Trophically Transmitted Parasites and Their Responses to Microbial Pathogens and Consumed Plastic Contaminants

Abstract

Trophically transmitted parasites, which move from one host to the next through host feeding activities, are subject to direct and indirect stressors within their hosts and the surrounding ecosystem. Infection success can be disrupted by host defenses and environmental conditions that exceed the tolerances of the parasites or their hosts. These interruptions can be caused by various factors, including host-derived antagonists, alterations in the host’s environment, exposure to toxic molecules, and disruption to the host’s microbial communities. Here, we present novel findings on the responses of intestinal helminth infracommunities to stressors associated with shifts in the microbiome due to bacterial infection and under a range of conditions where microplastics were consumed.

1. Introduction

1.1 Helminths are ubiquitous and important

Parasitism is one of the most common ways that organisms acquire critical resources. While the exact numbers are not fully resolved, several studies [1, 2] suggest that for every type of vertebrate host (free-living organisms), one or more species of parasitic helminths occur, and this only counts parasites of three phyla. Despite its commonness, parasitism remains an underexplored trophic strategy that may shape direct and indirect interactions among free-living organisms and other symbionts. Moreover, parasites are not passive players in this dynamic; they respond to the behaviors of their hosts that introduce other symbionts and foreign substances to their sites of infection, particularly the gastrointestinal tract (GIT).

The ecological consequences of parasitism include impacts on biodiversity through modifications of community structure, genetic diversity, and trait frequency distributions [3]. Further, parasites can influence niche development and host specialization by altering the behaviors of intermediate hosts and modulating trophic interaction strengths between predators and prey [4]. Because trophically transmitted parasites exploit predator-prey interactions, these parasites can be useful ecological indicators [5, 6]. For instance, Kuris et al. [7] used parasites to infer that shortfin mako sharks and sperm whales are common predators of oarfish, a teleost that is rarely observed in the wild and has no previously documented predators. The utility of parasites in shedding light on host interactions is, thus, impactful at scales that range from cellular processes to predator-prey interactions within ecosystems and beyond. Alterations to parasite community composition and structure can have nuanced yet sweeping consequences on individuals, populations, communities, and ecosystems.

Losses in parasite diversity can have major implications on overall biodiversity, considering that around half of Earth’s animals are parasitic [1]. Losses to parasite diversity result from intentional anthropogenic actions designed to eliminate human and veterinary diseases but also stem from losses in host diversity that occur from changing distributions, densities, and the carrying capacities of hosts’ habitats [8, 9]. As parasite communities respond to host distributions, their infections can adapt to new surroundings, sometimes becoming more threatening and sometimes rendered benign [10].

Trophically transmitted parasites will only infect hosts that have consumed them, and thus, can be used to learn about shifting diets in their hosts. For example, altered frequencies of the echinostomatid Drepanocephalus auritus in Double-crested Cormorants indicate the extent to which the trematode’s intermediate host (catfish) [11] are included or excluded from a bird’s diet [6, 12]. In some cases, these altered feeding habits occur as hosts migrate through areas where the parasite is absent [13] because the intermediate host is locally rare due to invasions or extirpations [14] or because the feeding preferences of individual hosts lead to niche partitioning and differentiation of parasite infracommunities [15]. Similarly, when intermediate host populations are threatened by anthropogenic landscape alterations, parasite populations with complex life cycles can be reduced or eliminated [16].

1.2 Parasites live in ever-changing conditions

Although parasitism costs the host, the severity and type of those consequences occur along a spectrum. Deadly outbreaks can occur when endemic pathogens mutate, as occurred with the Zika virus in an avian outbreak from 2014 to 2016 [17]. The host-parasite relationship becomes lethal when a parasite’s survival depends on the host’s death. Likewise, when direct contact is not needed because vectors or other transport agents spread propagules, deadly diseases can persist unchecked [18]. However, many such relationships are not sustainable through evolutionary time. Highly virulent diseases often remove the genes of high susceptibility from host populations, leaving a larger proportion of tolerant or resistant individuals [19]. Consequently, virulence in some contagions is reduced as the host-parasite interaction evolves [18, 20]. Benign infections are widespread for typical parasites infecting definitive hosts, as symbiont fitness increases with host longevity.

In established relationships between hosts and parasites, the host can tolerate some parasite load without experiencing detrimental impact [21]. This is likely more of a reflection of the faculties of parasites that manipulate host immune function and evade detection and immune responses. However, when parasites use cellular commodities produced by the host, the immune system can be activated by danger-associated molecular patterns (DAMPs) that occur when host tissue is damaged [22]. The innate and adaptive immune systems can eliminate parasitic propagules, but many parasites are well-defended and highly evolved to escape host defenses [23, 24]. Additionally, host chronic immune responses can be energetically costly, disrupting tissues with systemic inflammation and inducing lethal health problems like tumorigenesis, fibrosis, and sepsis [25]. Because animal predators cannot eliminate all infectious agents within their prey, they are exposed repeatedly to trophically transmitted parasites while feeding. Because immune responses are not feasible solutions to parasite avoidance, definitive hosts often tolerate infections and isolate damaged tissues rather than defend against parasites [26]. This tolerance and stability can change when climatic, environmental, and biotic conditions shift. Anthropogenic changes are currently the greatest drivers of change affecting local and global environments.

With an estimated 45% of the Earth’s habitable land used for agriculture, the widespread replacement of diverse communities with monocultures has important implications [27]. The expansion of agriculture pasture and turf at the expense of previously diverse communities profoundly impacts the forage base, feeding behavior, and the transfer of nutrients and energy in these systems. Agriculture as a primary land cover can prompt some avian species to adjust their migration patterns, impacting their regional abundances [28, 29]. In turn, ecosystem alteration, extensive retooling of landscapes for agriculture, and consequent changes in energy and nutrient transfer can alter parasite communities. Some of these changes could harm host organisms or agriculture [30]; however, the full extent of large-scale ecosystem change caused by agriculture on organisms and parasite communities remains unknown. One challenge for elucidating the impacts of landscape change on specific taxonomic groups, such as birds, arises from the diverse trophic positions occupied within the class Aves. Comparing bird species with varying dietary preferences, such as those consuming plants, freshwater invertebrates, marine invertebrates, freshwater fishes, marine fishes, and combinations across different ranges and seasons, presents analytical complexity. The dynamic nature of dietary needs and prey availability further complicates the assessment of avian feeding behaviors. An established approach to categorically assess these organisms involves evaluating their trophic position by analyzing stable isotope concentrations of elements like Carbon and Nitrogen.

Many elements can vary in their number of neutrons, affecting their atoms’ isotopic weight. Lighter isotopes move more freely in biological processes and chemical reactions, while heavier isotopes accumulate in tissues during slow metabolic processes [31]. The fractionation of heavy and light isotopes in organisms’ tissues correlates with the biology of the organism. For example, plants using the C₃ photosystem cycle tend to have a higher accumulation of the heavy carbon isotope (C¹³) compared to those using the C₄ photosystem, making it possible to differentiate between basal photosynthetic groups and those from which higher organisms derive [32]. Similarly, Nitrogen¹⁵, being heavier than N¹⁴, tends to accumulate in higher trophic level organisms due to its slower metabolic processing, representing a form of bioaccumulation [31]. Thus, we can learn about the implications of ecosystem change to hosts and parasites with stable isotope concentrations. Human-made products can also disturb parasite communities. Anthropogenic waste management introduces pollutants and other contaminants into the environment, affecting food webs. Plastic debris is a recently emerged concern, posing physical and chemical threats to wildlife, agriculture, and human health [33].

1.3 Helminths and contaminants

Parasites have long been recognized as valuable bioindicators for habitat and host disruption [34, 35, 36]. Unfortunately, the responses that different parasite taxa exhibit vary with the type of stressor [37, 38]. As biomarkers, monogeneans and acanthocephalans have been sensitive to changing concentrations of dissolved oxygen, fecal coliform, and total ammonia nitrogen in water [39]. Some trematodes and nematodes increase in prevalence when hosts are exposed to heavy metal contaminants [34], and reductions in acanthocephalans are documented in fishes downstream of pulp mill discharges [40]. However, some groups, like cestodes, tend to increase or decrease depending on the combination of contaminants [35].

Beyond the direct effects of contaminants on helminths in situ, polluted systems can also disrupt the life cycle of parasites. When these interferences include reduced densities or richness of obligate or facultative hosts, the cycle can contract to levels where it is no longer supported [41]. For example, the tapeworm Bothriocephalus acheilognathi has a 2-host life cycle that includes a copepod (Cyclops strenuus) and a freshwater fish. When exposed to cadmium pollution, copepod populations decline (especially when copepods are parasitized with tapeworms: [42]), and infections in the fish hosts become rare. This is problematic in highly dynamic and stochastic systems, where temporary removal of hosts via migration or extirpation could eliminate the parasite from the system or alter its host specificity and virulence [43].

Contaminants can also modulate the trophic interactions of organisms by changing their behaviors, physical conditions, and crypsis [44]. For example, fishes exposed to microplastic contaminants were more difficult to capture by a simulated predator than controls [45]. Contrastingly, Zebra Finches exposed to fluoxetine reduced avoidance of high-risk locations for predation and freezing behavior in the presence of a simulated predator, increasing the likelihood of predation [46]. The system-wide consequences of these altered trophic interactions can modify the transfer of energy and cycling of nutrients, indicators of ecological integrity [47].

Considering the migratory nature of many bird host species, there is an increased risk of their exposure to threats at large spatial and climactic scales [48]. Climate change contributes to the altered distributions of organisms as abiotic conditions vary spatially, and biotic communities respond to them. Within these new climate regimes, all organisms have the potential to respond to changed conditions, and the presence of additional contaminants can further modify the impacts that organisms experience. Of particular concern in contamination science is the added presence of plastics that represent persistent chemical compounds and durable physical composites that enter food webs [49].

Marine debris impacts the environment, wildlife, and human communities, especially in remote islands and coastal areas. These areas have experienced environmental degradation due to the accumulation of marine debris on their shorelines [50]. This issue interests community members who rely on subsistence species, such as seabirds and marine mammals, which can ingest or become entangled in marine debris [51, 52, 53].

Plastic debris does not degrade; it only gets smaller [54]. Some of the smallest components, microplastics (1–10 mm) and nanoplastics (<1 mm), are increasingly seen as serious contaminants in marine ecosystems. They have been implicated in altering physiological processes in the organisms that come into contact with them [55, 56, 57]. Furthermore, micro- and nano-plastics affect parasite presence [58, 59] and host-parasite interactions [60, 61] in marine phytoplankton [62], invertebrates [63], and vertebrates [57, 64]. Overall, it seems that micro- and nano-plastics have harmful effects on the interactions between living and non-living elements in host-parasite systems at all levels of the food chain. Therefore, the implications of plastic contamination on helminth communities occur at both large and small scales. In addition to helminths, the distributions of other symbiotic organisms, such as bacteria, also respond to human-caused environmental changes, climate change, and contamination.

1.4 Microbe-helminth interactions

The host microbiome is crucial in resistance to parasitic infections. It harbors symbiotic organisms, particularly mutualistic bacteria, pivotal for combating parasitic threats through various mechanisms such as antibiotic production, enhanced digestion efficiency, and pathogen resistance [65, 66]. These bacteria, specialized and sensitive to specific environmental conditions, are subject to influences from abiotic factors like the microclimate and pH of the infection site and biotic factors such as the host’s taxa and diet [67, 68].

Extensively studied, GIT microbiome composition varies among individuals and is shaped by sex, migration status, age, and the host’s physiological condition, including nutrition, stress hormones, and parasitic infections [69, 70]. Under normal circumstances, a delicate balance exists among microbial mutualists and commensals, ensuring sustained symbiotic benefits. Disruptions, like the reduction or loss of these beneficial microbes, may lead to dysbiosis, favoring colonization by other pathogens and exogenous microbes, thereby jeopardizing host health [71, 72, 73].

The microbiome extends its influence beyond the GIT to other host compartments, engaging in microbial exchanges between the skin, blood, and vectors, facilitating pathogen dissemination [74, 75]. Notably, helminthic parasites and microbial symbionts share infection sites and immune evasion strategies, with interactions shaping host defenses and microbial richness [76, 77]. While complimentary associations between microbes and helminths are well-documented, antagonistic dynamics underscore the complexity of these relationships [77, 78].

Mouse studies have further elucidated the intricate interplay between helminths and the gut microbiota, demonstrating how host defenses are modulated in response to infection and affect digestion efficiency and fecal composition [79, 80]. Perturbing the microbial community through dysbiosis or specific bacterial triggers can impede parasite colonization pathways, underscoring the interconnectedness of host-microbe-parasite dynamics and ecosystem health [81, 82]. Moreover, microbial metabolic reactions can activate virulence factors in pathogenic bacteria, impacting community structure and ecosystem resilience, thus highlighting the broader implications of microbial dysbiosis beyond host-parasite interactions [83, 84]. Understanding these interactions offers insights into managing host-parasite dynamics and underscores the broader ecological consequences of microbial disruptions.

While fewer instances document reductions of helminths by bacteria [78], the absence of Escherichia coli and Salmonella typhimurium, or dysbiosis that impedes their function, reduces the ability of nematodes like Trichuris muris to colonize a host successfully. E. coli and S. typhimurium are triggers that initiate the opening of T. muris eggs in the host intestine [81, 85]. Thus, treatments that disrupt the microbial community can lead to various pathways where parasites are reduced or eliminated from hosts [82, 86].

Here, we present findings from multiple bird collections in which avian hosts were examined for gastrointestinal helminths and ingestion of plastics. Some of the birds were sourced from an outbreak of Avian Botulism, where it is believed that the hosts perished due to botulinum neurotoxin (BoNT) poisoning and experienced distinct shifts in their surficial microbial communities. Although all the birds in the collection are waterbirds, they represent various taxonomic and trophic groups. As such, many birds were also analyzed for stable isotope concentrations. This allowed us to assess the effects of two stressors on the infracommunities of their internal parasitic helminths: contamination by microplastics and pathogenic bacteria capable of causing dysbiosis of the GIT.

2. Empirical evidence and hypothesis testing

2.1 Methodologies

Since 2015, we have simultaneously assessed the GITs for trophically transmitted parasites and consumed plastics of 26 species (n = 124 for whole birds and another 241 GITs for a total of 366 birds). All birds were collected on the water, at roost sites over water, or soon after death from natural causes, like Avian Botulism. Whole carcass assessments (n = 102) included quantitative necropsy, where morphometric weights of each primary organ system were measured and evaluated for condition. Although several organ systems besides the GIT were assessed, we focus on the helminth parasites of the gut here. Because the sources of birds varied in time and space, the handling of carcasses varied by agency, and the condition of GITs or their contents varied by project, we do not attempt to make conclusions regarding individual parasite species but instead use the community composition (=infracommunity) of each bird, using parasite morphotypes as operational “species.” These morphotypes were based on general morphological characteristics and were consistent at the host species level.

Most of the whole birds were sourced from natural populations of seabirds in the Western Aleutian Islands in cooperation with the U.S. Fish and Wildlife Service. We shot these birds on the water, froze them within 1 hour of collection, and wrapped them in aluminum foil to ensure minimal contact and contamination of plastics. We took body measurements and qualitatively assessed body condition [87], washed the external surfaces with soap and water, and retained the resulting “ectowash” (containing ectoparasites) before opening the birds for qualitative necropsy [88]. We sampled liver tissue during necropsy for stable isotope measurements and various tissues for plastic congener quantification. For these 124 samples, we compare the host trophic position with parasite metrics and the frequencies of consumed plastics and their chemical congeners.

Within the whole bird samples, we had a subset (n = 21) associated with a die-off event on Middleton Island, Alaska, in the summer of 2021. Black-legged Kittiwakes (Rissa tridactyla: BLKI) (n = 21) and Glaucous-winged Gulls (Larus glaucescens: GWGU) (n = 2), were frozen within hours of death and later were also fully processed with quantitative necropsy, gut parasitology, and stable isotope analyses. Further, we performed microbial assessments of the parasites from the ectowashes (lice, mites, ticks, and fleas) of the BLKI to learn whether there were differences between their microbiomes and those of the same species from the 2016 Aleutian Island collection [89].

In addition to the whole-body assessments, we include host data collected by the U.S. Department of Agriculture, Animal Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center (NWRC) on commercial aquaculture facilities or at roost sites near aquaculture. The first set of samples (n = 136) consisted of two bird species, Double-crested Cormorants (Nannopterum auritum) and Lesser Scaup (Aythya affinis), foraging on baitfish aquaculture farms in Arkansas and roosting near catfish aquaculture ponds in Mississippi in 2016 and 2017. These birds were shot in the field, individually wrapped and placed on ice, and transported to an NWRC lab for necropsy within 24 hours of collection. We emptied the gut contents into warm saline and transferred them into formalin, and liver tissue samples were taken for stable isotope analyses [12, 90]. The second sample set consisted of seven waterbird species (N = 105) shot on shrimp aquaculture farms in Alabama and Florida in 2020 and 2021. Gut contents were processed similarly to the former group, except half of the samples were preserved in ethanol and the other half in 10% buffered formalin.

Parasite analyses following quantitative necropsy were separated by the anatomical regions of the digestive system: the esophagus, stomach/gizzard, duodenum, jejunum, ileum, colon, and cloaca. For these assessments, we could evaluate the co-infections of parasites and infer the preferential infection sites for many parasites. In contrast, in gut content analyses from birds processed from aquaculture collections, all GIT sections were combined into a single sample. We could not ascertain infection localities beyond upper and lower GIT. While assessing digestate from samples, we scrutinized the liquid diluted with 0.04% saline (Instant Ocean) in square, gridded sorting under 4–40× total magnification (stereoscope). Any parasites or plastics were removed, placed into 9-well borosilicate glass plates, sorted into morphologically distinct groupings (i.e., “morphospecies”), and preserved in either 10% buffered formalin for long-term preservation or 90% ethanol for DNA conservation. Each morphospecies was counted for abundance estimates and plastic type (fiber, shard, etc.) was enumerated and recorded.

Data from each project were combined into a database where each row pertained to a given morphospecies present in the GIT. When assessments had previously split components of the GIT into several sections (n = 124), we summed the counts of each morphospecies to calculate a total infrapopulation for each bird. In addition, we summed the number of plastics found within each organ, when applicable, and for birds where we measured them, we included mean stable isotope values for each species, as well as mean plastic chemical congener concentrations.

We extracted the microbiome of diseased BLKI samples using a combination of 2.3 and 0.5 mm silica beads for homogenization. DNA extraction followed the Qiagen DNeasy Blood and Tissue Kit protocol with an extended 36-hour incubation period at 56°C [91]. 16S metagenomic sequencing was conducted on the Illumina MiSeq platform using standard Illumina primers for the hypervariable region V3-V4. Data processing occurred in QIIME2, denoising sequences with DADA2, and filtering based on quality scores and expected errors for chloroplasts, mitochondria, and unclassified sequences. Sequences were clustered to 99% similarity, and taxonomy was assigned using a combination of the Silva 138 and GreenGenes databases, resolving discrepancies in favor of higher confidence assignments [92, 93, 94, 95].

We used a sampling depth of 29k for the core metrics function in QIIME2 to analyze the microbial community composition. The Jaccard dissimilarity matrix was exported to R and assessed with Canonical Analyses of Principal Coordinates (CAP: vegan). The CAPs were constrained separately by helminthic parasite assemblage, host plastic consumption, and year. P-values were based on PERMANOVA with 999 permutations of each variable to determine statistical significance and the R² values indicate the variation attributable to that environmental variable.

2.2 Ecological differentiation of hosts

Using δC¹³ and δN¹⁵ isotopic ratios of the 11 assessed bird species, we grouped the hosts into five distinct trophic clusters (K-means clusters based on Principal Components, Figure 1). Two singleton clusters contain species that are high-order predators yet feed in distinctly different habitats: Double-crested Cormorants, piscivorous birds that breed primarily in freshwater habitats but also feed in coastal habitats during non-breeding seasons, and the Northern Fulmar (Fulmarus glacialis), a highly marine species that breeds on oceanic islands. While these species appear to share a similar trophic level, suggesting a highly piscivorous diet, the habitats in which they occur provide sufficient separation in trophic ordinate space, separating them through cluster analysis. The remaining three groups (Figure 2) include bird species that persist in low-to-intermediate trophic levels.

Figure 1.

Of the five clusters identified from distinct heavy nitrogen and carbon isotope concentrations, three multi-species clusters could be differentiated. These groups included invertebrate consumers (2: orange), coastal predators (4: green), and offshore predators (3: blue). BLKI = Black-legged Kittiwake, COMU = Common Murre, CRAU = Crested Auklet, GWGU = Glaucous-winged Gull, HOPU = Horned Puffin, LESC = Lesser Scaup, PECO = Pelagic Cormorant, PIGU = Pigeon Guillemot, TUPU = Tufted Puffin.

Figure 2.

Mean concentrations of total phthalates and the plastic prevalence (proportion of birds with plastic in gastrointestinal tract) of nine species of seabirds (colored by American Ornithological Union code) with sample sizes of 3 or more individuals. BLKI = Black-legged Kittiwake, COMU = Common Murre, CRAU = Crested Auklet, GWGU = Glaucous-winged Gull, HOPU = Horned Puffin, NOFU = Norther Fulmar, PECO = Pelagic Cormorant, PIGU = Pigeon Guillemot, TUPU = Tufted Puffin.

The invertebrate-specialist group (cluster 2) spans habitat types and includes freshwater and marine specialists. This group contains Lesser Scaup, a ubiquitous species that can consume a range of prey from plant seeds and small invertebrates to small fishes; the Crested Auklet (Aethia cristatella), a consumer of marine/pelagic arthropods and sometimes small fish; and the Horned Puffin (Fratercula corniculatus), a frequent consumer of small fishes, all of which might share similar trophic positions with predatory invertebrates in freshwater systems.

The coastal-specialist group (cluster 3) includes bird species that breed and forage near coastlines or shallow marine communities. It includes Pelagic Cormorants (Urile pelagicus), Pigeon Guillemots (Cepphus columba), and Glaucous-winged Gulls. While cormorants are often considered fish specialists (like the Double-crested Cormorant above), feeding on small coastal fish can shift their isotopic signatures to be similar to those of trophic generalists and coastal predators like gulls and guillemots [31].

The coastal-pelagic group (cluster 4) consists of species that typically feed in areas beyond the continental shelf, where certain groups of organisms (such as Gastropoda) are largely absent. By feeding on small pelagic fishes, krill, and intraguild predators of these prey, the isotope similarities indicate that Tufted Puffin (Fratercula cirrhata), Common Murre (Uria aalge), and Black-Legged Kittiwakes belong to a similar trophic guild. Despite all being waterbirds, their varied diets and geographic ranges make them valuable bioindicators for other avian groups at similar trophic levels.

2.3 Trends of plastic contamination in hosts

Some correlations between plastic abundances and their congeners were detected within the isotopic trophic groupings. Considering mean values for tissue total phthalates for each species where they were measured (n = 9 species, 110 hosts), as plastic consumption increased, so did phthalate concentrations (Figure 2). Within the trophic clusters of this sample set, plastic consumption increased in the invertebrate-consumers group (cluster 2), as did the concentration of DEHP, a plastic phthalate congener. This contaminant also increased in the coastal generalist/piscivore group, although the relative frequencies of plastics consumed were consistent. The offshore piscivorous birds showed consistent patterns for plastic frequencies (count), and the amount of plastic ingested by these birds was consistent with the DEHP plastic congener concentration in their muscle tissues. These results agree with previous works of plastic phthalate congeners of the same species [54], where the foraging strategy explained more variance in congener concentrations than geographic locality, size, or sex.

The relationship between plastic consumption and parasites has yet to be fully resolved [58, 94], yet some trends emerge from the entire dataset evaluated here (n = 356). From the individual host perspective (Figure 3), those that contained more helminths (higher intensities) also contained the most plastics. Thus, these trends appear among individuals and species. Similarly, from the host species’ perspective (Figure 4), we find that waterbirds that harbor higher maximum abundances of parasites also contain higher abundances of consumed plastics. Further, the maximum species richness of helminths increased with parasite load (abundance) and plastic frequencies at the host species level (Figure 5). The causality associated with this correlation has yet to be established, and it is unclear whether species that interact with higher frequencies of parasites also interact with more plastic debris. These are important considerations for future exploration, as contaminants and parasites can alter the probability of consumption for intermediate hosts [95], and whether they have confounding, additive, or synergistic effects on definitive host plastic consumption remains unknown.

Figure 3.

Species richness (left) and count data (right) for gut helminth infracommunities and consumed plastic microfibers for individuals from 22 species of waterbirds (colored by American Ornithological Union code). BEKI = Belted Kingfisher, BLKI = Black-legged Kittiwake, COMU = Common Murre, CRAU = Crested Auklet, DCCO = Double-crested Cormorant, GBHE = Great Blue Heron, GREG = Great Egret, GRHE = Green Heron, GWGU = Glaucous-winged Gull, HOPU = Horned Puffin, LBHE = Little Blue Heron, LEAU = Least Auklet, LESC = Lesser Scaup, NOFU = Northern Fulmar, PAAU = Parakeet Auklet, PBGR = Pied-billed Grebe, PECO = Pelagic Cormorant, PIGU = Pigeon Guillemot, RECO = Red-faced Cormorant, TBMU = Thick-billed Murre, TUPU = Tufted Puffin, WHAU = Whiskered Auklet.

Figure 4.

Count data for gut helminth infracommunities and consumed plastic microfibers for 18 species of waterbirds (colored by American Ornithological Union code) with sample sizes of 3 or more hosts. BEKI = Belted Kingfisher, BLKI = Black-legged Kittiwake, COMU = Common Murre, CRAU = Crested Auklet, DCCO = Double-crested Cormorant, GBHE = Great Blue Heron, GREG = Great Egret, GWGU = Glaucous-winged Gull, HOPU = Horned Puffin, LBHE = Little Blue Heron, LEAU = Least Auklet, LESC = Lesser Scaup, PAAU = Parakeet Auklet, PBGR = Pied-billed Grebe, PECO = Pelagic Cormorant, PIGU = Pigeon Guillemot, TBMU = Thick-billed Murre, TUPU = Tufted Puffin.

Figure 5.

The maximum species richness of intestinal helminths reported for a waterbird species compared to plastic frequencies (left = count of plastics for a species: middle = mean count of plastics within an organ) and total parasite intensity (load). Bird species are colored based on trophic cluster: large piscivores (1: black), invertebrate consumers (2: orange), coastal predators (4: green), and offshore predators (3: blue), offshore small piscivores (5: red).

As hosts migrate, they move from one foraging location to another and are exposed to differential compositions of infectious agents and contaminants. Double-crested Cormorants and Lesser Scaup collected in 2016 and 2017 were documented as consistently consuming microplastics; however, the prevalence of microplastics in our samples differed seasonally and by species (Figure 6). These birds were arriving in their southern non-breeding territories and using baitfish and catfish aquaculture facilities to feed opportunistically. In some instances, birds continued southward after stopping at the fishponds. In other instances, individual birds apparently alternated between inland and more coastal foraging habitats [96]. We observe higher frequencies of plastics in the GIT of cormorants as they arrived on the ponds from northern and southern trips, whereas just before leaving for their northern breeding grounds, fewer individuals contained plastics (R² = 0.20, df = 6, F = 3.02, p = 0.0109), but the plastic load did not differ (p = 0.3558).

Figure 6.

Monthly frequencies of plastics combined by month over 2 collections (2016–2018) of Double-crested Cormorants (DCCO) and Lesser Scaup (LESC) during the non-breeding seasons in Arkansas and Mississippi, USA.

Scaup showed a peak of plastic prevalence in January (R² = 0.111, df = 3, F = 2.846, p = 0.044); however, with only 4 months of collection data to compare (n = 71), we do not suggest that a seasonal change can be readily recognized from this analysis. Further, given that there are likely shifts in the loads of microplastic contaminants depending on when and where a host is, the individual and species-level trends we report above are compelling. As the distributions and collection times of the aggregated datasets represent what should amount to a considerable variation within a host, even moderate trends are telling of large-scale trends.

2.4 Impacts of bacterial dysbiosis on helminth communities

Birds associated with the summer 2021 die-off event on Middleton Island, Alaska, were infected with the pathogenic C. botulinum/phage combination, resulting in intoxication with BoNT/C. We note that none of the birds assessed here were directly tested for the toxin. However, following several tests, other birds from the die-off were found to contain the neurotoxin and were absent of any other disease agents and toxins [97]. As such, we consider the birds from Middleton as “diseased” birds and those we compare them to from the Western Aleutian Islands as “healthy” because there was no die-off occurring at those locations during that summer collection. The geographic distance between our two sites is more than 2000 km, and this, too, has the potential to influence the trophic interactions that a host experiences, whether that be from different prey populations, vectors, or pathogens present in the Pacific/Beringean oceanic realm (Aleutian samples) and those in the Gulf of Alaska (Middleton Island samples). Here, we present comparisons of the microbial and helminth communities of diseased and healthy birds from those collections.

We processed birds for the Middleton-Aleutian comparisons for C¹³ and N¹⁵. We found slight differences in isotopic signatures between kittiwakes and gulls but pronounced shifts in the signatures between the two seabird collections (Figure 7). Whether dysbiosis in the microbial community is influenced by C. botulinum infection or local microbe community contributions has yet to be definitively addressed; however, there appears to be distinct differentiation of isotopic signatures between both species and possibly health status.

Figure 7.

Stable isotopic heat maps indicating the trophic position of Black-legged Kittiwakes (BLKI) and Glaucus-winged Gulls (GWGU) for birds collected during non-die-off conditions (“Healthy”) between 2009 and 2019 from the western Aleutian Islands and birds of the same species collected during a presumed Avian Botulism outbreak on Middleton Island in 2021 (“Diseased”).

The endoparasites and plastics consumed by this subset of birds also differed based on location/year/disease status (Figure 8). Kittiwakes (hereafter BLKI, n = 42) from the Middleton Island die-off in 2021 contained larger counts (R² = 0.16, df = 1, p = 0.0216) and types (R² = 0.32, df = 1, p = 0.0008) of parasites; however, the number of organs infected within the GIT was lower (R² = 0.72, df = 1, p < 0.0001). Conversely, diseased birds contained fewer plastic particles when compared to healthy conspecifics collected from the western Aleutian Islands in 2016. With a smaller sample size (n = 12), Glaucus-winged Gulls exhibited similar parasite frequencies (p = 0.340), richness (p = 0.166), and plastic frequencies (0.680). However, like the BLKI, parasites were distributed among more organs in healthy birds compared to diseased conspecifics from the Middleton disease event (R² = 0.95, df = 1, p < 0.0001).

Figure 8.

Frequencies of parasites and plastics in two species of seabirds (Black-legged Kittiwakes [BLKI] and Glaucus-winged Gulls [GWGU]) from a die-off event in 2021 on Middleton Island (diseased) and the western Aleutian Island (healthy) of Alaksa. Metrics include the number of parasites found in a host (abundance), the number of parasite species in a host (richness), the mean number of host organs that were infected, and the frequency of consumed plastics observed (plastic number). Lines above paired bars indicate significant differences at 0.05 (*), 0.001 (**) and <0.0001 (***).

In addition to differences in the endoparasite communities of birds associated with a die-off event, similar trends in ectoparasite ecological metrics occurred in these birds [89]. Moreover, differences occurred in the microbiome of these hosts based on ecological metrics (alpha, beta, and gamma diversity) for BLKI [89]. Although the microbial communities were based on each bird’s ectoparasites and surficial symbionts, we document marginal differences in microbial composition based on Constrained Analysis of Principal Coordinates informed by helminth communities and year (Figure 9). We found differences between healthy and diseased birds (p = 0.046; R² = 0.67; CAP 1 = −0.32, CAP 2 = −0.58); however, we cannot conclusively say whether geographic, temporal, or disease status differences drove these differences. Further, no parasite metrics provided explanatory power of host separation in ordinate space (p = 0.298). Similarly, plastic ingestion did not impact the surficial microbial communities of the birds assessed here (p = 0.283).

Figure 9.

Ordination plot based on principle coordinates analysis of microbial communities of Black-legged Kittiwakes from a collection of birds taken in the Western Aleutian Islands in 2016 (blue) and a presumed Avian Botulism outbreak in the Gulf of Alaska in 2021 (red). Metadata for evaluation of ordinate separation based on parasite infracommunity compositions (left) and consumed microplastic frequencies (right).

Helminthic communities can drive microbial community alterations [83, 84, 85], so the interplay between microbial responses to helminthic parasites and helminths’ responses to microbial pathogens remains to be elucidated. This will likely require laboratory experiments where helminth and pathogen infection dynamics can be controlled.

3. Conclusions

The utility of endoparasites as ecological indicators for host foraging behavior allows researchers and managers to understand the consequences of land use change, expanding reaches of plastic contamination, and expanding ranges of pathogens like C. botulinum.

When assessing the complete suite of waterbirds included in these analyses, we found that the infracommunities of birds varied by location (Figure 8), season (Figure 6), and, most importantly, bird species (Figures 2–5). As waterbird hosts are also recognized as sentinels of ecosystem change [49], the prospect of parasite assessments to more fully evaluate alterations in trophic structure is compelling. Further, the differentiation resolution observed here confirms the utility of parasite intracommunity assessments in the niche divergences of birds that forage in similar habitats and at similar trophic levels (Figure 5).

Previous works demonstrate that trophic strategies can correlate with plastic contamination [54]; however, many of those studies report macroplastics as opposed to microplastics evaluated here and did not find that phthalate concentrations correlated with increased plastics consumption. Here, we provide evidence that this is the case so long as microplastics are also considered, as plastic consumption increased, so did phthalate concentrations in tissues, especially total phthalates and DEHP.

While the contaminant concentrations in host tissues were not reflected in parasite infracommunity metrics, plastic frequencies did correspond with higher intensities. However, causality between parasite burden and plastic consumption remains unclear. There is the potential for parasitized intermediate hosts to have an increased probability of plastic exposure or entanglement. Likewise, it could be that intermediate hosts interacting with plastic are more susceptible to parasitic infection. Further experimentation would help understand the dynamic relationships.

It is necessary for the prosperity and security of human populations to consider the impact of land use change and contamination from anthropogenic waste products, such as single-use plastics, on natural systems and organisms. We have discovered a notable positive correlation between parasites and plastic frequencies and have found that aquaculture facilities appear to be sinks rather than sources of plastic contamination. The interaction of human-created stressors on organisms and their symbionts undoubtedly responds to changes in these systems. There is an urgent need for highly replicated and robust research to determine their consequences.

Birds from Middleton Island, affected by a 2021 die-off, likely due to C. botulinum/phage exposure, had distinct parasite communities compared to healthy birds from the Western Aleutian Islands. Geographic distance between sites may affect trophic interactions, and the isotopic signatures of birds from the die-off suggest a substantial shift and contraction of dietary niche breadth in the diseased hosts. Middleton birds had more parasites but fewer plastics in their GI tracts than healthy Aleutian birds. Ectoparasite metrics and microbial diversity varied between diseased and healthy birds, possibly influenced by helminth communities. Further study is needed to understand microbial responses to helminths and vice versa.

The Bering Sea region faces numerous environmental threats impacting its health and the sustainability of its communities and fisheries, from global climate change to marine debris pollution. Bering Sea communities are working to maintain a healthy ecosystem in many ways, including taking leadership actions to clean up marine debris. Efforts to manage this ecosystem would be informed by a better understanding of the ecological and physiological relationships of seabirds with their prey, their parasites, and plastic contaminants.

Acknowledgments

We express our sincere gratitude to the decade of training and funding contributions that helped make the development and execution of these ideas possible. Agencies that contributed financial and additional resources include the National Science Foundation (USA), the U.S. Department of Agriculture/Animal Plant Health Inspection Service/Wildlife Services/National Wildlife Research Center (NWRC), and the National Oceanographic and Atmospheric Association. Institutional contributions include Frostburg State University, University of Alaska Anchorage, University of California, San Diego (UCSD) and Santa Barbara, Scripps Institute of Oceanography, University of Georgia Southeastern Cooperative for Wildlife Health Service, and Clemson University. We also thank collaborators at the New England and Cape Wildlife Centers for their expertise and donation of specimens.

Specimens were collected under permits ADFG 16-096, 17-093, 18-100, 19-152, 22-101; USFWS MB 795841; and IACUC protocols 1216862, 1216863, 1721620, 1721621.

Funding for specimen collection and processing came, in part, from NSF EEID (OCE-1115965), the Southern Regional Aquaculture Center Grant number 2016-38500-25752 from the U.S. Department of Agriculture National Institute of Food and Agriculture, a Frontiers of Innovation Scholars Program grant from UCSD, a 2022-2023 Research Publication Grant in Engineering, Medicine, and Science from the American Association of University Women, and cooperative agreements with the U.S. Department of Agriculture, Animal Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center 17-7428-1326-CA and 18-7428-1326-CA.

Thanks

We thank the dozens of university students and technicians who assisted in processing samples, data entry, diet analysis, and parasitology for the hundreds of birds included in our analyses. In particular, Dr. Veronica Padula’s hard work in tissue assessments for plastic congeners is most appreciated.