Reduction of Paraoxonase Expression Followed by Inactivation across Independent Semiaquatic Mammals Suggests Stepwise Path to Pseudogenization.

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation.

Evaluating Hair as a Predictor of Blood Mercury: The Influence of Ontogenetic Phase and Life History in Pinnipeds.

Mercury (Hg) biomonitoring of pinnipeds increasingly utilizes nonlethally collected tissues such as hair and blood. The relationship between total Hg concentrations ([THg]) in these tissues is not well understood for marine mammals, but it can be important for interpretation of tissue concentrations with respect to ecotoxicology and biomonitoring. We examined [THg] in blood and hair in multiple age classes of four pinniped species. For each species, we used paired blood and hair samples to quantify the ability of [THg] in hair to predict [THg] in blood at the time of sampling and examined the influence of varying ontogenetic phases and life history of the sampled animals. Overall, we found that the relationship between [THg] in hair and blood was affected by factors including age class, weaning status, growth, and the time difference between hair growth and sample collection. Hair [THg] was moderately to strongly predictive of current blood [THg] for adult female Steller sea lions (Eumetopias jubatus), adult female California sea lions (Zalophus californianus), and adult harbor seals (Phoca vitulina), whereas hair [THg] was poorly predictive or not predictive (different times of year) of blood [THg] for adult northern elephant seals (Mirounga angustirostris). Within species, except for very young pups, hair [THg] was a weaker predictor of blood [THg] for prereproductive animals than for adults likely due to growth, variability in foraging behavior, and transitions between ontogenetic phases. Our results indicate that the relationship between hair [THg] and blood [THg] in pinnipeds is variable and that ontogenetic phase and life history should be considered when interpreting [THg] in these tissues.

Crossing to safety: dispersal, colonization and mate choice in evolutionarily distinct populations of Steller sea lions, Eumetopias jubatus.

Population growth typically involves range expansion and establishment of new breeding sites, while the opposite occurs during declines. Although density dependence is widely invoked in theoretical studies of emigration and colonization in expanding populations, few empirical studies have documented the mechanisms. Still fewer have documented the direction and mechanisms of individual transfer in declining populations. Here, we screen large numbers of pups sampled on their natal rookeries for variation in mtDNA (n = 1106) and 16 microsatellite loci (n = 588) and show that new Steller sea lion breeding sites did not follow the typical paradigm and were instead colonized by sea lions from both a declining (Endangered) population and an increasing population. Dispersing individuals colonized rookeries in the distributional hiatus between two evolutionarily distinct (Φ¯(st) = 0.222, R¯(st) = 0.053, K = 2) metapopulations recently described as separate subspecies. Hardy-Weinberg, mixed-stock and relatedness analysis revealed levels of interbreeding on the new rookeries that exclude (i) assortative mating among eastern and western forms, and (ii) inbreeding avoidance as primary motivations for dispersal. Positive and negative density dependence is implicated in both cases of individual transfer. Migration distance limits, and conspecific attraction and performance likely influenced the sequence of rookery colonizations. This study demonstrates that resource limitation may trigger an exodus of breeding animals from declining populations, with substantial impacts on distribution and patterns of genetic variation. It also revealed that this event is rare because colonists dispersed across an evolutionary boundary, suggesting that the causative factors behind recent declines are unusual or of larger magnitude than normally occur.

Relating fibropapilloma tumor severity to blood parameters in green turtles Chelonia mydas.

Fibropapillomatosis is a neoplastic disease that is commonly found in the green turtles Chelonia mydas in tropical and subtropical regions of the world. In the current project, juvenile green turtles were captured with large-mesh tangle nets in the Indian River Lagoon and on nearshore reefs of Indian River County, Florida, USA, in 1998 and 1999. The purpose of the study was to determine the relationship between the severity of the disease and the general health of green turtles as indicated by blood parameters. All turtles were measured and examined, and the overall severity of the disease was rated by the size, number, and location of external fibropapilloma tumors. Hematocrit, total protein, and hemoglobin concentration were measured and compared with tumor scores (tumor severity appraisal). As the tumor score increased, the blood parameters of turtles decreased; for instance, the percentage of decrease in hematocrit for mildly afflicted, moderately afflicted, and severely afflicted groups were 2.6, 18.3, and 45.5%, respectively. Severely afflicted turtles suffered from anemia, while individuals with mild affliction did not.

Preliminary comparisons between a point-of-care ketometer and reference method using Steller sea lion pup whole blood and plasma.

We evaluated the Precision Xtra™ ketometer as part of a larger study categorizing fasting status of free-ranging Steller sea lion (Eumetopias jubatus; SSL) pups which necessitated the identification of plasma ß-hydroxybutyrate concentrations ([ß-HBA]) around a threshold of <0.3 and ≥0.3 mmol/l. Whole blood samples mixed with sodium heparin (NaHep) or ethylenediaminetetraacetic acid liquid anticoagulants were tested <10 minutes after collection (n = 14; triplicate technical replicates). Plasma (stored at -80°C, NaHep, Thaw1) measured via our laboratory's Reference Assay (Sigma Aldrich, St. Louis, MO, Kit #MAK041) served as the standard [ß-HBA] for ketometer comparisons. Our observed ß-HBA range (0.0-1.6 mmol/l), consistent with published [ß-HBA] of free-ranging Otariid pups, represented the lower 20% of the ketometer's range (0.0-8.0 mmol/l). The maximal coefficient of variation (%CV) of ketometer technical replicates was 9.1% (NaHep, whole blood). The majority of ketometer technical replicate sets (84%, including all matrices, anticoagulants and thawings) were identical (CV = 0%). We found linear relationships and agreement of ketometer [ß-HBA] between whole blood preserved with different anticoagulants and between whole blood and plasma (Thaw1) measurements. The ketometer produced results with linearity to the Reference Assay for both whole blood and plasma (Thaw1). We identified a non-linear relationship between plasma at Thaw1 and Thaw2 (tested four months apart, NaHep), as only samples with higher SSL [ß-HBA] decreased in concentration, and all others remained the same. With respect to categorizing SSL pup fasting in our larger study, the ketometer's %Accuracy, %Sensitivity and %Specificity for samples with Reference Assay ß-HBA <0.2 and >0.4 mmol/l were 100%. We adopted a modified procedure: plasma samples with mean ketometer concentrations ±0.1 mmol/l of 0.3 mmol/l ß-HBA were re-evaluated using the Reference Assay, improving measurement precision from tenths (ketometer) to thousandths (assay) mmol/l. The Precision Xtra™ ketometer was valuable to our application over the range of [ß-HBA] observed in SSL pup plasma and whole blood samples.

Total mercury concentrations in Steller sea lion bone: Variability among locations and elements.

Mercury is a global contaminant that bioaccumulates in a tissue-specific manner in long-lived predators such as Steller sea lions (SSL). Bone is a well-preserved material amenable for studying millennial scale trends; however, little is known about the distribution and variability of total mercury concentrations ([THg]) within individual bones and among bone elements in SSL. We assessed SSL bone [THg] variability with respect to physiologic age, bone type, longitudinally within a bone, and among bone elements. Pup bones (mean ± SD; 31.4 ± 13.58 ppb) had greater [THg] than adults (7.9 ± 1.91 ppb). There were greater and more variable [THg] within individual long bones near epiphyses compared to mid-diaphysis. Pup spongy bone in ribs (62.7 ± 44.79 ppb) had greater [THg] than long bones (23.5 ± 8.83 ppb) and phalanges (19.6 ± 10.78 ppb). These differences are likely due to variability in bone composition, growth, and turnover rate. This study informs standardized sampling procedures for [THg] in bone to improve interpretations of mercury variability over time and space.

Fasting durations of Steller sea lion pups vary among subpopulations-evidence from two plasma metabolites.

Geographic differences in population growth trends are well-documented in Steller sea lions (Eumetopias jubatus), a species of North Pacific pinniped listed under the U.S. Endangered Species Act in 1990 following a marked decline in population abundance that began during the 1970s. As population growth is intrinsically linked to pup production and survival, examining factors related to pup physiological condition provides useful information to management authorities regarding potential drivers of regional differences. During dam foraging trips, pups predictably transition among three fasting phases, distinguished by the changes in the predominant metabolic byproduct. We used standardized ranges of two plasma metabolites (blood urea nitrogen and ß-hydroxybutyrate) to assign pups to fasting categories (n = 1528, 1990-2016, 12 subpopulations): Recently Fed-Phase I (digestion/assimilation-expected hepatic/muscle glycogen usage), Phase II (expected lipid utilization), transitioning between Phases II-III (expected lipid utilization with increased protein reliance), or Phase III (expected protein catabolism). As anticipated, the majority of pups were classified as Recently Fed-Phase I (overall mean proportion = 0.72) and few pups as Phase III (overall mean proportion = 0.04). By further comparing pups in Short (Recently Fed-Phase II) and Long (all other pups) duration fasts, we identified three subpopulations with significantly (P < 0.03) greater proportions of pups dependent upon endogenous sources of energy for extended periods, during a life stage of somatic growth and development: the 1) central (0.27 ± 0.09) and 2) western (0.36 ± 0.13) Aleutian Island (declining population trend) and 3) southern Southeast Alaska (0.32 ± 0.06; increasing population trend) subpopulations had greater Long fast proportions than the eastern Aleutian Islands (0.10 ± 0.05; stabilized population). Due to contrasting population growth trends among these highlighted subpopulations over the past 50+ years, both density-independent and density-dependent factors likely influence the dam foraging trip duration, contributing to longer fasting durations for pups at some rookeries.

Total Mercury, Total Selenium, and Monomethylmercury Relationships in Multiple Age Cohorts and Tissues of Steller Sea Lions (Eumetopias jubatus).

Steller sea lion (Eumetopias jubatus) tissue mercury concentrations increasingly above thresholds of concern occur in regions of Alaska where lack of population recovery is noted. Selenium-monomethylmercury interactions may mitigate toxicosis but may also result in functional selenium deficiency, impacting essential selenium-dependent processes. Physiologically driven differences in tissue concentrations (organotropism) of total mercury ([THg]), total selenium ([TSe]), and monomethylmercury ([MeHg+ ]) confound interpretation for various age cohorts. Archived tissues from Alaska Steller sea lions (2002-2016) were used to compare [THg], [MeHg+ ], and [TSe] across age cohorts and tissue types. Liver [THg] ranged from 0.05 to 63.7 µg/g. Fetal and pup livers had significantly lower [THg] and [TSe], higher percentage MeHg+ , and greater range of molar TSe:THg than subadult and adult livers. Molar Se:MeHg+ ratios, including Se in excess of nonmethylmercury, were dependent on [MeHg+ ] in fetuses and pups. While [THg] varied significantly by muscle type (heart vs. skeletal) and anatomical location, concentrations were strongly correlated. Biomagnification and/or bioaccumulation of THg in liver of older animals confounded comparison with other tissues; however, in fetal and pup liver [THg] correlated with other tissues. In contrast, liver [MeHg+ ] correlated with other tissues across all age classes. Fetal and pup tissues, which reflect in utero exposure and are subject to limited bioaccumulation, are ideal for assessing mercury exposure related to dam diet, including intertissue comparison, and represent key cohorts of concern. Evaluating [MeHg+ ] and [TSe] in tissues from multiple age cohorts allows better intertissue comparison, providing insight into time courses, routes of exposure, and potential for adverse effects. Environ Toxicol Chem 2022;41:1477-1489. © 2022 SETAC.

Regional variations and relationships among cytokine profiles, white blood cell counts, and blood mercury concentrations in Steller sea lion (Eumetopias jubatus) pups.

The Steller sea lion (SSL) population west of 144°W longitude experienced a significant population decline. While there appears to be a stable or increasing population trend in rookeries in the Gulf of Alaska (GOA) and Southeast Alaska (SEA), some rookeries within the Aleutian Islands (AI) have failed to recover. Previous studies found regional differences in whole blood total mercury concentrations ([THg]) showing more than 20% of AI pups had [THg] above critical thresholds for increased risk of immunological effects and other adverse outcomes. Measurements of immune cell-signaling proteins can be used to evaluate the immune status of marine mammals in relation to [THg]. We compared serum cytokine and chemokine concentrations in pups among regions (AI, eastern GOA, SEA), and examined associations among cytokines, chemokines, white blood cell (WBC) counts, and [THg]. Considering liver is an important target organ for mercury and immune protein synthesis we additionally examined the relationship of [THg] with liver-related enzymes serum aspartate (AST) and alanine aminotransferase (ALT). We observed regional differences in cytokine and chemokine measurements and immune protein associations. There was a positive association between total WBC counts and [THg] in AI pups, whereas a negative association between lymphocytes and [THg] in SEA pups. These findings may indicate regional variation in proliferation and differentiation of hematopoietic cells, differences in immune system development, and/or a difference in antigenic stimuli. No associations between [THg] and cytokines, chemokines, AST or ALT were found. Observed regional differences in cytokine and chemokine milieu during gestational and early development in SSL pups could lead to an imbalance in cell differentiation that could impact immunological resiliency in juvenile and adult life stages. We report concentration ranges of a suite of cytokines and chemokines which may prove to be a useful metric for ecotoxicology and risk assessment studies in SSLs and other wildlife.

Assessment of clinical outcomes associated with mercury concentrations in harbor seal pups (Phoca vitulina richardii) in central California.

Monomethyl mercury (MeHg+) from the diet can cause mild to severe neurotoxicosis in fish-eating mammals. Chronic and low-level in utero exposure also can be neurotoxic, as documented in laboratory animal studies and epidemiologic investigations. In free-ranging animals, it is challenging to study low-level exposure related neurotoxicosis, and few studies have investigated the relationship between mercury (Hg) and adverse outcomes in wild populations. Relative to Hg concentrations on admission we evaluated different types of behaviors for 267 Pacific harbor seal (HS; Phoca vitulina richardii) pups at The Marine Mammal Center from 2015 to 2019 during rehabilitation after stranding and maternal separation. Admitted HS pups underwent a clinical exam; including sex and weight determination, and hair (partly lanugo grown in utero) and blood samples were collected for total Hg concentration ([THg]) determination. All pups were monitored weekly (behavior assessments included response to tactile stimulation, movement, swimming, interactions with other seals, hand feeding, and feeding independently), and days in rehabilitation and survival were recorded. There was a significant negative correlation between [THg] and responses to tactile stimulation and movements, measured in both hair and whole blood (p < 0.05). This relationship was found both during the intensive care unit (ICU) stage, and during the pool stage of rehabilitation. Additionally, there was a significant association between greater [THg] and number of days spent in rehabilitation, although there was no relationship between [THg] and survival. There was a significant sex difference, with greater [THg] in female pups, which contrasts with previously published findings in juvenile and adult harbor seals. Our findings support small, but significant associations between gestational THg exposure and clinical effects for tactile sensory response and movement, and longer rehabilitation durations for HS pups, although there was considerable variability among animals.

Whiskers as a novel tissue for tracking reproductive and stress-related hormones in North Pacific otariid pinnipeds.

Keratinized tissues, including whiskers, are ideal for acquiring a record of physiological parameters. Most tissues provide a snapshot of physiological status; however, whiskers may support longitudinal sampling for reproductive and stress-related hormones, if hormones are incorporated as whiskers grow and concentrations change with physiological state. Whiskers from female Steller sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus) were serially sectioned and pulverized and steroid hormones were extracted. Standard methods were used to validate enzyme immunoassay kits for cortisol, progesterone, 17ß-estradiol and testosterone. All hormones were measurable in whisker segments from both species with progesterone concentrations showing cyclical patterns, which appear to signify previous pregnancies or luteal phases. Yearly progesterone concentrations were greater in years a pup was produced compared with years when no pup was observed. Free-ranging female Steller sea lions had reproductive rates between 0 and 1.0 (0.53 ± 0.33, n = 12) using a yearly progesterone concentration of 30 pg/mg or greater to classify a reproductive year as producing a pup and below 30 pg/mg as non-reproductive. Cortisol concentrations were greater near the root and rapidly declined, lacking any obvious patterns, throughout the rest of the whisker. Progesterone and testosterone concentrations were able to help determine sex of unknown individuals. Immunohistochemistry revealed that steroid hormones most likely do not leach out of whiskers based on the deposition patterns of progesterone and cortisol being present throughout the whisker length. Overall, measuring steroid hormones in whiskers can reveal individual reproductive histories over multiple years in sea lions and fur seals. Cyclical patterns of δ15N were useful for identifying periods of up to ~10 years of growth within whiskers, and measuring both stable isotopes and hormones may be useful for differentiating periods of active gestation from diapause and potentially track multi-year reproductive histories of female otariids.

Methyl mercury (MeHg) in vitro exposure alters mitogen-induced lymphocyte proliferation and cytokine expression in Steller sea lion (Eumetopias jubatus) pups.

Steller sea lions (Eumetopias jubatus, SSLs) are managed as two distinct population segments within U.S. waters: the endangered western distinct population segment and the recently delisted eastern distinct population segment. Recent studies reported concentrations of mercury in several tissues collected from young SSLs in the Aleutian Islands that were at or above concentrations found to negatively impact health in other fish-eating mammals. However, there are limited studies which have investigated the range of mercury concentrations that may negatively influence the SSL immune system. This study assessed relationships between methyl mercury (MeHg+) concentrations and two immune functions, lymphocyte proliferation and cytokine expression. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from pups on three rookeries within the western distinct population segment: Chiswell Island, Ulak, and Agattu Islands. Lymphocyte proliferation and cytokine expression were assessed in vitro using thawed PBMCs with exposure to MeHg+ (unexposed control, 0.001, 0.01, and 0.1 µg/ml). Lymphocyte proliferation was measured without and with stimulation with a T cell mitogen (ConA) and B cell mitogen (LPS) and the concentration of cytokines was measured in the cell culture supernatant (with and without ConA or LPS). Spontaneous lymphocyte proliferation was significantly increased at 0.01 and 0.1 µg/ml. T lymphocyte proliferation was significantly increased at 0.001 µg/ml and 0.1 µg/ml, while B lymphocyte proliferation was decreased at 0.1 µg/ml. Cytokine concentrations for INFγ, IL-10, IL-6, and TNFα were reduced at 0.1 µg/ml upon either T or B cell mitogen stimulation, with the exception for IL-10, where 0.1 µg/ml reduced IL-10 concentration compared to unstimulated cells. These data suggest immune functions were affected by MeHg+ exposure requiring in vivo follow up investigations. The observed modulation of immune functions is of concern as any toxicant-induced modulation may adversely affect the health of individuals, particularly younger animals undergoing periods of critical development.

Assessing oxidative stress in Steller sea lions (Eumetopias jubatus): Associations with mercury and selenium concentrations.

Selenium (Se) bioavailability is required for synthesis and function of essential Se-dependent antioxidants, including the enzyme glutathione peroxidase (GPx). Strong interactions between monomethyl mercury and Se impair the critical antioxidant role of Se. Approximately 20% of Steller sea lion (Eumetopias jubatus, SSL) pups sampled in the western Aleutian Islands, Alaska, had total Hg concentrations ([THg]) measured in hair and whole blood above thresholds of concern for adverse physiologic effects in pinnipeds. Importantly, low molar ratios of TSe:THg, in some cases < 1 in several tissues (hair, liver, pelt, muscle, spleen, intestine, heart, lungs, brain) were documented for one SSL pup with [THg] above threshold of concern, which may lead to antioxidant deficiency. Our aim with this study was to evaluate the relationship between circulating [THg], [MeHg+], [TSe] and TSe:THg molar ratio status relative to oxidative stress and antioxidants measured during general anesthesia in free-ranging SSL. We captured, anesthetized and sampled newborn SSL pups at rookeries located in the Aleutian Islands or Gulf of Alaska. Biomarkers analyzed for oxidative stress included 4-hydroxynenonal and thiobarbituric acid reactive substances (4-HNE and TBARS, respectively, lipid peroxidation), protein carbonyl content (PCC, protein oxidation), and GPx activity as a key indicator for Se-dependent antioxidant defense levels. We found a negative association between TBARS and [TSe], and SSL with low [TSe] had higher concentrations of 4-HNE than those with intermediate [TSe]. These results suggest that SSL with lower [TSe] experience increased lipid peroxidation potentially associated with [THg] status.

Organochlorine contaminant concentrations in blubber of young Steller sea lion (Eumetopias jubatus) are influenced by region, age, sex, and lipid stores.

Contaminant exposure is particularly important for species and populations of conservation concern, such as the Steller sea lion (Eumetopias jubatus). We used blubber samples (n = 120) to determine organochlorine concentrations, including polychlorinated biphenyls (PCBs) and dichloro-diphenyl-trichloroethane (DDTs), and blood samples (n = 107) to estimate total body lipids based on the hydrogen isotope dilution method. We assessed the influence of age, sex, condition, and geographic area on contaminant concentrations in blubber and contaminant body load. The concentration of ΣPCBs was highest in pups (<6 months) from the Aleutian Islands, and the concentrations in males were higher than females in all regions. The ΣPCBs and ΣDDTs concentrations and loads decreased with increasing mass in pups, however, there were no regional or sex differences in contaminant load. Within each of the five age classes, the concentrations of ΣPCBs and ΣDDTs decreased with increasing mass, but overall these OCs increased with age. Further, accounting for the lipid content, a potential proxy for energy balance, in the animal load reduced or removed the regional and sex effects present in age models for contaminants. We propose, that adjusting OCs concentration by the lipid content of the blubber sample alone may not fully account for the variability in OC concentrations associated with differences in condition or energy states between young Steller sea lions.

Mercury concentrations in marine species from the Aleutian Islands: Spatial and biological determinants.

Several species found in the Bering Sea show significant spatial variation in total mercury concentrations ([THg]) longitudinally along the Aleutian Island chain. We assessed [THg] in other members of the Bering Sea food web to better understand the factors shaping regional differences. [THg] and stable carbon and nitrogen isotope ratios (δ15N and δ13C values) were measured in muscle tissue from 1052 fishes and cephalopods from parts of the Bering Sea and North Pacific Ocean adjacent to the Aleutian Islands. The spatial distribution of the samples enabled regional comparisons for 8 species of fish and one species of cephalopod. Four species showed higher mean length-standardized [THg] in the western Aleutian Islands management area. [THg] in yellow Irish lord were very different relative to those observed in other species and when included in multi-species analyses drove the overall regional trends in mean [THg]. Multi-species analyses excluding measurements for yellow Irish lord showed mean length-standardized [THg] was greater in the western Aleutian Islands than in the central Aleutian Islands management area. Linear regression of [THg] and δ15N values showed a significant and positive relationship across all species, varying between regions and across species. Isotopic space of all species was significantly different between the western Aleutian Islands and central Aleutian Islands, driven largely by δ13C values. Stable isotope values observed follow the same regional trend of lower trophic taxa reported in the literature, with significantly lower δ13C values in the western Aleutian Islands. We conclude that there are regional differences in carbon and nitrogen stable isotope ecology, as well as species-specific feeding ecology that influence [THg] dynamics in part of the marine food web along the Aleutian Island chain. These regional differences are likely contributors to the observed regional variations of [THg] in some high-level predators found in these regions.

REGIONAL AND AGE-RELATED VARIATIONS IN HAPTOGLOBIN CONCENTRATIONS IN STELLER SEA LIONS ( EUMETOPIAS JUBATUS) FROM ALASKA, USA.

Varying concentrations of the highly conserved acute phase response protein, haptoglobin, can indicate changes to the health and disease status of mammals, including the Steller sea lion (SSL; Eumetopias jubatus). To better understand factors relating to acute phase response in SSLs, circulating haptoglobin concentrations (Hp) were quantified in plasma collected from 1,272 individuals sampled near rookeries and haulouts off the coast of Alaska, US. We compared Hp in SSLs between sexes and among different age classes (young pups, young-of-the-year, yearlings, subadults, and adults) sampled within distinct regions in Alaska (Aleutian Islands, Gulf of Alaska, Southeast Alaska). Regional and age-related differences were observed, particularly in younger SSLs. No sex-related differences were detected. We identified weakly significant relationships between Hp and hematology measurements including white blood cell counts and hematocrit in young pups from the Aleutian Islands and Southeast Alaska. No relationship between Hp and body condition was found. Lastly, a nonlinear relationship of plasma Hp and whole blood total mercury concentrations (THg) was observed in SSLs from the endangered western distinct population segment in Alaska. These results demonstrated that regional variation in Hp, especially in younger SSLs, may reflect regional differences in health and circulating THg.

Enhanced quantification of serum immunoglobulin G from a non-model wildlife species, the Steller sea lion (Eumetopias jubatus), using a protein A ELISA.

Immunoglobulins (Ig) are proteins that preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring Ig in non-model wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide reliable results especially when lacking species-specific reagents. We modified and optimized existing ELISA methodology utilizing the binding properties of Staphylococcus-derived Protein A (PrtA) to immunoglobulin G (IgG). We enhanced the assay for quantifying IgG in Steller sea lion (SSL) serum using critical quality control measures including dilution linearity, spike and percent recoveries, and internal controls. Of the modifications made, heat treatment of SSL serum enhanced accuracy and precision of IgG measurements by improving linearity and percent recovery in parallel dilutions and serum spikes. Purified canine IgG standard was not affected by heat inactivation. These results support that confounding serum proteins interfere with binding of PrtA with IgG demonstrating the need for heat treatment of serum to optimize IgG quantification using the PrtA-ELISA. Further, essential validation measures ensure proper assay performance. Consequently, the improved PrtA-ELISA provides species-independent IgG detection with validation criteria to enhance accuracy and precision for addressing future immunological questions in non-model wildlife in clinical, ecological, and conservation contexts.

Chlorinated fatty acids in lipid class fractions from cardiac and skeletal muscle of Chinook salmon.

Analysis of chlorinated fatty acids (CFA) in tissues can be difficult because of their low concentrations. This task becomes even more difficult when samples are from organisms living in remote locations with very little exposure to environmental contamination. Therefore, enrichment of CFA is necessary prior to analysis. In this study, CFA were enriched from fractionated lipid classes of cardiac and skeletal muscle of Chinook salmon (Oncorhynchus tshawytscha) to determine CFA distribution among lipid classes and tissue types and to demonstrate the sensitivity of this method to detect CFA at trace concentrations. The lipids extracted from cardiac and skeletal muscle of O. tshawytscha were separated into fractions containing TAG, FFA, and phospholipids. After transesterification, the FAME derivatives from each lipid class were analyzed by GC with a halogen-selective detector (XSD) to determine the concentrations of dichlorostearic acid and dichloropalmitic acid. Other chlorinated compounds detected by GC-XSD were analyzed by GC-MS. CFA were observed in all lipid classes in both cardiac and skeletal muscle tissues. However, the highest concentrations of CFA were found in the phospholipids of both tissue types, about 1-2 mg/g lipid. It was also shown that dichloropalmitic acid concentrated in cardiac phospholipids whereas dichlorostearic acid was found primarily in the phospholipids of skeletal tissue. CFA concentrations in TAG and FFA fractions were below 150 mg/g lipid. This study demonstrates a small-scale approach to the study of CFA at trace concentrations and their distribution among lipid classes.

Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry.

The determination of FAME by GC is among the most commonplace analyses in lipid research. Quantification of FAME by GC with FID has been effectively performed for some time, whereas detection with MS has been used chiefly for qualitative analysis of FAME. Nonetheless, the sensitivity and selectivity of MS methods advocate a quantitative role for GC-MS in FAME analysis-an approach that would be particularly advantageous for FAME determination in complex biological samples, where spectrometric confirmation of analytes is advisable. To assess the utility of GC-MS methods for FAME quantification, a comparative study of GC-FID and GC-MS methods has been conducted. FAME in prepared solutions as well as a biological standard reference material were analyzed by GC-FID and GC-MS methods using both ion trap and quadrupole MS systems. Quantification by MS, based on total ion counts and processing of selected ions, was investigated for FAME ionized by electron impact. Instrument precision, detection limits, calibration behavior, and response factors were investigated for each approach, and quantitative results obtained by each technique were compared. Although there were a number of characteristic differences between the MS methods and FID with respect to FAME analysis, the quantitative performance of GC-MS compared satisfactorily with that of GC-FID. The capacity to combine spectrometric examination and quantitative determination advances GC-MS as a powerful alternative to GC-FID for FAME analysis.

Assessment of mercury and selenium tissular concentrations and total mercury body burden in 6 Steller sea lion pups from the Aleutian Islands.

Concentrations of total mercury ([THg]) and selenium ([TSe]) were measured in several tissue compartments in Steller sea lion (Eumetopias jubatus) pups; in addition we determined specific compartment and body burdens of THg. Compartmental and body burdens were calculated by multiplying specific compartment fresh weight by the [THg] (summing compartment burdens equals body burden). In all 6 pup tissue sets (1) highest [THg] was in hair, (2) lowest [THg] was in bone, and (3) pelt, muscle and liver burdens contributed the top three highest percentages of THg body burden. In 5 of 6 pups the Se:Hg molar ratios among compartments ranged from 0.9 to 43.0. The pup with the highest hair [THg] had Se:Hg molar ratios in 9 of 14 compartments that were ⩽ 0.7 potentially indicating an inadequate [TSe] relative to [THg].