahalanobis distance together with the largest value of p(F) positively identified hairs from each species. The 6-D analysis was more accurate and applied to hairs found in the leopard seal scats. The majority of prey species were identified as crabeater seals, which are a known prey item of the leopard seal.     

The origin and genetic relationships of the Baikal seal, Phoca sibirica, by restriction analysis of mitochondrial DNA

The origin and genetic relationships of the Baikal seal, Phoca sibirica, were studied by restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA). Using 17 different six-base recognition restriction endonucleases, we examined 98 Baikal seals, and two other related species, the ringed seal, P. hispida, (n=87), and the Caspian seal, P. caspica, (n=94). Analysis revealed the existence of 87 mtDNA haplotypes in the total of 279 specimens. The haplotypes of each species were divided into different clusters on a dendrogram obtained by UPGMA based on haplotype frequency and mtDNA base substitution. No common haplotypes were found among the species examined. The Baikal seal is much more closely related to the ringed seal than the Caspian seal. The amount of divergence suggested that an ancestor of the Baikal seal came down to the lake approximately 0.4 million years ago as was previously indicated by paleontological studies. The seals examined here showed lower variabilities.     

Fatty-acyl esters of retinol (vitamin A) in the liver of the harp seal (Phoca groenlandica), hooded seal (Cystophora cristata), and California sea lion (Zalophus californianus).

The fatty-acid composition of retinyl esters in the livers of two species of phocid seal, the harp seal (Phoca groenlandica, n = 20) and the hooded seal (Cystophora cristata, n = 15), and one species of otariid seal, the California sea lion (Zalophus californianus, n = 6), was determined. Vitamin A ranged in concentration from 4 to 1024 nmol retinol/g liver for the phocids and from 381 to 979 nmol/g liver for the otariids. In most of the livers, retinyl palmitate was not the principal ester, and the palmitate + stearate + oleate trio of retinyl esters represented less than 50% of the total. In all samples, the retinyl esters contained 20:1, 20:4, 20:5, and 22:6 in unusually large amounts. Retinyl esters tended to be richer than whole-liver lipids in 20:5 + 22:6, whereas whole-liver lipids were richer in 18:0 and 18:2. Therefore, the pool of acyl donors used for the esterification of retinol may be distinct from that used for other lipids. Birth-to-weaning changes were seen only in the harp seals. In the pups, the hepatic vitamin-A concentration increased 454%, while the proportion of 18:0 and 20:1 in the retinyl esters rose and that of 14:0 + 16:1 and 20:4 fell. Concomitantly, in their mothers, the proportion of 20:4 increased but that of 16:0 and 18:0 decreased.     

Dinucleotide microsatellite markers from the Antarctic seals and their use in other Pinnipeds

Twenty‐four microsatellite loci were isolated from three species of Antarctic seals (Subfamily Monachinae, Tribe Lobodontini). Eleven loci were cloned from Weddell seal, Leptonychotes weddellii, seven from leopard seal, Hydrurga leptonyx, and six from crabeater seal, Lobodon carcinophagus. Variability was assessed in Weddell seals collected in McMurdo Sound, leopard seals from Bird Island, South Georgia, and crabeater seals sampled in the eastern Ross Sea. All loci were variable in the three species used for cloning and 22 of these loci amplified variable products in the Ross seal, Ommatophoca rossii. Cross‐species amplification was largely successful, with an average of 19 loci amplifying products in other phocids.     

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.     

Pinniped phylogeny and a new hypothesis for their origin and dispersal

The relationships and the zoogeography of the three extant pinniped families, Otariidae (sea lions and fur seals), Odobenidae (one extant species, the walrus), and Phocidae (true seals), have been contentious. Here, we address these topics in a molecular study that includes all extant species of true seals and sea lions, four fur seals and the walrus. Contrary to prevailing morphological views the analyses conclusively showed monophyletic Pinnipedia with a basal split between Otarioidea (Otariidae+Odobenidae) and Phocidae. The northern fur seal was the sister to all remaining otariids and neither sea lions nor arctocephaline fur seals were recognized as monophyletic entities. The basal Phocidae split between Monachinae (monk seals and southern true seals) and Phocinae (northern true seals) was strongly supported. The phylogeny of the Phocinae suggests that the ancestors of Cystophora (hooded seal) and the Phocini (e.g. harp seal, ringed seal) adapted to Arctic conditions and ice-breeding before 12 MYA (million years ago) as supported by the white natal coat of these lineages. The origin of the endemic Caspian and Baikal seals was dated well before the onset of major Pleistocene glaciations. The current findings, together with recent advances in pinniped paleontology, allow the proposal of a new hypothesis for pinniped origin and early dispersal. The hypothesis posits that pinnipeds originated on the North American continent with early otarioid and otariid divergences taking place in the northeast Pacific and those of the phocids in coastal areas of southeast N America for later dispersal to colder environments in the N Atlantic and the Arctic Basin, and in Antarctic waters.     

Population genetic structure of the spotted seal Phoca largha along the Coast of Hokkaido,based on mitochondrial DNA sequences

Population genetic structure of the spotted seal, Phoca largha, along coastal regions of Hokkaido was investigated, using mitochondrial DNA (mtDNA) sequences. A 571-bp fragment of the mtDNA control region and adjacent threonine and proline transfer RNA genes was sequenced from 66 seals. We categorized all individuals into three groups considering sampling area and season: twenty-four seals from the Sea of Okhotsk in winter, 11 seals from the Sea of Okhotsk coast in fall, and 31 seals from the Sea of Japan coast in winter. From the 66 animals, 57 haplotypes were identified. Compared with the harbor seal sequence, all spotted seals examined shared two deletions in the control region, which distinguished between the two species. Forty-nine haplotypes were represented by a single individual, and haplotypes shared by more than two animals were generally restricted to same sampling-groups. Phylogenetic trees did not indicate clear geographic differences among the three groups. An Analysis of molecular variances (AMOVA) did not showed any significant population genetic structure in Hokkaido spotted seals (Phi(st )= -0.003). Our results showed a high level of diversity but no genetic structure, and did not deny the possibility that seals in the Okhotsk breeding concentration mainly stayed in the fall Okhotsk and also inhabited in the winter Sea of Japan.     

DISPERSAL and BYCATCH MORTALITY IN GRAY, HALICHOERUS GRYPUS, AND HARBOR, PHOCA VITULINA, SEALS TAGGED AT THE NORWEGIAN COAST

Between 1975 and 1998, 3,571 gray and 630 harbor seal pups were tagged along the Norwegian coast, and 259 (7%) gray and 80 (13%) harbor seal tags were returned. Incidental mortality, mainly in bottom-set nets, accounted for the majority of deaths (79% in gray and 48% in harbor seals, respectively). Seals were most vulnerable to incidental mortality in fishing gear during the first three months after birth, but high incidental mortality prevailed during the first 8–10 mo. Gray seals dispersed more widely (mean distance: 120 km) than harbor seals (mean distance: 69 km). Both species dispersed most widely during the two first months after tagging. The maximum distance moved was 739 km for gray and 463 km for harbor seals. Strong fidelity for their place of birth was observed in adult gray seals during breeding season. No significant difference in incidental mortality was detected between the areas of tagging. However, for 37 harbor seals tagged in a 724 km nature reserve no returns were reported.     

Detection of Cryptosporidium sp. in two new seal species, Phoca vitulina and Cystophora cristata, and a novel Cryptosporidium genotype in a third seal species, Pagophilus groenlandicus, from the Gulf of Maine

Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium.     

Laminae in the teeth of the Cape fur seal used for age determination

A sample of 40 known age Cape fur seal, $\text{Arctocephalus pusillus}$ canine teeth were histologically and manually sectioned and examined for laminae to determine if these growth layers could be used for age determination in this species of seal. Results of this investigation indicate that two laminae (one opaque and one translucent) are deposited per annum and that these structures provide an accurate method for aging seals 2 years old or less; agreement between age and laminae was also obtained for seals up to 8 years of age, but the small number (N = 3) of known-aged canines older than two years does not permit a definite conclusion as to the applicability of the method throughout the life span of the Cape fur seal.     

Amphibian metallothionein. Induction in the frogs Rana japonica, R. nigromaculata and Rhacophorus schlegelii

Cadmium (Cd) was injected intramuscularly into three species of frogs to examine the number of isoforms in the induced metallothionein (MT). The induced MT was shown to consist of a single isoform in the three species of frogs Rana japonica, R. nigromaculata and Rhacophorus schlegelii. Native MT in the livers of R. japonica and R. nigromaculata was copper (Cu)-MT and the induced MT in the three species was Cd, Cu-MT, the Cd/Cu ratio in the induced MT being different from species to species. The effect of Cd injections on the concentrations of 10 elements in the livers and kidneys of the Rana species was not significant except for Cu.     

Feeding habits of three seal species at the Danco Coast, Antarctica: a re-assessment

The analysis of prey overlap among Weddell, Antarctic fur and leopard seals was conducted using fecal samples collected at the Danco Coast, Antarctic Peninsula, in 1998 and 2000. The re-occurrence of prey species was moderate in samples collected in 1998, and low in 2000, and reflects resource partitioning among seal species. Prey species that mostly co-occurred in seals’ diet were the Antarctic krill Euphausia superba, bivalves, and the myctophids Gymnoscopelus nicholsi and Electrona antarctica. A dietary similarity index of prey overlap has been calculated and demonstrates evident fluctuations in pairwise comparisons between the seal species. The highest and lowest values of prey overlap were observed between Antarctic fur seals and leopard seals, and between Weddell seals and leopard seals, respectively. Prey overlap between Antarctic fur seals and Weddell seals was moderate in both seasons.     

Genetically effective population sizes of Antarctic seals estimated from nuclear genes

We analyzed eight nuclear microsatellite loci in three species of Antarctic seals; Weddell seal (Leptonychotes weddellii; mean N = 163), crabeater seal (Lobodon carcinophaga; 138) and Ross seal (Ommatophoca rossii; 35). We estimated genetic diversity (Θ) and effective population size (N _E) for each species. Autosomal microsatellite based N _E estimates were 151,200 for Weddell seals, 880,200 for crabeater seals, and 254,500 for Ross seals. We screened one X-linked microsatellite (Lw18), which yielded similar N _E estimates to the autosomal loci for all species except the Ross seals, where it was considerably larger (~103 times). Microsatellite N _E estimates were comparable with previously published N _E estimates from mitochondrial DNA, but both are substantially lower than direct estimates of population size in all species except the Ross seals. The ratio of maternally versus biparentally derived estimates of N _E for Ross seals was not consistent with the hypothesis that they are a polygynous species. We found no sign of a recent, sustained genetic bottleneck in any of the species.     

First documentation of leopard seal predation of South Georgia pintail duck

Leopard seals are regular winter visitors to Bird Island, South Georgia, where they mostly prey on fur seals and penguins, and to a lesser extent on Antarctic krill and fish. Leopard seals can exploit many different species, but there are no records of predation on flying shorebirds in the wild. On 4 October 2008, an individually identified juvenile leopard seal female was observed killing and eating a South Georgia Pintail duck. It also preyed on Antarctic fur seals and gentoo and macaroni penguins during its 2-month temporary residency around the island. The varied diet of this seal exemplifies the generalist prey utilization typical of its species. Long-term diet studies at Bird Island and the published record suggest that predation on ducks is a rather exceptional finding; individual ducks are more likely to escape leopard seal attacks than penguins and provide a far less substantial ration. This note documents the first observation of this species of duck in the diet of leopard seals.