Transcriptomics reveal immune downregulation of newts overwhelmed by chytrid co-infection.

What happens when two emergent diseases infect the same host? In a From the Cover article in this issue of Molecular Ecology, McDonald et al. (2020) compare transcriptomic responses to co-infection by the two chytrid fungi in the skin, liver and spleen of Eastern newts (Notophthalmus viridescens). Novel molecular tools, such as high-throughput DNA sequencing for genome discovery and transcriptomics, have revolutionized our understanding of host-pathogen interactions and disease ecology (Güimil et al. 2005; Rosenblum et al. 2012). For example, epidemiologists are using genomic data to track the spread of the emergent SARS-CoV-2 in real time, both locally and globally. RNA sequencing (RNA-Seq) is routinely employed to study response to disease in humans, improving disease diagnostics, profiling and development of intervention strategies. Transcriptomic profiles may be particularly informative for emergent diseases, whose pathologies and effect on host phenotype are poorly known. Fungal pathogens increasingly threaten a variety of wild and domesticated organisms (Fisher et al. 2012), and two chytrid fungi attacking amphibians are causing one of the worst losses of vertebrate biodiversity ever recorded (Scheele et al. 2019).

Modulation of Iberian Ribbed Newt Complement Component C3 by Stressors Similar to those Encountered during a Stay Onboard the International Space Station.

The complement system plays an important role in inflammation, innate and acquired immunity, as well as homeostasis. Despite these functions, the effects of spaceflight conditions on the complement system have not yet been intensively studied. Consequently, we investigated the effects of five types of chronic stressors, similar to those encountered during a stay onboard the International Space Station, on C3 expression in larvae of the urodele amphibian Pleurodeles waltl. We focused on C3 because it is a critical component of this system. These studies were completed by the analysis of adult mice exposed to two models of inflight stressors. Our data show that simulating space radiation, or combining a modification of the circadian rhythm with simulated microgravity, affects the amount of C3 proteins. These results suggest that C3 expression could be modified under real spaceflight conditions, potentially increasing the risk of inflammation and associated tissue damage.

Mycolactone-producing Mycobacterium marinum infection in captive Hong Kong warty newts and pathological evidence of impaired host immune function.

A mass mortality event of captive Hong Kong warty newts Paramesotriton hongkongensis with non-granulomatous necrotic lesions occurred in Taipei Zoo, Taiwan, in 2014. Clinically, the sick newts were lethargic and often covered with water mold Saprolegnia sp. on the skin of the body trunk or extremities. Predominant pathological findings were multifocal non-granulomatous necrotic lesions in the liver, spleen, and kidneys and severe skin infection with Saprolegnia sp., with deep invasion and involvement of underlying muscles. The possibility of ranavirus infection was ruled out by negative PCR results. Unexpectedly, abundant intralesional acid-fast positive bacilli were found in the necrotic lesions of the liver, spleen, and kidney in all 14 sick newts. PCR targeting the hsp65, ITS region, and partial 16S rRNA genes was performed, and the sequence identity from amplified amplicons of hsp65 and partial 16S rRNA genes was 100% identical to that of the corresponding gene fragment of Mycobacterium marinum. Further molecular investigations demonstrated that the current M. marinum was a mycolactone-producing mycobacterium with the presence of esxA/esxB genes. Mycolactone is a plasmid-encoded, immunosuppressive, and cytotoxic toxin. The possible immunosuppression phenomenon characterized by systemic non-granulomatous necrotic lesions caused by M. marinum and the unusual deep invasive infection caused by water mold might be associated with the immunosuppressive effect of mycolactone. Therefore, it should be noted that non-granulomatous necrotic lesions in amphibians can be caused not only by ranavirus infection but also by mycobacteriosis.

Physiological Responses to Salinity Vary with Proximity to the Ocean in a Coastal Amphibian.

Freshwater organisms are increasingly exposed to elevated salinity in their habitats, presenting physiological challenges to homeostasis. Amphibians are particularly vulnerable to osmotic stress and yet are often subject to high salinity in a variety of inland and coastal environments around the world. Here, we examine the physiological responses to elevated salinity of rough-skinned newts (Taricha granulosa) inhabiting a coastal stream on the Pacific coast of North America and compare the physiological responses to salinity stress of newts living in close proximity to the ocean with those of newts living farther upstream. Although elevated salinity significantly affected the osmotic (body weight, plasma osmolality), stress (corticosterone), and immune (bactericidal ability) responses of newts, animals found closer to the ocean were generally less reactive to salt stress than those found farther upstream. Our results provide possible evidence for some physiological tolerance in this species to elevated salinity in coastal environments. As freshwater environments become increasingly saline and more stressful, understanding the physiological tolerances of vulnerable groups such as amphibians will become increasingly important to our understanding of their abilities to respond, to adapt, and, ultimately, to survive.

Condition-dependent trade-offs between sexual traits, body condition and immunity: the effect of novel habitats.

BACKGROUND: The optimal allocation of resources to sexual signals and other life history traits is usually dependent on an individual's condition, while variation in the expression of sexual traits across environments depends on the combined effects of local adaptation, mean condition, and phenotypic responses to environment-specific cues that affect resource allocation. A clear contrast can often be drawn between natural habitats and novel habitats, such as forest plantations and urban areas. In some species, males seem to change their sexual signals in these novel environments, but why this occurs and how it affects signal reliability is still poorly understood. RESULTS: The relative size of sexual traits and level of immune responses were significantly lower for male palmate newts Lissotriton helveticus caught in pine and eucalyptus plantations compared to those caught in native forests, but there was no habitat-dependent difference in body condition (n = 18 sites, 382 males). The reliability with which sexual traits signalled body condition and immune responses was the same in all three habitats. Finally, we conducted a mesocosm experiment in which males were maintained in pine, eucalypt or oak infused water for 21 days. Males in plantation-like water (pine or eucalypt) showed significantly lower immune responses but no change in body condition. This matches the pattern seen for field-caught males. Unlike field-caught males, however, there was no relationship between water type and relative sexual trait size. CONCLUSIONS: Pine and eucalyptus plantations are likely to be detrimental to male palmate newt because they are associated with reduced immune function and smaller sexual traits. This could be because ecological aspects of these novel habitats, such as high water turbidity or changes in male-male competition, drive selection for reduced investment into sexual traits. However, it is more probable that there are differences in the ease of acquisition, hence optimal allocation, of resources among habitats. Our mesocosm experiment also provides some evidence that water toxicity is a causal factor. Our findings offer insights into how plantations affect amphibian life histories, and how novel habitats might generate long-term selection for new resource allocation strategies in native species.

Experimental evidence for a cost of resistance to the fungal pathogen, Batrachochytrium dendrobatidis, for the palmate newt, Lissotriton helveticus.

BACKGROUND: Batrachochytrium dendrobatidis (Bd), the causative agent of chytridiomycosis, is decimating amphibians worldwide. Unsurprisingly, the majority of studies have therefore concentrated on documenting morbidity and mortality of susceptible species and projecting population consequences as a consequence of this emerging infectious disease. Currently, there is a paucity of studies investigating the sub-lethal costs of Bd in apparently asymptomatic species, particularly in controlled experimental conditions. Here we report the consequences of a single dose of B. dendrobatidis zoospores on captive adult palmate newts (Lissotriton helveticus) for morphological and behavioural traits that associate with reproductive success. RESULTS: A single exposure to ~2000 zoospores induced a subclinical Bd infection. One week after inoculation 84% of newts tested positive for Bd, and of those, 98% had apparently lost the infection by the day 30. However, exposed newts suffered significant mass loss compared with control newts, and those experimental newts removing higher levels of Bd lost most mass. We found no evidence to suggest that three secondary sexual characteristics (areas of dorsal crest and rear foot webbing, and length of tail filament) were reduced between experimental versus control newts; in fact, rear foot webbing was 26% more expansive at the end of the experiment in exposed newts. Finally, compared with unexposed controls, exposure to Bd was associated with a 50% earlier initiation of the non-reproductive terrestrial phase. CONCLUSIONS: Our results suggest that Bd has measureable, but sub-lethal effects, on adult palmate newts, at least under the laboratory conditions presented. We conclude that the effects reported are most likely to be mediated through the initiation of costly immune responses and/or tissue repair mechanisms. Although we found no evidence of hastened secondary sexual trait regression, through reducing individual body condition and potentially, breeding season duration, we predict that Bd exposure might have negative impacts on populations of palmate newts through reducing individual reproductive success and adult recruitment.

New(t)s and views from hybridizing MHC genes: introgression rather than trans-species polymorphism may shape allelic repertoires.

One of the key features of major histocompatibility complex (MHC) genes is the frequent occurrence of trans-species polymorphism, that is 'the passage of allelic lineages from ancestral to descendant species' (Klein et al. 2007). Selectively maintained ancestral polymorphism may, however, be hard to distinguish from introgression of MHC alleles between hybridizing species (Fig. 1). In this issue of Molecular Ecology, Nadachowska-Brzyska et al. (2012) present data that suggest that the latter can be observed in two closely related species of newts, Lissotriton vulgaris (Lv) and L. montandoni (Lm) from south-east Europe. Strikingly, allelic MHC variation displayed more structure between geographically separated populations of L. vulgaris than across species in the hybrid zone. This suggests that high MHC variation in L. montadoni may result from mainly unidirectional gene flow between species, while differentiation between northern and southern populations of L. vulgaris might reflect local adaptation.

Chronic transplantation immunity in newts: temperature susceptibility of an effector phase in allo-skin graft rejection.

Urodele amphibians are unique due to their greatly reduced immune responsiveness compared to bony fishes, which show acute immune responsiveness. In newts, the mean survival time of allogenic skin grafts in the transplantation immunity was 48.8 ± 8.3 days at 25°C, suggesting that it occurs in a chronic manner. The graft rejection process was categorized into three stages: a latent stage with frequent blood circulation, or the immune induction phase; a vascular stoppage stage with dominant infiltrating cells of T cells; and a rejection stage showing the change of the dominant cells to monocytes/macrophages, probably as effector cells, tetntatively referred to as the immune effector phase. The immune induction phase is susceptible to the cyclophosphamide (CY) mitosis inhibitor, but not to a temperature shift from 18 to 27°C, while the immune effector phase is susceptible to temperature shifts, but not CY-treatment, although the temperature shift failed to shorten the graft survival time to less than 25 days, which nearly equals that of the secondary set of grafts where the lack of complete blood circulation is remarkable and graft rejection is resistant to CY-treatment. In contrast, a very low temperature (5-10°C) completely prevented effector generation in newts; in frogs, however, it is reported that such low temperatures did not prevent the generation of effectors. Taken together, these data suggest that chronic responses in newts are due to effector cells other than cytotoxic T cells; possible effector cells are discussed.

Long-term survival of a urodele amphibian despite depleted major histocompatibility complex variation.

Depletion of polymorphism at major histocompatibility complex (MHC) genes has been hypothesized to limit the ability of populations to respond to emerging pathogens, thus putting their survival at risk. As pathogens contribute substantially to the global amphibian decline, assessing patterns of MHC variation is important in devising conservation strategies. Here, we directly compare levels of MHC class II and neutral variation between multiple populations of the great crested newt (Triturus cristatus) from refugial (REF: Romania) and postglacial expansion (PGE: Germany, Poland and UK) areas. REF populations harboured high levels of adaptive variation (24 expressed alleles), exhibiting clear signatures of historical positive selection, which points to the overall importance of MHC class II variation in this species. On the other hand, PGE populations were extremely depauperate (two alleles) but nevertheless have survived for c. 10,000 years, since the postglacial expansion. Variation in putative MHC class II pseudogenes, microsatellites and allozymes also showed a significant southern richness-northern purity pattern. The populations in the postglacial expansion area thus provide the clearest example to date of the long-term survival of populations in which MHC variation, historically under positive selection, has been depleted.

Isolation of the third component of complement and its derivative with anaphylatoxin-like activity from the plasma of the newt Cynops pyrrhogaster.

The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta-chains of C3 were 120,000 and 70,000, respectively) linked by disulfide bonds. The alpha-chain retained an internal thiolester bond that was cleaved with methylamine, and the N-terminal amino acid sequence of the alpha-chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results indicate that newt C3 retains structural and functional properties shared with mammalian C3.

A developmentally regulated wound epithelial antigen of the newt limb regenerate is also present in a variety of secretory/transport cell types.

The role of the wound epithelium in amphibian limb regeneration is not understood. We showed previously that monoclonal antibody (mAb) WE3 stains the wound epithelium but not skin epidermis, suggesting that the WE3 antigen may be a marker for, or be important in, the function of the wound epithelium. In the present study, we conducted an extensive immunohistochemical survey of adult newt tissues to define the distribution of the WE3 antigen. The results show that the antigen is most commonly found in tissues specialized in macromolecular secretion and/or ion transport. Since the enzyme, carbonic anhydrase, serves as a useful marker for a variety of specialized transporting cell types, we examined whether this enzyme was present in WE3-reactive cells. Of the tissues examined, a striking degree of colocalization of carbonic anhydrase and the WE3 antigen was observed, further strengthening the view that the WE3 antigen is an important constituent of specialized transporting cells. A preliminary biochemical characterization suggests that the antigen is probably a glycoprotein, which elutes during gel filtration as a species of over 660 kDa. Possible implications for the function of the wound epithelium are discussed.

Appearance and regulation of an antigen associated with limb regeneration in Notophthalmus viridescens.

Previous reports from this laboratory have documented a cellular antigen, 22/18, associated with regeneration of forelimbs in the newt Notophthalmus viridescens (Kintner and Brockes: Nature 308:67-69, 1984; Journal of Embryology and Experimental Morphology 89:37-55, 1985; Brockes: Science 225:1280-1287, 1984; Kintner et al.: Molecular Basis of Neural Development, 1985). The antigen is expressed at various stages of regeneration in several types of cells including Schwann cells and muscle cells. We report here that, in addition to the previous results, the expression of 22/18 occurs very early in the regeneration process and is initially confined to an apparently nonmuscle, non-neural population of cells. The well-defined context of the antigen's first appearance has allowed us to investigate the regulation of its expression. In particular, injury is sufficient to elicit the appearance of 22/18. It can appear prior to mitosis and despite denervation. In circumstances where regeneration is inhibited, expression of 22/18 can persist.

The substitution of carrier priming of helper function in the common American newt, Notophthalmus viridescens by lectins and human lymphokines.

There is no clear evidence that helper function is thymus dependent in the Common American newt, Notophthalmus viridescens. Here we test the capacity of concanavalin A, wheat germ agglutinin, human rIL-1 and rIL-2, reagents which stimulate T cell activities in other species, to substitute for carrier priming in the newt. Cytofluorimetric analyses have been used to demonstrate specific IL-2 receptor binding sites on newt splenocytes. Competitive pre-binding with rIL-2 tested whether anti-IL-2 receptor antibody binding sites would bind rIL-2. While Con A can substitute for carrier priming in the newt only when it is presented on Sepharose or agarose particles, wheat germ agglutinin cannot, even when it is injected in particulate form. Additionally, human rIL-1 can serve as an effective substitute for carrier priming, but rIL-2 cannot. The cytofluorimetric data are in agreement with the functional data in that they suggest that human rIL-2 may not bind newt splenocytes. Our data which show shared lectin specificities with T cell regulated helper function in another amphibian species are consistant with the possibility that T-like cells are responsible for helper function in this species.

The phylogeny of macrophage function: antigen uptake and degradation by peritoneal exudate cells of two amphibian species and CAF1 mice.

Equal numbers of thioglycollate mobilized peritoneal exudate cells (PEC) of the newt, Notophthalmus viridescens, the South African clawed toad, Xenopus laevis, and CAF1 mice were compared with respect to their capacity to take up and degrade soluble 14C-ovalbumin (OVA). PEC of the newt failed to take up the labeled antigen, while those of the toad incorporated only one-half as much as those of the mice. Moreover, the toad PEC degraded only 42% of the immunogen which was taken up, while PEC of the mice degraded 78% of the immunogen they had ingested during the 60-min period. Paraformaldehyde treatment of the PEC prevented antigen uptake, while chloroquine treatment prevented degradation with both species, and thus, active processes were involved. While newt PEC were unable to ingest soluble OVA, they were able to ingest and degrade OVA conjugated to sepharose during the same time period. The failure of primitive vertebrates to respond immunologically to soluble proteins appears to be due to their failure to ingest soluble immunogen.

Monoclonal antibodies to the cells of a regenerating limb.

Monoclonal antibodies were raised against differentiated cells, and blastemal cells from regenerating limbs of adult newts (Notophthalmus viridescens) and screened for specific staining by immunocytochemistry. In addition to antibodies that identify muscle, Schwann cells and cartilage, two reagents were specific for subpopulations of blastemal cells. One of these latter antibodies, termed 22/18, has provided new evidence about the origin of blastemal cells from Schwann cells and myofibres, and also identifies blastemal cells whose division is persistently dependent on the nerve supply.

Comparative immunology as a research tool.

Only very few immunological publications are based on investigations using non-mammalian vertebrates as experimental animals. Nevertheless, some important immunological problems, such as dichotomy of T and B lymphocytes or immune tolerance, were first discovered or even solved using such species. The usefulness of comparative investigations with species of different vertebrate classes is shown, referring to our own investigations of organ transplantations in the 1950s and of the regulation of the IgM antibody response in the last years.