Scanning Electron Microscopy and EDX Spectroscopy of Commercial Swabs Used for COVID-19 Lateral Flow Testing.

The chemical composition of COVID test swabs has not been examined beyond the manufacturer's datasheets. The unprecedented demand for swabs to conduct rapid lateral flow tests and nucleic acid amplification tests led to mass production, including 3D printing platforms. Manufacturing impurities could be present in the swabs and, if so, could pose a risk to human health. We used scanning electron microscopy and energy dispersive X-ray (EDX) spectroscopy to examine the ultrastructure of seven assorted brands of COVID test swabs and to identify and quantify their chemical elements. We detected eight unexpected elements, including transition metals, such as titanium and zirconium, the metalloid silicon, as well as post-transition metals aluminium and gallium, and the non-metal elements sulphur and fluorine. Some of the elements were detected as trace amounts, but for others, the amount was close to reported toxicological thresholds for inhalation routes. Experimental studies have shown that the detrimental effects of unexpected chemical elements include moderate to severe inflammatory states in the exposed epithelium as well as proliferative changes. Given the massive testing still being used in the context of the COVID pandemic, we urge caution in continuing to recommend repeated and frequent testing, particularly of healthy, non-symptomatic, individuals.

Bacterial dysbiosis and epithelial status of the California sea lion (Zalophus californianus) in the Gulf of California.

Despite the high incidence of urogenital carcinoma (UGC) in California sea lions stranded along California, no UGC has been reported in other areas of their distribution; however, cell morphologies typical of premalignant states have been found. Risk factors for UGC include high of organochlorines and infection with a gammaherpesvirus, OtHV-1, but the importance of the bacteriome for epithelial status remains unknown. We characterized the genital bacteriome of adult female California sea lions along their distribution in the Gulf of California and examined whether the diversity and abundance of the bacteriome varied spatially, whether there were detectable differences in the bacteriome between healthy and altered epithelia, and whether the bacteriome was different in California sea lions infected with OtHV-1 or papillomavirus. We detected 2270 ASVs in the genital samples, of which 35 met the criteria for inclusion in the core bacteriome. Fusobacteriia and Clostridia were present in all samples, at high abundances, and Actinobacteria, Alphaproteobacteria, and Campylobacteria were also well-represented. Alpha diversity and abundance of the California sea lion genital bacteriome varied geographically. The abundance of bacterial ASVs varied depending on the genital epithelial status and inflammation, with differences driven by classes Fusobacteriia, Clostridia, Campylobacteria and Alphaproteobacteria. Alpha diversity and abundance were lowest in samples in which OtHV-1 was detected, and highest those with papillomavirus. Our study is the first investigation of how the bacteriome is related to epithelial status in a wild marine species prone to developing cancer.

Phylogenomic Discordance in the Eared Seals is best explained by Incomplete Lineage Sorting following Explosive Radiation in the Southern Hemisphere.

The phylogeny and systematics of fur seals and sea lions (Otariidae) have long been studied with diverse data types, including an increasing amount of molecular data. However, only a few phylogenetic relationships have reached acceptance because of strong gene-tree species tree discordance. Divergence times estimates in the group also vary largely between studies. These uncertainties impeded the understanding of the biogeographical history of the group, such as when and how trans-equatorial dispersal and subsequent speciation events occurred. Here, we used high-coverage genome-wide sequencing for 14 of the 15 species of Otariidae to elucidate the phylogeny of the family and its bearing on the taxonomy and biogeographical history. Despite extreme topological discordance among gene trees, we found a fully supported species tree that agrees with the few well-accepted relationships and establishes monophyly of the genus Arctocephalus. Our data support a relatively recent trans-hemispheric dispersal at the base of a southern clade, which rapidly diversified into six major lineages between 3 and 2.5 Ma. Otaria diverged first, followed by Phocarctos and then four major lineages within Arctocephalus. However, we found Zalophus to be nonmonophyletic, with California (Zalophus californianus) and Steller sea lions (Eumetopias jubatus) grouping closer than the Galapagos sea lion (Zalophus wollebaeki) with evidence for introgression between the two genera. Overall, the high degree of genealogical discordance was best explained by incomplete lineage sorting resulting from quasi-simultaneous speciation within the southern clade with introgresssion playing a subordinate role in explaining the incongruence among and within prior phylogenetic studies of the family. [Hybridization; ILS; phylogenomics; Pleistocene; Pliocene; monophyly.].

The demographic decline of a sea lion population followed multi-decadal sea surface warming.

The population growth of top predators depends largely on environmental conditions suitable for aggregating sufficient and high-quality prey. We reconstructed numerically the size of a resident population of California sea lions in the Gulf of California during 1978-2019 and its relation with multi-decadal sea surface temperature anomalies. This is the first multi-decadal examination of the sea surface temperature of the Gulf of California and of one of its major predators. A three-decade sustained warming explained the population's trend accounting for 92% of the variance, including a 65% decline between 1991 and 2019. Long-term warming conditions started in the late 80s, followed by the population's decline from 43,834 animals (range 34,080-58,274) in 1991 to only 15,291 (range 11,861-20,316) in 2019. The models suggested a century-scale optimum sea surface habitat occurring in mildly temperate waters, from 0.18 to 0.39 °C above the 100-year mean. The mechanistic links of this relation are still untested, but apparent diversification of pelagic fish catches suggests a reduction of high quality prey. We propose this population should be considered vulnerable to any disturbance that could add to the negative effects of the current warm sea surface conditions in the Gulf of California.

Gut Bacterial Families Are Associated with Body Composition and Metabolic Risk Markers in School-Aged Children in Rural Mexico.

Background: Differences in gut microbiota composition have been associated with obesity and metabolic alterations in children. The aim of this study was to analyze the abundance of the main bacterial families of the gut among children according to their body composition and metabolic markers. Methods: A cross-sectional study was conducted with 93 school-aged children (8.4 ± 1.6 years old). Anthropometric and body composition variables were measured and a blood sample was collected to determine glucose, insulin, lipid profile, C-reactive protein, leptin, and cytokines [interleukin 6, interleukin 10 (IL-10), tumor necrosis factor α (TNFα)]. DNA was extracted from stool samples and the abundance of bacterial families (Bacteroidaceae-Porphyromonadaceae-Prevotellaceae, Lactobacillaceae, Enterococcaceae, and Lachnospiraceae-Ruminococcaceae) was determined by qPCR assays. Results: Children with obesity and high waist/height ratio had lower Bacteroidaceae-Porphyromonadaceae-Prevotellaceae and higher abundance of Lactobacillaceae when compared with normal-weight children. TNFα was negatively associated and IL-10 was positively associated with Bacteroidaceae-Porphyromonadaceae-Prevotellaceae. Triglycerides showed a positive relationship with Lachnospiraceae-Ruminococcaceae whereas high-density lipoprotein-cholesterol was negatively associated with Lactobacillaceae. Conclusion: In rural Mexican school-aged children, a low abundance of Bacteroidaceae-Porphyromonadaceae-Prevotellaceae and a high abundance of Lactobacillaceae are associated with obesity and metabolic disturbances.

Filarial infections in California sea lions vary spatially within the Gulf of California, Mexico.

At least two species of filarial worms, Dirofilaria immitis and Acanthocheilonema (Dipetalonema) odendhali, infect otariid pinnipeds, including the California sea lion (Zalophus californianus). To date, evidence of infection in sea lions has come from dead or captive animals, and little is known about filariasis in free-living populations. We sampled 45 California sea lion adults and 197 pups captured at 12 rookeries from different ecological regions within the Gulf of California and detected and quantified D. immitis and A. odendhali microfilariae in blood smears. We investigated differences in prevalence and parasite load (intensity of infection) among ecological regions. Microfilariae were detected in the blood of 35 of the 45 (77.78%) adult females and in 1 of the 197 (0.51%) pups examined. The average burden of A. odendhali per microlitre of blood was nearly twice that of D. immitis. Prevalence and intensity of infection differed significantly among regions, being highest for colonies within the northern and northcentral regions and lowest in the southern region. Dirofilaria immitis and A. odendhali infections displayed a similar spatial pattern of prevalence. Colony density inversely predicted the prevalence of microfilariae. Based on the clinical parameters typically associated with filarial infections in carnivores and physical examinations, none of the sea lions appeared to have evidence of disease. This is a first approximation to investigate the prevalence of microfilaria infections in free-ranging California sea lions and to explore their relevance to population health.

Does sociality drive diversity and composition of airway microbiota in cetaceans?

The number of social contacts of mammals is positively correlated with the diversity of their gut microbes. There is some evidence that sociality also affects microbes in the respiratory tract. We tested whether the airway microbiota of cetacean species differ depending on the whales' level of sociality. We sampled the blow of blue (Balaenoptera musculus), grey (Eschrichtius robustus), humpback (Megaptera novaeangliae) and long-finned pilot whales (PWs) (Globicephala melas) and analysed the blow microbiota by barcode tag sequencing targeting the V4 region of the bacterial 16S rRNA gene. Humpback whales (HWs) show higher levels of sociality than blue (BW) and grey (GW), while PWs are the most gregarious among the four species. The blow samples of the HWs showed the highest richness and diversity. HWs were also the only species with a species-specific clustering of their microbial community composition and a relatively large number of core taxa. Therefore, we conclude that it cannot be sociality alone shaping the diversity and composition of airway microbiota. We suggest the whale species' lung volume and size of the plume of exhaled air as an additional factor impacting the transmission potential of blow microbiota from one individual whale to another.

Early sexual dimorphism in the developing gut microbiome of northern elephant seals.

The gut microbiome is an integral part of a species' ecology, but we know little about how host characteristics impact its development in wild populations. Here, we explored the role of such intrinsic factors in shaping the gut microbiome of northern elephant seals (Mirounga angustirostris) during a critical developmental window of 6 weeks after weaning, when the pups stay ashore without feeding. We found substantial sex differences in the early-life gut microbiome, even though males and females could not yet be distinguished morphologically. Sex and age both explained around 15% of the variation in gut microbial beta diversity, while microbial communities sampled from the same individual showed high levels of similarity across time, explaining another 40% of the variation. Only a small proportion of the variation in beta diversity was explained by health status, assessed by full blood counts, but clinically healthy individuals had a greater microbial alpha diversity than their clinically abnormal peers. Across the post-weaning period, the northern elephant seal gut microbiome was highly dynamic. We found evidence for several colonization and extinction events as well as a decline in Bacteroides and an increase in Prevotella, a pattern that has previously been associated with the transition from nursing to solid food. Lastly, we show that genetic relatedness was correlated with gut microbiome similarity in males but not females, again reflecting early sex differences. Our study represents a naturally diet-controlled and longitudinal investigation of how intrinsic factors shape the early gut microbiome in a species with extreme sex differences in morphology and life history.

Physiological Thresholds in the Context of Marine Mammal Conservation.

There is growing evidence of the adverse effects of global environmental change on marine mammals, particularly in terms of changes in abundance, distribution, habitat use, migratory phenology, feeding habits, risk of infectious diseases, bioaccumulation of contaminants, declines in reproductive success, and reductions in genetic diversity. These anthropogenic stressors have led to an evident conservation crisis: a quarter of the extant marine mammal species that have been assessed and now considered at risk of extinction. However, we still know very little about the subtle, subclinical effects of environmental stressors on various aspects of physiology that could impacts their survival and long-term fertility, or that could impact the performance of future generations. This chapter argues for the need of considering physiological thresholds when examining how drivers of global environmental change can impact marine mammal populations. It also poses questions regarding our understanding of individual phenotypic plasticity and resilience of species in the face of environmental stressors, particularly under abrupt and unpredictable environmental changes. Specifically, I present evidence of how drivers of environmental change can exert effects at different levels of biological organization to influence the viability of individuals and populations, and discuss which aspects of environmental change could be the most likely to shift the physiological thresholds of different marine mammal species. Finally, the chapter identifies areas of further research in marine mammal conservation physiology in the current context of global environmental change.

Transcriptional Profiles of California Sea Lion Peripheral NK and CD+8 T Cells Reflect Ecological Regionalization and Infection by Oncogenic Viruses.

The California sea lion is one of the few wild mammals prone to develop cancer, particularly urogenital carcinoma (UGC), whose prevalence is currently estimated at 25% of dead adult sea lions stranded along the California coastline. Genetic factors, viruses and organochlorines have been identified as factors that increase the risk of occurrence of this pathology. Given that no cases of UGC have as yet been reported for the species along its distribution in Mexican waters, the potential relevance of contaminants for the development of urogenital carcinoma is highlighted even more as blubber levels of organochlorines are more than two orders of magnitude lower in the Gulf of California and Mexican Pacific than in California. In vitro studies have shown that organochlorines can modulate anti-viral and tumor-surveillance activities of NK and cytotoxic T-cells of marine mammals, but little is known about the activity of these effectors in live, free-living sea lions. Here, we examine leukocyte transcriptional profiles of free-ranging adult California sea lions for eight genes (Eomes, Granzyme B, Perforin, Ly49, STAT1, Tbx21, GATA3, and FoxP3) selected for their key role in anti-viral and tumor-surveillance, and investigate patterns of transcription that could be indicative of differences in ecological variables and exposure to two oncogenic viruses: sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1) and systemic inflammation. We observed regional differences in the expression of genes related to Th1 responses and immune modulation, and detected clear patterns of differential regulation of gene expression in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus or for simultaneous infections, similar to what is known about herpesvirus and papillomavirus infections in humans. Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions and their association with ecological regions and oncogenic viruses. The observed results add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution.

Hepatic DNA damage in harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines.

One level at which persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons PAHs) can exert damage is by causing DNA strand-breaks or nucleotide base modifications, which, if unrepaired, can lead to embryonic mutations, abnormal development and cancer. In marine ecosystems, genotoxicity is expected to be particularly strong in long-lived apex predators due to pollutant bioaccumulation. We conducted 32 P-postlabeling analyses optimized for the detection and quantification of aromatic/hydrophobic DNA adducts in the livers of 40 sexually-mature North Atlantic harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines. We examined hepatic tissue to search for inflammatory and preneoplastic lesions and examine their association with adduct levels. Adducts were found in all porpoises (mean: 17.56 ± 11.95 per 108 nucleotides), and were higher than levels reported for marine vertebrates from polluted sites. The pollutants causing the induced DNA adducts could not be further characterized. Hepatic DNA damage did not correlate with levels of blubber POP concentrations (including total polychlorinated biphenyl [PCBs], dichlorodiphenyltrichloroethane [DDT] and dieldrin); PAH concentrations were not available for the present study. However, DNA damage predicted occurrence of inflammatory and preneoplastic lesions. Further, our data showed a reduction in hepatic DNA adduct levels with age in the 40 animals examined while POP concentrations, particularly PCBs, increased with age. Using a different dataset of 145 mature male harbour porpoises confirmed that higher contaminant levels (total PCBs, DDT and dieldrin) are found in older animals. The reduction in hepatic DNA adduct levels in older animals was in accordance with other studies which show that suppression of hepatic CYP1A enzyme activity at high PCB concentrations might impact on CYP1A-mediated DNA adduct formation of PAHs which are ubiquitous environmental pollutants and readily metabolized by CYP1A to species binding to DNA. In summary, our study shows that pollutant-induced DNA damage is prevalent in harbour porpoises from UK waters and may lead to detectable sub-lethal hepatic damage. Environ. Mol. Mutagen. 59:613-624, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

MHC class II DRB diversity predicts antigen recognition and is associated with disease severity in California sea lions naturally infected with Leptospira interrogans.

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.

Atypical Red Blood Cells Are Prevalent in California Sea Lion Pups Born during Anomalous Sea Surface Temperature Events.

To date, there is limited knowledge of the effects that abnormal sea surface temperature (SST) can have on the physiology of neonate pinnipeds. However, maternal nutritional deficiencies driven by alimentary restrictions would expectedly impact pinniped development and fitness, as an adequate supply of nutrients is essential for growth and proper functioning of all body systems, including red blood cell synthesis and clearance. Here, we investigated red blood cell morphology of California sea lion (CSL) pups from the San Benito Archipelago born during the 2014 and 2015 anomalously high SST events recorded in the northeastern Pacific Ocean. We examined whether atypical erythrocyte morphologies were more common in 2015, when the high SST event was more pronounced, and whether the stable isotope signature of pup fur, as an indicator of maternal feeding strategies, accounted for the number of atypical cells. Various atypical erythrocyte morphologies were more prevalent and more abundant than reference values. Evidence of iron deficiency was found in both years, and only pups born in 2014 showed evidence of active erythropoiesis. Microcytes and reticulocytes were more common in pups with higher isotopic δ13C and lower δ15N values, suggesting a probable relationship between maternal feeding strategies and the effect of climatic anomalies on red blood cell physiology of their pups. As developing pinnipeds require increased oxygen storage capacity for diving and foraging, the presence of atypical erythrocytes could be relevant to CSL pup fitness if the underlying cause is not reverted. This study is a first step to explore the effects that climatic alterations in the marine environment can have on the blood physiology of developing individuals.

Climatic anomaly affects the immune competence of California sea lions.

The past decades have been characterized by a growing number of climatic anomalies. As these anomalies tend to occur suddenly and unexpectedly, it is often difficult to procure empirical evidence of their effects on natural populations. We analysed how the recent sea surface temperature (SST) anomaly in the northeastern Pacific Ocean affects body condition, nutritional status, and immune competence of California sea lion pups. We found that pup body condition and blood glucose levels of the pups were lower during high SST events, although other biomarkers of malnutrition remained unchanged, suggesting that pups were experiencing early stages of starvation. Glucose-dependent immune responses were affected by the SST anomaly; specifically, pups born during high SST events had lower serum concentrations of IgG and IgA, and were unable to respond to an immune challenge. This means that not only were pups that were born during the SST anomaly less able to synthesize protective antibodies; they were also limited in their ability to respond rapidly to nonspecific immune challenges. Our study provides empirical evidence that atypical climatic conditions can limit energetic reserves and compromise physiological responses that are essential for the survival of a marine top predator.

Melanin granules melanophages and a fully-melanized epidermis are common traits of odontocete and mysticete cetaceans.

BACKGROUND: The cellular mechanisms used to counteract or limit damage caused by exposure of marine vertebrates to solar ultraviolet (UV) radiation are poorly understood. Cetaceans are vulnerable because they lack protective skin appendages and are obliged to surface continuously to breathe, thus being exposed repeatedly to UV light. Although molecular mechanisms of photoprotection of cetaceans have been studied, there is limited knowledge about their epidermal structure and photoprotective effectors. OBJECTIVE: To describe and compare the epidermis of mysticete and odontocete cetaceans and identify potentially photoprotective traits. ANIMALS: Twenty eight free-living individuals belonging to six cetacean species were sampled in the Mexican Central Pacific and Gulf of California. Species sampled were the bottlenose dolphin, pantropical spotted dolphin, spinner dolphin, Bryde's whale, fin whale and humpback whale. METHODS: Histological and cytological evaluation of skin biopsy tissue collected in the field between 2014 and 2016. RESULTS: All cetaceans had only three epidermal layers, lacking both the stratum granulosum and stratum lucidum. A relatively thick stratum corneum with a parakeratosis-like morphology was noted. Melanin was observed within keratinocytes in all epidermal layers, including the stratum corneum and apical melanin granules obscured the keratinocyte nucleus. Keratinocytes had a perinuclear halo. Keratinocyte diameter differed between cetacean suborders and amongst species. Melanophage clusters were common in most cetacean species. CONCLUSIONS: The widespread presence of melanin and the unexpectedly high number of melanophages may constitute a unique photoprotective trait of cetaceans and could reflect primitive adaptations to their environment and to their obligate marine-bound life.

MHC class II transcription is associated with inflammatory responses in a wild marine mammal.

Inflammation is one of the most important non-specific and rapid responses that a vertebrate can elicit in response to damage or a foreign insult. To date, despite increasing evidence that the innate and adaptive branches of immunity are more intricately related than previously thought, few have examined interactions between the Major Histocompatibility Complex (MHC, a polymorphic region of the vertebrate genome that is involved with antigen presentation) and inflammation, and even less is known about these interactions in an eco-immunological context. Here, we examined the effect of MHC class II DRB gene multiplicity and transcription on phytohemagglutinin (PHA)-induced inflammation during the early stages of development of California sea lions. Neither constitutive nor expressed ZacaDRB diversity was found to be associated with pup responses to PHA at any of the stages of pup development. However, for two-month-old pups, those with a specific MHC-DRB locus (ZacaDRB-A) tended to have less efficient responsive inflammation. Transcription of distinct MHC-DRB loci was also linked to PHA-induced inflammation, with patterns that varied markedly between ages, and that suggested that ongoing infectious processes could limit the capacity to respond to a secondary challenge. Life history constraints and physiological processes associated with development of California sea lions, in conjunction with their changing pathogenic environment could explain the observed effects of MHC class II transcription on PHA-induced inflammation. To our knowledge, ours is the first study to examine the importance of expressed vs. constitutive MHC loci on inflammation in a natural population.

Impact of the 2015 El Niño-Southern Oscillation on the Abundance and Foraging Habits of Guadalupe Fur Seals and California Sea Lions from the San Benito Archipelago, Mexico.

The abundance of California sea lions (Zalophus californianus) (CSLs) and Guadalupe fur seals (Arctocephalus philippii townsendi) (GFSs) from the San Benito Archipelago (SBA) was determined through nine monthly surveys in 2014-2015. Assessment of their foraging habits was examined based on the isotopic analysis of pups (maternal indicators) (SIAR/SIBER-R). Environmental variability between 2014 and 2015 was also analyzed, in terms of sea surface temperature (SST) and chlorophyll (Chl-a) concentration. Both otariids reached their highest abundance in July of both years; however, relative to 2014, the 2015 survey showed a 59.7% decline in the total GFS abundance and a 42.9% decrease of GFS pups, while total CSL abundance decreased 52.0% and CSL pup presence decreased in 61.7%. All monthly surveys for both otariids showed a similar trend (>50% decrease in 2015). Compared to 2014, the 2015 GFSs isotopic niche was three times larger (2.0 in 2015, 0.6 in 2014) and the δ13C was significantly lower. CSLs also showed significantly lower δ13C and higher δ15N in 2015. Interannual segregation was greater for CSLs, and their pup body mass was also significantly lower during the 2015 breeding season (mean = 8.7 kg) than in the same season of 2014 (mean = 9.9 kg). The decrease in δ13C for both otariids reflected a more oceanic foraging; most likely associated with the decline in primary productivity in surrounding areas to the SBA, related to a higher SST caused by the 2015 ENSO, with a subsequent increase in foraging effort. These would explain the fewer observed individuals on land, especially pups, which showed diminished body condition (CSLs). This study highlights the importance of marine mammals as sentinel species that respond dynamically to changes in environment, providing valuable information on the effect of ENSO on pinnipeds in Mexican waters.

Transformation of the genital epithelial tract occurs early in California sea lion development.

An unusually high prevalence of metastatic urogenital carcinoma has been observed in free-ranging California sea lions stranded off the coast of California in the past two decades. No cases have been reported for sea lions in the relatively unpolluted Gulf of California. We investigated occurrence of genital epithelial transformation in 60 sea lions (n=57 pups and 3 adult females) from the Gulf of California and examined whether infection by a viral pathogen previously found to be associated with urogenital carcinoma accounted for such alterations. We also explored the contribution of MHC class II gene expression on transformation. Cellular alterations, such as squamous cell atypia (ASC), atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions were observed in 42% of the pups and in 67% of the adult females. Normal genital epithelium was more common in male than female pups. ASC was five times more likely to occur in older pups. Epithelial alterations were unrelated to infection by the potentially oncogenic otarine type I gammaherpesvirus (OtHV-1), but ASCUS was more common in pups with marked and severe inflammation. Expression of MHC class II DRB loci (Zaca DRB-D) by peripheral antigen-presenting leucocytes showed a slightly 'protective' effect for ASC. We propose that transformation of the California sea lion genital epithelium is relatively common in young animals, increases with age and is probably the result of infection by an unidentified pathogen. Expression of a specific MHC class II gene, suggestive of presentation of specific antigenic peptides to immune effectors, appears to lower the risk of transformation. Our study provides the first evidence that epithelial transformation of the California sea lion genital tract is relatively common, even from an early age, and raises questions regarding differences in sea lion cancer-detection and -repair success between geographical regions.

Context-dependent associations between heterozygosity and immune variation in a wild carnivore.

BACKGROUND: A multitude of correlations between heterozygosity and fitness proxies associated with disease have been reported from wild populations, but the genetic basis of these associations is unresolved. We used a longitudinal dataset on wild Galapagos sea lions (Zalophus wollebaeki) to develop a relatively new perspective on this problem, by testing for associations between heterozygosity and immune variation across age classes and between ecological contexts. RESULTS: Homozygosity by locus was negatively correlated with serum immunoglobulin G production in pups (0-3 months of age), suggesting that reduced genetic diversity has a detrimental influence on the early development of immune defence in the Galapagos sea lion. In addition, homozygosity by locus was positively correlated with total circulating leukocyte concentration in juveniles (6-24 months of age), but only in a colony subject to the anthropogenic environmental impacts of development, pollution and introduced species, which suggests that reduced genetic diversity influences mature immune system activity in circumstances of high antigen exposure. CONCLUSIONS: These findings demonstrate the environmental context-dependency of the phenotypic expression of immune variation, which is implicit in the theory of ecoimmunology, but which has been rarely demonstrated in the wild. They also indicate that heterozygosity may be linked to the maintenance of heterogeneity in mammalian immune system development and response to infection, adding to the body of evidence on the nature of the mechanistic link between heterozygosity and fitness.

Activation of an inflammatory response is context-dependent during early development of the California sea lion.

Variations in immune function can arise owing to trade-offs, that is, the allocation of limited resources among costly competing physiological functions. Nevertheless, there is little information regarding the ontogeny of the immune system within an ecological context, and it is still unknown whether development affects the way in which resources are allocated to different immune effectors. We investigated changes in the inflammatory response during early development of the California sea lion (Zalophus californianus) and examined its association with body condition, as a proxy for the availability of energetic resources. We found that the relationship between inflammation and body condition varied according to developmental stage and circulating levels of leucocyte populations, a proxy for current infection. Body condition was related to the magnitude of the inflammatory response during two of the three developmental periods assessed, allowing for the possibility that the availability of pup energetic reserves can limit immune function. For older pups, the ability to mount an inflammatory response was related to their circulating levels of neutrophils and the neutrophil to lymphocyte ratio, implying that the infection status of an individual will influence its ability to respond to a new challenge. Our results suggest that trade-offs may occur within the immune system and highlight the importance of taking into account ontogeny in ecoimmunological studies.