Batrachochytrium dendrobatidis continues to cause declines in amphibian populations worldwide, and it remains unclear why skin defenses often fail to control the infection. Although amphibians have a complex, multifunctional immune system, the chytridiomycosis agent seems to have evolved countermeasures that enable it to survive and eventually impair critical skin functions. Previous studies show that B. dendrobatidis cells or cell-free supernatants inhibit lymphocytes by inducing apoptosis, suggesting impaired local cell killing. However, there is little evidence of lymphocyte recruitment to chytrid-infected skin, implying the fungus may also inhibit the functions of antigen-presenting cells. Here, we demonstrate that phagocytosis by peritoneal macrophages is significantly reduced by co-culture with live or heat-killed B. dendrobatidis zoosporangia, freeze-thawed zoospores, fungal cell-free supernatants, or cell-wall fragments. The phagocytic capacity of frog bone marrow-derived macrophages, differentiated by colony-stimulating factor-1 (CSF-1) or interleukin-34 (IL-34) (key macrophage growth factors), as well as immortalized mammalian macrophages, is also impaired. Inhibition of mammalian macrophages suggests that these inhibitory factors are not restricted to amphibian cells. Overall, these studies indicate that B. dendrobatidis cells and their components can hinder the recognition and function of macrophages that reside in or enter the skin to clear infections. This disabling of host phagocytosis is undoubtedly central to how B. dendrobatidis prevents effective innate and adaptive immune responses in the skin.

Strategies to improve survival of wildlife impacted by emerging infectious disease are needed for species of conservation concern. We investigated how prior infection by the skin fungal pathogen, Batrachochytrium dendrobatidis (Bd), affected subsequent disease outcomes in a highly susceptible and critically endangered toad, Atelopus glyphus. Our experimental design consisted of: (1) Bd infection followed by itraconazole clearance and Bd re-infection (Bd-Bd n = 20), (2) mock infection followed by itraconazole treatment and Bd infection (mock-Bd n = 20), (3) Bd infection followed by itraconazole clearance and mock infection (Bd-mock n = 10), (4) mock infection followed by itraconazole treatment and mock infection (mock-mock n = 10), (5) untreated animals (untreated n = 10). Prior infection shifted the skin bacterial and fungal communities to a new state enriched in putatively anti-Bd bacteria. However, prior infection worsened disease outcomes, accelerating Bd infection and mortality in prior infected animals compared to naïve animals. Prior infection animals not re-infected (Bd-mock) showed immune gene downregulation compared to re-infected Bd-Bd animals suggesting that even after Bd clearance, Bd caused sustained immunosuppression in genes that respond to Bd; this likely led to lowered immune efficacies when re-infected. Upon re-infection, as Bd load increased, Bd became the primary driver of both immune expression and bacterial composition changes regardless of infection history. Observed bacterial community differences were closely tied to host immune gene expression. The fungal community did not shift in response to re-infection, suggesting that the initial shift was due to itraconazole treatment. Prior infected Bd-Bd animals did not show any novel shifts in immune gene expression, rather they had similar expression patterns that were just more pronounced compared to naïve mock-Bd animal. This suggests that the infection-clearance-infection treatment was ineffective in improving animal survival because there was limited long-lasting effects on immune genes once the pathogen was re-encountered. Unlike previous studies demonstrating protective effects following Bd clearance in other species, this highly susceptible amphibian showed no evidence of acquired resistance. Our results underscore the species-specific nature of disease outcomes and the value of integrative approaches for examining strategies to reduce pathogen susceptibility in endangered wildlife.

Mast cells are tissue-resident granulocytes that are often viewed through a negative lens due to their roles in allergies and hypersensitivity. While central to these conditions, mast cells have broader roles in vertebrate immune systems that are often overlooked. Whereas most mast cell research to date has been conducted in mammalian models, mast-like cells are found across vertebrates, presumably with conserved and divergent functions in disparate immune systems. Our past studies defined a role of amphibian (Xenopus laevis) mast cells in skin antifungal defenses but stopped short of functionally characterizing these immune effectors. Presently, to further elucidate the biology of amphibian mast cells and to delineate evolutionarily conserved and divergent mast cell characteristics, we developed in vitro X. laevis mast cell cultures from peritoneal-derived and bone marrow-derived precursors. The phenotypes, transcriptomes and degranulation capacities were examined and compared between these frog mast cell cultures and to those of frog neutrophils, a distinct granulocyte lineage. Our results indicate that X. laevis mast cells are distinct from frog neutrophils and share many canonical features that are associated with mammalian mast cells. Akin to their mammalian counterparts, frog mast cells are capable of degranulation and release potent immune proteins and lipid mediators. Notably, although many characteristics appear to be shared between frog and mammalian mast cells, our findings also indicate unique aspects to these frog leukocytes. Future studies of nonmammalian mast cells will grant unique perspectives of the evolutionary pressures that shape mast cell defenses and associated pathologies.

IntroductionThe California purple sea urchin, Strongylocentrotus purpuratus, relies exclusively on an innate immune system to survive in its pathogen rich marine environment. Central to this defense is the SpTransformer (SpTrf) gene family that is unique to the euechinoid group of echinoderms. These genes were initially identified based on their striking upregulation in response to immune challenge. The SpTrf gene family encodes structurally similar proteins with a wide range of sequence diversity within and among individual sea urchins. A recombinant (r)SpTrf protein interacts specifically with a variety of non-self targets. Other rSpTrf proteins cross-linked to inert beads show distinct functions for cell binding and augmenting phagocytosis . However, whether the rSpTrf proteins bind to sea urchin phagocytes, and the cellular consequences of binding are largely unexplored. MethodsrSpTrf protein binding to, and responses by phagocytes was investigated by cytology, flow cytometry, binding competitions using In-cell ELISA, and gene expression analyses. ResultsSoluble rSpTrf proteins bind specifically and exclusively to both live and fixed polygonal and small phagocytes. The different rSpTrf proteins appear to bind shared receptor(s) or other form of cell surface binding site. The phagocyte response to bound rSpTrf proteins culminates in modulated expression of the SpTrf gene family as well as other immune-related genes. ConclusionsThese findings underscore the multifaceted and dynamic functions of SpTrf proteins within the innate immune system of the purple sea urchin. Their varied functions enable a robust immune response while also providing a unique modulatory mechanism by which response levels are controlled and adjusted to the level of the foreign threat.

The chytrid fungus, Batrachochytrium dendrobatidis (Bd), infects amphibian skin, causing chytridiomycosis, which is a contributing cause of worldwide declines and extinctions of amphibians. Relatively little is known about the roles of amphibian skin-resident immune cells, such as macrophages, in these antifungal defenses. Across vertebrates, macrophage differentiation is controlled through the activation of colony-stimulating factor-1 (CSF1) receptor by CSF1 and interleukin-34 (IL34) cytokines. While the precise roles of these respective cytokines in macrophage development remain to be fully explored, our ongoing studies indicate that frog (Xenopus laevis) macrophages differentiated by recombinant forms of CSF1 and IL34 are functionally distinct. Accordingly, we explored the roles of X. laevis CSF1- and IL34-macrophages in anti-Bd defenses. Enriching cutaneous IL34-macrophages, but not CSF1-macrophages, resulted in significant anti-Bd protection. In vitro analysis of frog macrophage-Bd interactions indicated that both macrophage subsets phagocytosed Bd. However, IL34-macrophages cocultured with Bd exhibited greater pro-inflammatory gene expression, whereas CSF1-macrophages cocultured with Bd showed greater immunosuppressive gene expression profiles. Concurrently, Bd-cocultured with CSF1-macrophages, but not IL34-macrophages, possessed elevated expression of genes associated with immune evasion. This work marks a step forward in our understanding of the roles of frog macrophage subsets in antifungal defenses.

Probiotic therapies have been suggested for amelioration efforts of wildlife disease such as chytridiomycosis caused by Batrachochytrium spp. in amphibians. However, there is a lack of information on how probiotic application affects resident microbial communities and immune responses. To better understand these interactions, we hypothesized that probiotic application would alter microbial community composition and host immune expression in Xenopus laevis. Accordingly, we applied three amphibian-derived and anti-Batrachochytrium bacteria strains (two Pseudomonas spp. and one Stenotrophomonas sp.) to X. laevis in monoculture and also as a cocktail. We quantified microbial community structure using 16S rRNA gene sequencing. We also quantified genes involved in X. laevis immune responses using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and skin transcriptomics over 1 and 3-week periods. All probiotic treatments successfully colonized X. laevis skin for 3 weeks, but with differential amplicon sequence variant (ASV) sequence counts over time. Bacterial community and immune gene effects were most pronounced at week 1 post-probiotic exposure and decreased thereafter. All probiotic treatments caused initial changes to bacterial community alpha and beta diversity, including reduction in diversity from pre-exposure anti-Batrachochytrium bacterial ASV relative abundance. Probiotic colonization by Pseudomonas probiotic strain RSB5.4 reduced expression of regulatory T cell marker (FOXP3, measured with RT-qPCR) and caused the greatest gene expression changes detected by transcriptomics. Single bacterial strains and mixed cultures, therefore, altered amphibian microbiome-immune interactions. This work will help to improve our understanding of the role of the microbiome-immune interface underlying both disease dynamics and emergent eco-evolutionary processes.IMPORTANCEAmphibian skin microbial communities have an important role in determining disease outcomes, in part through complex yet poorly understood interactions with host immune systems. Here we report that probiotic-induced changes to the Xenopus laevis frog skin microbial communities also result in significant alterations to these animals' immune gene expression. These findings underscore the interdependence of amphibian skin immune-microbiome interactions.

Disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), are compounding global amphibian declines. Bd infections are confined to amphibian skin, and there are many published studies describing the roles of amphibian skin commensals and skin-produced antimicrobial components in anti-Bd defenses. In contrast, relatively little is known about the roles of skin-resident immune cells, such as mast cells, in amphibian antifungal immunity. Indeed, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Thus, we investigated the roles of frog (Xenopus laevis) mast cells during Bd infections. Our findings indicate that frog mast cells confer antifungal protection by reducing neutrophil infiltration into Bd-infected skins, ablating inflammation-associated damage therein and promoting mucus production by cutaneous mucus glands. Akin to mammalian mast cells, which produce copious amounts of the pleiotropic interleukin-4 (IL4) cytokine, frog mast cells confer their anti-Bd protection at least in part through IL4 production. Together, our findings underscore the importance of skin-resident immune cells in amphibian anti-Bd defenses and suggest a new direction to explore host-chytrid pathogen interactions. Support: NSF 2147466, NSF 2131061 (LG) and NSF 2147467 (LR-S). Veterinary and Comparative Immunology (VET)

Xenopus laevis juvenile frogs regenerate wounded skin without scarring, yet the underlying mechanisms driving this process remain poorly defined. Macrophages are critical to wound repair across vertebrates, and our results indicate a transient influx of macrophages into regenerating frog wounds. The colony stimulating factor-1 (CSF1) and interleukin-34 (IL34) growth factors control macrophage development. Through RNA in situ hybridization studies, we found that csf1 gene expression peaked early during juvenile frog wound responses, whereas il34 expression increased later in the repair process. Our past studies indicate that X. laevis CSF1- and IL34-differentiated macrophages are functionally distinct. Presently, we treated frog wounds with recombinant (r)CSF1 and rIL34 to determine the roles of the corresponding macrophage subsets in wound repair. Using a combination of RNA in situ hybridization, RNA sequencing and histology, we demonstrated that wounds skewed towards greater proportions of rCSF1-macrophages exhibited greater infiltration of leukocytes, chiefly amongst them neutrophils. These wounds also possessed robust expression of inflammatory genes and transcripts associated with granulation and fibrosis. By contrast, rIL34-treated frog wounds exhibited greater fibroblast activation concurrent with greater type I/III collagen ratios and expression of genes typically seen at later phases of wound repair. Together, we propose that while CSF1-macrophages are likely more prominently involved in the inflammatory phase of X. laevis wound repair, IL34-macrophages predominate the later reparative phase of these responses.

Amphibians continue to experience global population declines due to chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Although amphibians have robust immune defenses, this pathogen appears to have evolved counter defenses that permit it to survive in amphibian skin. Previous studies showed that Bd cells or cell-free supernatants inhibited lymphocytes by induction of apoptosis, suggesting impaired local cell killing. However, there is little evidence for lymphocyte recruitment to chytrid-infected skin, suggesting that the fungus may also inhibit functions of antigen presenting cells. The capacity of peritoneal leukocytes (PLs) enriched in macrophages (Mfs) and neutrophils to phagocytose pHrodo™ Green Zymosan BioParticles™ was significantly reduced by co-culture with Bd cells. Cell-free supernatants produced by Bd also inhibited uptake of pHrodo™ Red Staph aureus BioParticles™. The phagocytic capacities of frog bone marrow-derived Mfs, differentiated by colony stimulating-factor-1- or interleukin-34 (key Mf growth factors), as well as a mammalian Mf cell line, were also impaired by Bd. Inhibition of mammalian Mfs suggests that the functions of the inhibitory factors are not restricted to amphibian cells. These findings suggest that Bd cells and cell-derived factors can inhibit recognition by Mfs that reside in or would enter the skin compartment to begin the process of clearing a Bd infection. Support: NSF 2147466 (LG) and 2147467 (to LR-S). Supported by NSF 2147466 ( to LG) and 2147467 (to LR-S) Microbial, Parasitic, and Fungal Immunology (MPF)

Amphibian metamorphosis represents a dramatic example of post-embryonic development. In the anuran Xenopus laevis frog, this process involves extensive changes to larval tissues, structures, and physiology to produce its adult form. As a long-standing model to study tissue remodeling, both amphibian metamorphosis and mammalian development are under the control of thyroid hormone. Successful remodeling though, also requires precise temporospatial regulation of immune activation. Yet there is much to learn about the immune components linked to metamorphosis. In turn, granulocytes are a class of innate immune cells recently touted for their participation in processes beyond classical immune defenses, including in pathological and non-pathological tissue remodeling. In this manuscript, we explore the roles of granulocytes in perhaps the most conspicuous anuran metamorphic event: tadpole tail reabsorption. We characterize granulocyte infiltration into the tail as metamorphosis progresses. Although some granulocyte subpopulations exist in both Xenopus and mammals, our previous work has identified additional Xenopus-specific populations. Thus, here we further explored subpopulation dynamics through distinct stages of natural metamorphosis, their likely roles during this process, and their relationship with thyroid hormone. As endocrine disruptors continue to threaten species across the animal kingdom, the work described here offers much-needed insight into immune contributions to endocrine-linked development.

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Global amphibian declines are compounded by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis ( Bd ). Much has been learned about the roles of amphibian skin-produced antimicrobial components and microbiomes in controlling Bd , yet almost nothing is known about the roles of skin-resident immune cells in anti- Bd defenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers anti- Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. This includes a significant reduction in infiltration of Bd -infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventing Bd -mediated changes to skin microbiomes. Mammalian mast cells are known for their production of the pleiotropic interleukin-4 (IL4) cytokine and our findings suggest that the X. laevis IL4 plays a key role in manifesting the effects seen following cutaneous mast cell enrichment. Together, this work underscores the importance of amphibian skin-resident immune cells in anti- Bd defenses and illuminates a novel avenue for investigating amphibian host–chytrid pathogen interactions.

AbstractRanaviruses (RV, family Iridoviridae) infect fish, amphibians, and reptiles, raising considerable ecological and commercial concerns due to the escalating infection prevalence and the resulting die-offs of wild and aquacultural species. Notably, ranaviruses exhibit uncanny capacities to cross host species barriers, likely owing to their potent immune evasion mechanisms. In turn, the species infected by these pathogens possess immune systems that are less well understood than those of mammals and often encode unique antiviral genes or multiple orthologs of single hallmark mammalian immune factors. Thus, garnering insight into ranavirus infection strategies is largely contingent on gaining greater understanding of host immune barriers faced by these emerging infectious agents. Accordingly, here we coalesce and update the current state of understanding of the distinct facets of ectothermic vertebrate immune responses to ranaviral infections and underline the most current perspectives of the evasion strategies by which these pathogens circumvent host defenses.

IntroductionThe California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis.MethodsTo address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. ResultsThe rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. ConclusionThese findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.

Macrophage (Mϕ)-lineage cells are integral to the immune defences of all vertebrates, including amphibians. Across vertebrates, Mϕ differentiation and functionality depend on activation of the colony stimulating factor-1 (CSF1) receptor by CSF1 and interluekin-34 (IL34) cytokines. Our findings to date indicate that amphibian (Xenopus laevis) Mϕs differentiated with CSF1 and IL34 are morphologically, transcriptionally and functionally distinct. Notably, mammalian Mϕs share common progenitor population(s) with dendritic cells (DCs), which rely on fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation while X. laevis IL34-Mϕs exhibit many features attributed to mammalian DCs. Presently, we compared X. laevis CSF1- and IL34-Mϕs with FLT3L-derived X. laevis DCs. Our transcriptional and functional analyses indicated that indeed the frog IL34-Mϕs and FLT3L-DCs possessed many commonalities over CSF1-Mϕs, including transcriptional profiles and functional capacities. Compared to X. laevis CSF1-Mϕs, the IL34-Mϕs and FLT3L-DCs possess greater surface major histocompatibility complex (MHC) class I, but not MHC class II expression, were better at eliciting mixed leucocyte responses in vitro and generating in vivo re-exposure immune responses against Mycobacterium marinum. Further analyses of non-mammalian myelopoiesis akin to those described here, will grant unique perspectives into the evolutionarily retained and diverged pathways of Mϕ and DC functional differentiation. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Granulocyte-lineage cells are important innate immune effectors across all vertebrates. Named for conspicuous secretory granules, granulocytes have historically been studied for their antimicrobial roles. Although versions of these cells are found in all vertebrate species examined to date, disparate environmental and physiological pressures acting on distinct vertebrate classes have shaped many of the facets dictating granulocyte biology. Immune pressures further determine granulopoietic constraints, ultimately governing granulocyte functions. For amphibians that inhabit pathogen-rich aquatic environments for some or all their lives, their unique granulocyte biologies satisfy many of their antimicrobial needs. Amphibians also occupy an intermediate position in the evolution of vertebrate immune systems, using combinations of primitive (e.g., subcapsular liver) and more recently evolved (e.g., bone marrow) tissue sites for hematopoiesis and specifically, granulopoiesis. The last decade of research has revealed vertebrate granulocytes in general, and amphibian granulocytes in particular, are more complex than originally assumed. With dynamic leukocyte phenotypes, granulocyte-lineage cells are being acknowledged for their multifaceted roles beyond immunity in other physiological processes. Here we provide an overview of granulopoiesis in amphibians, highlight key differences in these processes compared to higher vertebrates, and identify open questions.

Xenopus is a genus of African clawed frogs including two species, X. tropicalis and X. laevis that are extensively used in experimental biology, immunology, and biomedical studies. The availability of fully sequenced and annotated Xenopus genomes is strengthening genome-wide analyses of gene families and transgenesis to model human diseases. However, inaccuracies in genome annotation for genes involved in the immune system (i.e., immunome) hamper immunogenetic studies. Furthermore, advanced genome technologies (e.g., single-cell and RNA-Seq) rely on well-annotated genomes. The annotation problems of Xenopus immunome include a lack of established orthology across taxa, merged gene models, poor representation in gene pages on Xenbase, misannotated genes and missing gene IDs. The Xenopus Research Resource for Immunobiology in collaboration with Xenbase and a group of investigators are working to resolve these issues in the latest versions of genome browsers. In this review, we summarize the current problems of previously misannotated gene families that we have recently resolved. We also highlight the expansion, contraction, and diversification of previously misannotated gene families.