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Oceans are changing and these changes are affecting the animals that live there. Animals respond differently to changes in water temperature, food availability, and contaminants. Those responses can be seen in their genes. A technique called transcriptomics allows scientists to see the response of an animal’s genes to its environment. We used transcriptomics to compare two populations of Pacific razor clams in Alaska (United States): one that has lots of clams and one that used to have lots but does not anymore. We were surprised when we did not find any differences in their gene responses! So, we had to think about what else might be influencing the number of clams in these two populations. As we “dug” for answers, we found out that there are differences between the populations that do not influence their genes but may impact their numbers, such as being eaten by predators.

Migrating adult Pacific salmon (Oncorhynchus spp.) are sensitive to warm water (>18 °C), with a range of consequences from decreased spawning success to early mortality. We examined the proportion of Yukon River Chinook salmon (O. tshawytscha) exhibiting evidence of heat stress to assess the potential that high temperatures contribute to freshwater adult mortality in a northern Pacific salmon population. Water temperatures greater than 18 °C have occurred almost annually in the Yukon River and correspond with low population abundance since the 1990s. Using gene transcription products and heat shock protein 70 biomarkers validated by field experiment, we identified heat stress in half of Chinook salmon examined (54%, n = 477) across three mainstem locations and three tributaries in 2016–2017. Biomarkers tracked wide variation in water temperature (14–23 °C) within a tributary. The proportion of salmon with heat stress differed between years at four of the six locations, with more prevalent heat stress in the warmer year. This work demonstrates that warming water temperatures are currently affecting northern populations of Pacific salmon.

Increasing risk of pathogen spillover coupled with overall declines in wildlife population abundance in the Anthropocene make infectious disease a relevant concern for species conservation worldwide. While emerging molecular tools could improve our diagnostic capabilities and give insight into mechanisms underlying wildlife disease risk, they have rarely been applied in practice. Here, employing a previously reported gene transcription panel of common immune markers to track physiological changes, we present a detailed analysis over the course of both acute and chronic infection in one wildlife species where disease plays a critical role in conservation, bighorn sheep (Ovis canadensis). Differential gene transcription patterns distinguished between infection statuses over the course of acute infection and differential correlation (DC) analyses identified clear changes in gene co-transcription patterns over the early stages of infection, with transcription of four genes—TGFb, AHR, IL1b and MX1—continuing to increase even as transcription of other immune-associated genes waned. In a separate analysis, we considered the capacity of the same gene transcription panel to aid in differentiating between chronically infected animals and animals in other disease states outside of acute disease events (an immediate priority for wildlife management in this system). We found that this transcription panel was capable of accurately identifying chronically infected animals in the test dataset, though additional data will be required to determine how far this ability extends. Taken together, our results showcase the successful proof of concept and breadth of potential utilities that gene transcription might provide to wildlife disease management, from direct insight into mechanisms associated with differential disease response to improved diagnostic capacity in the field.

Health diagnostics of wildlife have historically relied on the evaluation of select serum biomarkers and the identification of a contaminant or pathogen burden within specific tissues as an indicator of a level of insult. However, these approaches fail to measure the physiological reaction of the individual to stressors, thus limiting the scope of interpretation. Gene-based health diagnostics provide an opportunity for an alternate, whole-system, or holistic assessment of health, not only in individuals or populations but potentially in ecosystems. Seabirds are among the most threatened marine taxonomic groups in the world, with ~25% of this species currently listed as threatened or considered of special concern; among seabirds, the penguins (Family Spheniscidae) are the most threatened seabird Family. We used gene expression to develop baseline physiological indices for wild penguins in the Falkland-Malvinas Islands, and captive zoo penguins. We identified the almost complete statistical separation of penguin groups (gentoo Detroit Zoo, gentoo Falkland-Malvinas Islands, rockhopper Detroit Zoo, and rockhopper Falkland-Malvinas Islands) based on gene expression profiles. Implementation of long-term longitudinal studies would allow for the assessment of temporal increases or decreases of select transcripts and would facilitate interpretation of the drivers of change.

With rapidly changing marine ecosystems, shifts in abundance and distribution are being documented for a variety of intertidal species. We examined two adjacent populations of Pacific razor clams (Siliqua patula) in lower Cook Inlet, Alaska. One population (east) supported a sport and personal use fishery, but this has been closed since 2015 due to declines in abundance, and the second population (west) continues to support commercial and sport fisheries. We used gene expression to investigate potential causes of the east side decline, comparing razor clam physiological responses between east and west Cook Inlet. The target gene profile used was developed for razor clam populations in Alaska based on physiological responses to environmental stressors. In this study, we identified no differences of gene expression between east and west populations, leading to two potential conclusions: (1) differences in factors capable of influencing physiology exist between the east and west and are sufficient to influence razor clam populations but are not detected by the genes in our panel, or (2) physiological processes do not account for the differences in abundance, and other factors such as predation or changes in habitat may be impacting the east Cook Inlet population.

Increasing risk of pathogen spillover coupled with overall declines in wildlife population abundance in the Anthropocene make infectious disease a relevant concern for species conservation worldwide. While emerging molecular tools could improve our diagnostic capabilities and give insight into mechanisms underlying wildlife disease risk, they have rarely been applied in practice. Here, employing a previously reported gene transcription panel of common immune markers to track physiological changes, we present a detailed analysis over the course of both acute and chronic infection in one wildlife species where disease plays a critical role in conservation, bighorn sheep (Ovis canadensis). Differential gene transcription patterns distinguished between infection statuses over the course of acute infection and differential correlation (DC) analyses identified clear changes in gene co-transcription patterns over the early stages of infection, with transcription of four genes—TGFb, AHR, IL1b and MX1—continuing to increase even as transcription of other immune-associated genes waned. In a separate analysis, we considered the capacity of the same gene transcription panel to aid in differentiating between chronically infected animals and animals in other disease states outside of acute disease events (an immediate priority for wildlife management in this system). We found that this transcription panel was capable of accurately identifying chronically infected animals in the test dataset, though additional data will be required to determine how far this ability extends. Taken together, our results showcase the successful proof of concept and breadth of potential utilities that gene transcription might provide to wildlife disease management, from direct insight into mechanisms associated with differential disease response to improved diagnostic capacity in the field.

Immune function plays an important role in an animal's defense against infectious disease. In reptiles, immune responses may be complex and counterintuitive, and diagnostic tools used to identify infection, such as induced antibody responses are limited. Recent studies using gene transcription profiling in tortoises have proven useful in identifying immune responses to various intrinsic and extrinsic stressors. As part of a larger experiment with Mojave desert tortoises (Gopherus agassizii), we facilitated the transmission of the pathogenic bacteria, Mycoplasma agassizii (Myag), to naïve adults and measured innate and induced immune reactions over time. Specifically, we evaluated clinical condition, presence of Myag in the nasal/oral cavity, induced antibody responses specific to Myag, and measured molecular reactions (gene transcript profiles) in 15 captive tortoises classified as naïve, exposed, or infected and 14 wild tortoises for comparison. Myag was confirmed inside the nasal/oral cavity in exposed tortoises within 30–60 days of introduction to infected animals, yet we did not detect Myag specific induced antibody responses in these individuals until 420–595 days post exposure. Surprisingly, we found no overall differences in the gene transcript profiles between our experimental treatment groups throughout this study. This work highlights the complexities in assessing immune function and diagnosing pathogen related infections in tortoises and other reptiles.

Chinook salmon (Oncorhynchus tshawytscha) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3′-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted.

An emerging approach to ecosystem monitoring involves the use of physiological biomarker analyses in combination with gene transcription assays. For the first time, we employed these tools to evaluate the Pacific razor clam (Siliqua patula), which is important both economically and ecologically, as a bioindicator species in the northeast Pacific. Our objectives were to (1) develop biomarker and gene transcription assays with which to monitor the health of the Pacific razor clam, (2) acquire baseline biomarker and gene transcription reference ranges for razor clams, (3) assess the relationship between physiological and gene transcription assays and (4) determine if site-level differences were present. Pacific razor clams were collected in July 2015 and 2016 at three sites within each of two national parks in southcentral Alaska. In addition to determining reference ranges, we found differences in biomarker assay and gene transcription results between parks and sites which indicate variation in both large-scale and local environmental conditions. Our intent is to employ these methods to evaluate Pacific razor clams as a bioindicator of nearshore ecosystem health. Links between the results of the biomarker and gene transcription assays were observed that support the applicability of both assays in ecosystem monitoring. However, we recognize the need for controlled studies to examine the range of responses in physiology and gene transcripts to different stressors.

A portable experimental system was developed for remote field settings, capable of holding large fish for several hours, while controlling for precise water temperatures. This system was used to conduct a thermal challenge experiment using adult Chinook salmon along the Yukon River in Alaska, USA., Manipulative experiments provide stronger evidence for identifying cause-and-effect relationships than correlative studies, but protocols for implementing temperature manipulations are lacking for large species in remote settings. We developed an experimental protocol for holding adult Chinook salmon (Oncorhynchus tshawytscha) and exposing them to elevated temperature treatments. The goal of the experimental protocol was to validate heat stress biomarkers by increasing river water temperature from ambient (~14°C) to a treatment temperature of 18°C or 21°C and then maintain the treatment temperature over 4 hours within a range of ±1.0°C. Our protocol resulted in a mean rate of temperature rise of 3.71°C h-1 (SD = 1.31) to treatment temperatures and mean holding temperatures of 18.0°C (SD = 0.2) and 21.0°C (SD = 0.2) in the low- and high-heat treatments, respectively. Our work demonstrated that manipulative experiments with large, mobile study species can be successfully developed in remote locations to examine thermal stress.

Chinook salmon (Oncorhynchus tshawytscha) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3′-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted.

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus . We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012–2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

We examined the associations between California sea lion MHC class II DRB (Zaca-DRB) configuration and diversity, and leptospirosis. As Zaca-DRB gene sequences are involved with antigen presentation of bacteria and other extracellular pathogens, we predicted that they would play a role in determining responses to these pathogenic spirochaetes. Specifically, we investigated whether Zaca-DRB diversity (number of genes) and configuration (presence of specific genes) explained differences in disease severity, and whether higher levels of Zaca-DRB diversity predicted the number of specific Leptospira interrogans serovars that a sea lion's serum would react against. We found that serum from diseased sea lions with more Zaca-DRB loci reacted against a wider array of serovars. Specific Zaca-DRB loci were linked to reactions with particular serovars. Interestingly, sea lions with clinical manifestation of leptospirosis that had higher numbers of Zaca-DRB loci were less likely to recover from disease than those with lower diversity, and those that harboured Zaca-DRB.C or -G were 4.5 to 5.3 times more likely to die from leptospirosis, regardless of the infective serovars. We propose that for leptospirosis, a disadvantage of having a wider range of antigen presentation might be increased disease severity due to immunopathology. Ours is the first study to examine the importance of Zaca-DRB diversity for antigen detection and disease severity following natural exposure to infective leptospires.

Coastal regions worldwide face increasing management concerns due to natural and anthropogenic forces that have the potential to significantly degrade nearshore marine resources. The goal of our study was to develop and test a monitoring strategy for nearshore marine ecosystems in remote areas that are not readily accessible for sampling. Mussel species have been used extensively to assess ecosystem vulnerability to multiple, interacting stressors. We sampled bay mussels (Mytilus trossulus) in 2015 and 2016 from six intertidal sites in Lake Clark and Katmai National Parks and Preserves, in south-central Alaska. Reference ranges for physiological assays and gene transcription were determined for use in future assessment efforts. Both techniques identified differences among sites, suggesting influences of both large-scale and local environmental factors and underscoring the value of this combined approach to ecosystem health monitoring.

Populations of wildlife species worldwide experience incidents of mass morbidity and mortality. Primary or secondary drivers of these events may escape classical detection methods for identifying microbial insults, toxin exposure, or additional stressors. In 2012, 28% of polar bears sampled in a study in the southern Beaufort Sea region of Alaska had varying degrees of alopecia that was concomitant with reduced body condition. Concurrently, elevated numbers of sick or dead ringed seals were detected in the southern Beaufort, Chukchi, and Bering seas in 2012, resulting in the declaration of an unusual mortality event (UME) by the National Oceanic and Atmospheric Administration (NOAA). The primary and possible ancillary causative stressors of these events are unknown, and related physiological changes within individual animals have been undetectable using classical diagnostic methods. Here we present an emerging technology as a potentially guiding investigative approach aimed at elucidating the circumstances responsible for the susceptibility of certain polar bears to observed conditions. Using transcriptomic analysis we identified enhanced biological processes including immune response, viral defense, and response to stress in polar bears with alopecia. Our results support an alternative mechanism of investigation into the causative agents that, when used proactively, could serve as an early indicator for populations and species at risk. We suggest that current or classical methods for investigation into events of unusual morbidity and mortality can be costly, sometimes unfocused, and often inconclusive. Advances in technology allow for implementation of a holistic system of surveillance and investigation that could provide early warning of health concerns in wildlife species important to humans.

The analysis of blood constituents is a widely used tool to aid in monitoring of animal health and disease. However, classic blood diagnostics (i.e. hematologic and plasma biochemical values) often do not provide sufficient information to determine the state of an animal’s health. Field studies on wild tortoises and other reptiles have had limited success in drawing significant inferences between blood diagnostics and physiological and immunological condition. However, recent research using gene transcription profiling in the threatened Mojave desert tortoise (Gopherus agassizii) has proved useful in identifying immune or physiologic responses and overall health. To improve our understanding of health and immune function in tortoises, we evaluated both standard blood diagnostic (body condition, hematologic, plasma biochemistry values, trace elements, plasma proteins, vitamin A levels) and gene transcription profiles in 21 adult tortoises (11 clinically abnormal; 10 clinically normal) from Clark County, NV, USA. Necropsy and histology evaluations from clinically abnormal tortoises revealed multiple physiological complications, with moderate to severe rhinitis or pneumonia being the primary cause of morbidity in all but one of the examined animals. Clinically abnormal tortoises had increased transcription for four genes (SOD, MyD88, CL and Lep), increased lymphocyte production, biochemical enzymes and organics, trace elements of copper, and decreased numbers of leukocytes. We found significant positive correlations between increased transcription for SOD and increased trace elements for copper, as well as genes MyD88 and Lep with increased inflammation and microbial insults. Improved methods for health assessments are an important element of monitoring tortoise population recovery and can support the development of more robust diagnostic measures for ill animals, or individuals directly impacted by disturbance.

Increasingly, population- and ecosystem-level health assessments are performed using sophisticated molecular tools. Advances in molecular technology enable the identification of synergistic effects of multiple stressors on the individual physiology of different species. Brown bears (Ursus arctos) are an apex predator; thus, they are ideal candidates for detecting potentially ecosystem-level systemic perturbations using molecular-based tools. We used gene transcription to analyze 130 brown bear samples from three National Parks and Preserves in Alaska. Although the populations we studied are apparently stable in abundance and exist within protected and intact environments, differences in transcript profiles were noted. The most prevalent differences were among locations. The transcript patterns among groups reflect the influence of environmental factors, such as nutritional status, disease, and xenobiotic exposure. However, these profiles also likely represent baselines for each unique environment by which future measures can be made to identify early indication of population-level changes due to, for example, increasing Arctic temperatures. Some of those environmental changes are predicted to be potentially positive for brown bears, but other effects such as the manifestation of disease or indirect effects of oceanic acidification may produce negative impacts.

Habitat disturbance from development, resource extraction, off-road vehicle use, and energy development ranks highly among threats to desert systems worldwide. In the Mojave Desert, United States, these disturbances have promoted the establishment of nonnative plants, so that native grasses and forbs are now intermixed with, or have been replaced by invasive, nonnative Mediterranean grasses. This shift in plant composition has altered food availability for Mojave Desert tortoises (Gopherus agassizii), a federally listed species. We hypothesized that this change in forage would negatively influence the physiological ecology, immune competence, and health of neonatal and yearling tortoises. To test this, we monitored the effects of diet on growth, body condition, immunological responses (measured by gene transcription), and survival for 100 captive Mojave tortoises. Tortoises were assigned to one of five diets: native forbs, native grass, invasive grass, and native forbs combined with either the native or invasive grass. Tortoises eating native forbs had better body condition and immune functions, grew more, and had higher survival rates (>95%) than tortoises consuming any other diet. At the end of the experiment, 32% of individuals fed only native grass and 37% fed only invasive grass were found dead or removed from the experiment due to poor body conditions. In contrast, all tortoises fed either the native forb or combined native forb and native grass diets survived and were in good condition. Health and body condition quickly declined for tortoises fed only the native grass (Festuca octoflora) or invasive grass (Bromus rubens) with notable loss of fat and muscle mass and increased muscular atrophy. Bromus rubens seeds were found embedded in the oral mucosa and tongue in most individuals eating that diet, which led to mucosal inflammation. Genes indicative of physiological, immune, and metabolic functions were transcribed at lower levels for individuals fed B. rubens, indicating potential greater susceptibility to disease or other health-related problems. This study highlights the negative indirect effects of invasive grasses, such as red brome, in desert ecosystems, and provides definitive evidence of a larger negative consequence to health, survival, and ultimately population recruitment for Mojave Desert tortoises than previously understood.

Using a panel of genes stimulated by oil exposure in a laboratory study, we evaluated gene transcription in blood leukocytes sampled from sea otters captured from 2006–2012 in western Prince William Sound (WPWS), Alaska, 17–23 years after the 1989 Exxon Valdez oil spill (EVOS). We compared WPWS sea otters to reference populations (not affected by the EVOS) from the Alaska Peninsula (2009), Katmai National Park and Preserve (2009), Clam Lagoon at Adak Island (2012), Kodiak Island (2005) and captive sea otters in aquaria. Statistically, sea otter gene transcript profiles separated into three distinct clusters: Cluster 1, Kodiak and WPWS 2006–2008 (higher relative transcription); Cluster 2, Clam Lagoon and WPWS 2010–2012 (lower relative transcription); and Cluster 3, Alaska Peninsula, Katmai and captive sea otters (intermediate relative transcription). The lower transcription of the aryl hydrocarbon receptor (AHR), an established biomarker for hydrocarbon exposure, in WPWS 2010–2012 compared to earlier samples from WPWS is consistent with declining hydrocarbon exposure, but the pattern of overall low levels of transcription seen in WPWS 2010–2012 could be related to other factors, such as food limitation, pathogens or injury, and may indicate an inability to mount effective responses to stressors. Decreased transcriptional response across the entire gene panel precludes the evaluation of whether or not individual sea otters show signs of exposure to lingering oil. However, related studies on sea otter demographics indicate that by 2012, the sea otter population in WPWS had recovered, which indicates diminishing oil exposure.

We measured biomass and frequency of herbaceous species before and 12 years after removal of feral sheep from a grassland on Santa Cruz Island, California. Native grasses, especially Stipa spp., increased dramatically following sheep removal whereas all exotic grasses except Avena spp. decreased, probably reflecting the competitive ability of native grasses as well as differential vulnerability to grazing. Native forbs showed a mixed response, with some species increasing but many decreasing, perhaps because some native forbs benefit from grazing. Almost all exotic forbs decreased, likely because these species benefit from herbivores and their activities. Our results provide encouraging evidence that an island grassland dominated by exotic species has the potential for recovery from severe overgrazing.

SummaryReal-time quantitation of cytokine mRNA is a routine immunologic technique, especiallyfitting for use in those species for which monoclonal antibodies are not available.Quantitative gene expression assays were developed to assist in the immunologicassessment of three cetacean species including bottlenosed dolphins, Pacific white-sideddolphins and beluga whales. Nine cytokine genes (IL-2, -4, -10, -12, -13, -18, TNFa, TGFband IFNg) and Cox-2 were selected for analysis. Most mitogen-induced mononuclearleukocyte responses were similar between the three cetacean species with either up- ordown-regulation of cytokine genes. IL-10 expression was highly variable between species.No T H /1T H 2 polarization was evident. Cytokine gene analysis has the potential to identifyimmune system perturbations induced by environmental insult as well as providingdiagnostic tools for characterizing immune responses to environmental antigens andvaccines.& 2008 Elsevier Ltd. All rights reserved. Introduction

Free-ranging sea otters are subject to hydrocarbon exposure from a variety of sources, both natural and anthropogenic. Effects of direct exposure to unrefined crude oil, such as that associated with the Exxon Valdez oil spill, are readily apparent. However, the impact of subtle but pathophysiologically relevant concentrations of crude oil on sea otters is difficult to assess. The present study was directed at developing a model for assessing the impact of low concentrations of fuel oil on sea otters. Quantitative PCR was used to identify differential gene expression in American mink that were exposed to low concentrations of bunker C fuel oil. A total of 23 genes, representing 10 different physiological systems, were analyzed for perturbation. Six genes with immunological relevance were differentially expressed in oil-fed mink. Interleukin-18 (IL-18), IL-10, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and complement cytolysis inhibitor (CLI) were down-regulated while IL-2 was up-regulated. Expression of two additional genes was affected; heat shock protein 70 (HSP70) was up-regulated and thyroid hormone receptor (THR) was down-regulated. While the significance of each perturbation is not immediately evident, we identified differential expression of genes that would be consistent with the presence of immune system-modifying and endocrine-disrupting compounds in fuel oil. Application of this approach to identify effects of petroleum contamination on sea otters should be possible following expansion of this mink model to identify a greater number of affected genes in peripheral blood leukocytes.

Characterization of the polygenic and polymorphic features of the Steller sea lion major histo- compatibility complex (MHC) provides an ideal window for evaluating immunologic vigor of the population and identifying emergence of new genotypes that reflect ecosystem pressures. MHC genotyping can be used to measure the potential immunologic vigor of a population. However, since ecosystem-induced changes to MHC genotype can be slow to emerge, measurement of differential expression of these genes can potentially provide real-time evidence of immunologic perturbations. MHC DRB genes were cloned and sequenced using peripheral blood mononuclear leukocytes derived from 10 Steller sea lions from Southeast Alaska, Prince William Sound, and the Aleutian Islands. Nine unique DRB gene sequences were represented in each of 10 animals. MHC DRB gene expression was measured in a subset of six sea lions. Although DRB in genomic DNA was identical in all individuals, relative levels of expressed DRB mRNA was highly variable. Selective sup- pression of MHC DRB genes could be indicative of geographically disparate environmental pressures, thereby serving as an immediate and sensitive indicator of population and ecosystem health.

Pacific salmon species including the U.S. federally endangered coho salmon (Oncorhynchus kisutch) and the U.S. federally threatened steelhead trout (Oncorhynchus mykiss) have declined at an alarming rate in the last 40 years. Two of the main causes for the decline in coastal coho populations include increases in temperature and contaminant loads in coastal watersheds. Zinc, in particular, is one of the most common contaminants in aquatic systems. Using an experimental mesocosm design, we examined physiological, biochemical, and behavioral responses of coho salmon to excess dietary zinc and increased temperatures, with the ultimate goal of relating results to wild populations of coho salmon and steelhead in the Navarro River, California. Fish were obtained from a hatchery and divided into four treatments: low water temperature-no dietary zinc, high temperature-no zinc, low temperature-zinc, and high temperature-zinc. Each treatment had four replicate tanks. Zinc concentrations in liver increased during exposure to a high zinc diet. Iron concentrations in liver increased during simultaneous exposure to high zinc diet and increased temperature, and growth was reduced in this experimental treatment. Expression of hsp-70 was not significantly different between treatments, but showed decreasing trends with high dietary zinc and high temperature. Feeding rate increased with exposure to a high zinc diet. Comparison with steelhead trout samples from the Navarro River, California, showed levels of zinc, iron, and hsp-70 greater than those found in the experimental Coho salmon. All comparisons between the hatchery coho salmon and wild steelhead should be viewed with caution due to the differences between species, the laboratory and natural environment, and the genetic differences between wild and hatchery fish.

The ectoparasite fauna for island foxes (Urocyon littoralis) on Santa Cruz Island (California, USA) in April (wet season) and September (dry season) 1998 was evaluated. Three taxa of ectoparasites were identified. These were fleas (Pulex irritans), lice (Neotrichodectes mephitidis), and ticks (Ixodes pacificus). Ectoparasite abundances varied seasonally. Typical of insular endemic species, island foxes may be especially vulnerable to the introduction of novel disease organisms and their vectors.

Islands are of special conservation importance because island ecosystems typically support unique biotic communities rich in endemic taxa (Mueller-Dombois 1981). Species endemic to islands, however, have suffered a disproportionate share of extinctions worldwide since 1600 (Atkinson 1989; Olson 1989; Reid & Miller 1989; Case et al. 1992). Exotic predators and herbivores introduced by humans are believed to be a major cause of extinctions on islands; herbivorous mammals in particular cause severe damage when introduced to island ecosystems (Vitousek 1988; Atkinson 1989). Insular endemic plants are believed to be vulnerable to introductions of herbivores because mammalian herbivores were absent from most islands until the arrival of humans (Atkinson 1989). Most plants have defenses against herbivory (Marquis 1991). Plants evolve defenses in direct proportion to the risk of herbivory (Rhoades 1979; Marquis 1991); because defenses are costly, production decreases when herbivores are absent (Rhoades 1979). Consequently, insular endemic plants that evolved in the absence of large mammalian herbivores may lack defenses against herbivory (Thorne 1969; Carlquist 1974; Coblentz 1978). This hypothesis, although widely accepted (Vitousek 1988; Atkinson 1989; Primack 1993; Paulay 1994), has never been tested. Santa Cruz Island (249 km2), 30 km from mainland southern California, is the largest of the eight California Channel Islands. Santa Cruz supports 650 plant taxa organized into 10 or more communities (Junak et al. 1995). Overall, the Santa Cruz Island flora is similar to nearby species and communities on the mainland. Fortyfive island taxa, however, are endemic to the Channel Islands and eight are endemic to Santa Cruz Island (Junak et al. 1995). Endemic plants on Santa Cruz exhibit both

Tortoises are susceptible to a wide variety of environmental stressors, and the influence of human disturbances on health and survival of tortoises is difficult to detect. As an addition to current diagnostic methods for desert tortoises, we have developed the first leukocyte gene transcription biomarker panel for the desert tortoise (Gopherus agassizii), enhancing the ability to identify specific environmental conditions potentially linked to declining animal health. Blood leukocyte transcript profiles have the potential to identify physiologically stressed animals in lieu of clinical signs. For desert tortoises, the gene transcript profile included a combination of immune or detoxification response genes with the potential to be modified by biological or physical injury and consequently provide information on the type and magnitude of stressors present in the animal's habitat. Blood from 64 wild adult tortoises at three sites in Clark County, NV, and San Bernardino, CA, and from 19 captive tortoises in Clark County, NV, was collected and evaluated for genes indicative of physiological status. Statistical analysis using a priori groupings indicated significant differences among groups for several genes, while multidimensional scaling and cluster analyses of transcription C T values indicated strong differentiation of a large cluster and multiple outlying individual tortoises or small clusters in multidimensional space. These analyses highlight the effectiveness of the gene panel at detecting environmental perturbations as well as providing guidance in determining the health of the desert tortoise.

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a 'reference' range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell-cell adhesion. The cycle threshold (C(T)) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.