Unraveling the gut microbiota of Mexican pinnipeds: the dominance of life histories over phylogeny.

Studying how phylogeny influences the composition and functions of microbiotas within animal hosts is essential for gaining insights into the connection between genetics, ecology, and health in the animal kingdom. However, due to limited comprehensive studies, this influence remains unclear for many wild mammals, including Mexican pinnipeds. We employed 16S rRNA gene deep-sequencing to investigate the impact of phylogeny on the gut microbiota of four pinniped species inhabiting Mexican shores: the Pacific harbor seal (Phoca vitulina richardii), the northern elephant seal (Mirounga angustirostris), the California sea lion (Zalophus californianus), and the Guadalupe fur seal (Arctocephalus philippii townsendi). Our results indicated that factors such as diets and shared life histories exerted more influence on microbiota composition than phylogeny alone. Notably, otariid species sharing similar life histories displayed greater microbiota similarity than phocids, which have distinct life histories and fewer microbiota similarities. Furthermore, harbor seals have more microbial similarities with the two otariid species than with elephant seals. Of particular concern, we observed a higher abundance of potentially pathogenic bacteria (e.g., Photobacterium damselae and Clostridium perfringens) in harbor seals and Guadalupe fur seals compared to other pinnipeds. This finding could pose health threats to these species and nearby human populations.IMPORTANCEPinnipeds in Mexico host microbial communities that remain understudied. While several factors can influence microbiota composition, the role of phylogenetic relationships among these pinnipeds remains unclear due to limited knowledge of the microbiota in certain species. This study aimed to fill this gap by characterizing the composition and function of the gut microbiota in the four pinniped species that occur in Mexico. Our analysis reveals that shared diets and life histories contribute to similarities in the composition of gut microbial communities. This study also highlights the potential differences in the metabolic capabilities and adaptations within the gut microbiota of pinnipeds. Understanding how phylogeny impacts microbial communities enhances our insights into the evolutionary dynamics of marine mammals.

Age as a primary driver of the gut microbial composition and function in wild harbor seals.

Dietary changes are the major variation cause in the composition of the gut microbiota. The short lactation phase in phocids provides an exceptional opportunity to explore the microbiota's response to a quick transition from a milk-based to a solid diet. We investigated the effects of age and sex on the gut microbiota of harbor seals in Mexico using rectal and fecal samples from pups and adults. 16S gene sequencing revealed age explains most of the observed variations in microbial composition. Individuals with frequent contact (pups-female adults) have major microbial similarities than those with little or no contact (pups-male adults). Overall, adults and females (regardless of sex and age, respectively) have a greater microbial richness; as seals grow, the core microbiome shrinks, and microbial diversity increases. We found pathways related to milk and chitin digestion in pups' microbiomes, indicating pups were transitioning to a solid diet. An enrichment of routes related to dramatic weight loss and body mass indicated higher metabolic stress in pups in late breeding season, when they are weaned and start intermittent fasting. Our findings highlight the host-microbiome interaction in harbor seals during late breeding season in response to food shifts and metabolic stress.

Human activities disturb haul out and nursing behavior of Pacific harbor seals at Punta Banda Estuary, Mexico.

Humans frequently interact with Pacific harbor seals (Phoca vitulina richardii) at Punta Banda Estuary, Baja California, Mexico, due to the high incidence of recreational activities people undertake there. The immediate effect of these interactions is that seals flush to the water, reducing their time on land and, probably, increasing their energy expenditure. On-land observations were used to study the impact of different sources of disturbance on seal behavior and evaluate their effect on the amount of time dedicated to nursing over three pupping seasons, (2015-2017), with 0.58-0.81 disturbance events/hour recorded over the entire sampling period. Terrestrial vehicles were the source with the highest disturbance rate (number of disturbance events/h), followed closely by pedestrians. However, the proportion of seals affected was highest when pedestrians were the disturbance source. Recovery events (seals hauling out after flushing) occurred after 34% of disturbance events, after less than half of which the same number of hauled-out seals as there were prior to the disturbance were observed. Recovery time varied among the years studied, of which 2017 saw the longest recovery time. In addition, pedestrians were the disturbance source with the longest recovery time. Given that resting on land is essential for pup survival, which depends on both the establishment of the mother-pup bond from birth and its maintenance throughout nursing, flushing behavior may have significant implications for the entire colony during the nursing season. We recorded a decrease in nursing duration, which did not return to the same level even after recovery and the resumption of nursing. Terrestrial vehicles were found to be the disturbance source that shortened nursing events most significantly.

Fetal mercury concentrations in central California Pacific harbor seals: Associated drivers and outcomes.

Mercury (Hg) is a well-known toxicant in wildlife and humans. High total Hg concentrations ([THg]) have been reported in central California harbor seals Phoca vitulina richardii. We evaluated the effects of presence/absence of early natal coat (lanugo), year (2012 to 2017), sex, stranding location, and trophic ecology (ẟ13C and ẟ15N values) on hair [THg] along coastal central California. Also examined were [THg] effects on growth rates of pups in rehabilitation and probability of release (e.g., successful rehabilitation). The [THg] ranged from 0.46-81.98 mg kg-1 dw, and ẟ15N and ẟ13C ranged from 13.6-21.5‰, and -17.2 to -13.0‰, respectively. Stranding location, year, and presence of lanugo coat were important factors explaining variation in [THg]. Seals from Sonoma and San Mateo County had higher [THg] than other locations. Seals with full or partial lanugo coat had lower [THg]. Seals from 2016 and 2017 had higher [THg] than those from 2015. Hair [THg] exceeded lower and upper toxicological thresholds (>20 mg kg-1 by year (5.88% to 23.53%); >30 mg kg-1 (0% to 12.31%)) with a pronounced increase from 2015 to 2016. Pups in 2017 had significantly higher odds ratio of [THg] above 20 mg kg-1 than pups of 2015, and pups in 2016 had significantly higher odds ratio than those from 2013 and 2015 (similar when using 30 mg kg-1). Pups in Sonoma County had the highest odds ratio for [THg] in lanugo above 20 mg kg-1. ẟ15N values were higher in 2015-2017, particularly relative to 2014, probably associated with the El Niño event. The [THg] was not a good predictor for probability of release and mass-specific growth rates in captivity. Further investigation of temporal trends of [THg] in harbor seals is warranted given the relatively high percentage of samples exceeding threshold values, particularly in the most recent sampling years.

Evaluating the performance of the Bayesian mixing tool MixSIAR with fatty acid data for quantitative estimation of diet.

We test the performance of the Bayesian mixing model, MixSIAR, to quantitatively predict diets of consumers based on their fatty acids (FAs). The known diets of six species, undergoing controlled-feeding experiments, were compared with dietary predictions modelled from their FAs. Test subjects included fish, birds and mammals, and represent consumers with disparate FA compositions. We show that MixSIAR with FA data accurately identifies a consumer's diet, the contribution of major prey items, when they change their diet (diet switching) and can detect an absent prey. Results were impacted if the consumer had a low-fat diet due to physiological constraints. Incorporating prior information on the potential prey species into the model improves model performance. Dietary predictions were reasonable even when using trophic modification values (calibration coefficients, CCs) derived from different prey. Models performed well when using CCs derived from consumers fed a varied diet or when using CC values averaged across diets. We demonstrate that MixSIAR with FAs is a powerful approach to correctly estimate diet, in particular if used to complement other methods.

Trophic ecology of Mexican Pacific harbor seal colonies using carbon and nitrogen stable isotopes.

There is limited information that provides a comprehensive understanding of the trophic ecology of Mexican Pacific harbor seal (Phoca vitulina richardii) colonies. While scat analysis has been used to determine the diet of some colonies, the integrative characterization of its feeding habits on broader temporal and spatial scales remains limited. We examined potential feeding grounds, trophic niche width, and overlap, and inferred the degree of dietary specialization using stable carbon and nitrogen isotope ratios (δ13C and δ15N) in this subspecies. We analyzed δ13C and δ15N on fur samples from pups collected at five sites along the western coast of the Baja California Peninsula, Mexico. Fur of natal coat of Pacific harbor seal pups begins to grow during the seventh month in utero until the last stage of gestation. Therefore pup fur is a good proxy for the mother's feeding habits in winter (~December to March), based on the timing of gestation for the subspecies in this region. Our results indicated that the δ13C and δ15N values differed significantly among sampling sites, with the highest mean δ15N value occurring at the southernmost site, reflecting a well-characterized north to south latitudinal 15N-enrichment in the food web. The tendency identified in δ13C values, in which the northern colonies showed the most enriched values, suggests nearshore and benthic-demersal feeding habits. A low variance in δ13C and δ15N values for each colony (<1‰) and relatively small standard ellipse areas suggest a specialized foraging behavior in adult female Pacific harbor seals in Mexican waters.

The Pacific harbor seal gut microbiota in Mexico: Its relationship with diet and functional inferences.

Diet is a primary driver of the composition of gut microbiota and is considered one of the main routes of microbial colonization. Prey identification is fundamental for correlating the diet with the presence of particular microbial groups. The present study examined how diet influenced the composition and function of the gut microbiota of the Pacific harbor seal (Phoca vitulina richardii) in order to better understand the role of prey consumption in shaping its microbiota. This species is a good indicator of the quality of the local environment due to both its foraging and haul-out site fidelity. DNA was extracted from 20 fecal samples collected from five harbor seal colonies located in Baja California, Mexico. The V4 region of 16S rRNA gene was amplified and sequenced using the Illumina technology. Results showed that the gut microbiota of the harbor seals was dominated by the phyla Firmicutes (37%), Bacteroidetes (26%) and Fusobacteria (26%) and revealed significant differences in its composition among the colonies. Funtional analysis using the PICRUSt software suggests a high number of pathways involved in the basal metabolism, such as those for carbohydrates (22%) and amino acids (20%), and those related to the degradation of persistent environmental pollutants. In addition, a DNA metabarcoding analysis of the same samples, via the amplification and sequencing of the mtRNA 16S and rRNA 18S genes, was used to identify the prey consumed by harbor seals revealing the consumption of prey with mainly demersal habits. Functional redundancy in the seal gut microbiota was observed, irrespective of diet or location. Our results indicate that the frequency of occurrence of specific prey in the harbor seal diet plays an important role in shaping the composition of the gut microbiota of harbor seals by influencing the relative abundance of specific groups of gut microorganisms. A significant relationship was found among diet, gut microbiota composition and OTUs assigned to a particular metabolic pathway.

Do marine reserves increase prey for California sea lions and Pacific harbor seals?

Community marine reserves are geographical areas closed to fishing activities, implemented and enforced by the same fishermen that fish around them. Their main objective is to recover commercial stocks of fish and invertebrates. While marine reserves have proven successful in many parts of the world, their success near important marine predator colonies, such as the California sea lion (Zalophus californianus) and the Pacific harbor seal (Phoca vitulina richardii), is yet to be analyzed. In response to the concerns expressed by local fishermen about the impact of the presence of pinnipeds on their communities' marine reserves, we conducted underwater surveys around four islands in the Pacific west of the Baja California Peninsula: two without reserves (Todos Santos and San Roque); one with a recently established reserve (San Jeronimo); and, a fourth with reserves established eight years ago (Natividad). All these islands are subject to similar rates of exploitation by fishing cooperatives with exclusive rights. We estimated fish biomass and biodiversity in the seas around the islands, applying filters for potential California sea lion and harbor seal prey using known species from the literature. Generalized linear mixed models revealed that the age of the reserve has a significant positive effect on fish biomass, while the site (inside or outside of the reserve) did not, with a similar result found for the biomass of the prey of the California sea lion. Fish biodiversity was also higher around Natividad Island, while invertebrate biodiversity was higher around San Roque. These findings indicate that marine reserves increase overall fish diversity and biomass, despite the presence of top predators, even increasing the numbers of their potential prey. Community marine reserves may help to improve the resilience of marine mammals to climate-driven phenomena and maintain a healthy marine ecosystem for the benefit of both pinnipeds and fishermen.

Sequence variation and gene duplication at the MHC DRB loci of the spotted seal Phoca largha.

The major histocompatibility complex (MHC) is one of the most important genetic systems associated with resistance to infectious diseases in vertebrates. The spotted seal (Phoca largha) is one of the most endangered species in China. In this study, we present the first step in the molecular characterization of a DRB-like locus in the spotted seal by analyzing the nucleotide sequence of the polymorphic exon 2 segments, a 288-nucleotide sequence. By examining the segment from a group of 41 individuals, 28 alleles were identified. No deletion, insertion, or exceptional stop codon was detected, suggesting that these alleles could be functional in vivo. The nucleotide and amino acid sequences of the segment both showed a relatively high level of similarity (nucleotides 97%; amino acids 98%) to those of Meles meles and Zalophus californianus. The high level of spotted seal MHC-DRB polymorphism revealed in the present study has not been reported for the Phocidae and could be a consequence of the small spotted seal population adapting to the Bohai Sea, which probably has a relatively high level of pathogens.

Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy.

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L.

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface-related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.