Do changes in body mass alter white blood cell profiles and immune function in Australian cane toads (Rhinella marina)?

Variation in food resources can result in dramatic fluctuations in the body condition of animals dependent on those resources. Decreases in body mass can disrupt patterns of energy allocation and impose stress, thereby altering immune function. In this study, we investigated links between changes in body mass of captive cane toads (Rhinella marina), their circulating white blood cell populations, and their performance in immune assays. Captive toads that lost weight over a three-month period had increased levels of monocytes and heterophils and reduced levels of eosinophils. Basophil and lymphocyte levels were unrelated to changes in mass. Because individuals that lost mass had higher heterophil levels but stable lymphocyte levels, the ratio of these cell types was also higher, partially consistent with a stress response. Phagocytic ability of whole blood was higher in toads that lost mass, owing to increased circulating levels of phagocytic cells. Other measures of immune performance were unrelated to mass change. These results highlight the challenges faced by invasive species as they expand their range into novel environments which may impose substantial seasonal changes in food availability that were not present in the native range. Individuals facing energy restrictions may shift their immune function towards more economical and general avenues of combating pathogens. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Transposable elements expression in Rhinella marina (cane toad) specimens submitted to immune and stress challenge.

Transposable elements (TEs) are important components of eukaryotic genomes and compose around 30% of the genome of Rhinella marina, an invasive toad species. Considering the possible role of TEs in the adaptation of populations, we have analyzed the expression of TEs in publicly available spleen tissue transcriptomic data generated for this species after immune and stress challenge. By analyzing the transcriptome assembly, we detected a high number of TE segments. Moreover, some distinct TE families were differentially expressed in some conditions. Our result shows that several TEs are capable of being transcribed in R. marina and they could help to generate a rapid response of specimens to the environment. Also, we can suggest that these TEs could be activated in the germinative cells as well producing variability to be selected and shaped by the evolutionary processes behind the success of this invasive species. Thus, the TEs are important targets for investigation in the context of R. marina adaptation.

Stress and immunity: Field comparisons among populations of invasive cane toads in Florida.

Cane toads (Rhinella marina) were introduced worldwide and have become invasive in multiple locations, representing a major driver of biodiversity loss through competition (food, shelter, territory), predation, and the poisoning of native species. These toads have been used in Australia as a model for studies concerning invasion biology and ecoimmunology, as longer-established (core) and invasion front (edge) populations show altered stress and immune response profiles. Although cane toads were also introduced into the United States in the 1950s, these patterns have yet to be evaluated for the populations spanning Florida. Toads introduced into Florida have dispersed primarily northward along a latitudinal gradient, where they encounter cooler temperatures that may further impact stress and immune differences between core and edge populations. In this study, we sampled cane toads from nine different locations spanning their invasion in Florida. Cane toads from southern populations showed higher plasma bacterial killing ability and natural antibody titers than the toads from the northern populations, indicating they have a better immune surveillance system. Also, southern toads were more responsive to a novel stressor (1 hr restraint), showing a higher increase in corticosterone levels. These results indicate that possible trade-offs have occurred between immune and stress responses as these toads have become established in northern cooler areas in Florida.

Innate immunity of Florida cane toads: how dispersal has affected physiological responses to LPS.

Physiological tradeoffs occur in organisms coping with their environments, which are likely to increase as populations reach peripheries of established ranges. Invasive species offer opportunities to study tradeoffs that occur, with many hypotheses focusing on how immune responses vary during dispersal. The cane toad (Rhinella marina) is a well-known invasive species. Populations near the expanding edge of the Australian invasion have altered immune responses compared to toads from longer-established core populations, although this has not been well-documented for Florida populations. In this study, cane toads from a northern edge [New Port Richey (NPR)] and southern core (Miami) population in Florida were collected and injected with lipopolysaccharide (LPS) to compare immune responses. Core population individuals injected with LPS showed greater metabolic increases compared to their baseline rates that were higher compared to those from the edge population. In addition, LPS-injected core individuals had different circulating leukocyte profiles compared to saline-injected cane toads while edge individuals did not. There was a significant interaction between plasma bacteria-killing capability (BKA) and treatment, such that BKA decreased with time in saline compared to LPS-injected individuals, and saline-injected toads from the edge population had lower BKA compared to LPS-injected edge toads at 20 h post-injection. There was also a significant interaction between location and time on circulating corticosterone (CORT) levels following injections with saline or LPS, with CORT decreasing more with time in core population toads. The differential CORT response indicates that differential stress responses contribute to the tradeoffs observed with immunity and dispersal.

Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina).

The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.

Invasion history alters the behavioural consequences of immune system activation in cane toads.

Acute activation of the immune system often initiates a suite of behavioural changes. These "sickness behaviours"-involving lethargy and decreased activity-may be particularly costly on invasion fronts, where evolutionary pressures on dispersal favour individuals that move large distances. We used a combination of field and laboratory studies to compare sickness behaviours of cane toads from populations differing in invasion history. To do this we stimulated immune system activation by injecting lipopolysaccharide (LPS) to mimic bacterial infection. We predicted that LPS would result in less severe sickness behaviour in toads from range-edge populations because they had undergone selection for rapid and sustained dispersal (activities in conflict with lethargy and decreased activity). Contrary to our prediction, LPS injection caused a greater reduction in dispersal-relevant traits in invasion-front individuals than in conspecifics from the range-core. Our data suggest that the rapid invasion of cane toads through tropical Australia has seen an evolutionary shift in the magnitude of sickness behaviour elicited by pathogen infection. The increased sickness behaviour among range-edge toads suggests a shift away from pathogen tolerance (seen in range-core populations) towards resistance to pathogen attack. But as a consequence, when pathogens do become successfully established, toads from invasion-front populations may have less capacity to tolerate their ill-effects.

Characterisation of major histocompatibility complex class I in the Australian cane toad, Rhinella marina.

The Major Histocompatibility Complex (MHC) class I is a highly variable gene family that encodes cell-surface receptors vital for recognition of intracellular pathogens and initiation of immune responses. The MHC class I has yet to be characterised in bufonid toads (Order: Anura; Suborder: Neobatrachia; Family: Bufonidae), a large and diverse family of anurans. Here we describe the characterisation of a classical MHC class I gene in the Australian cane toad, Rhinella marina. From 25 individuals sampled from the Australian population, we found only 3 alleles at this classical class I locus. We also found large number of class I alpha 1 alleles, implying an expansion of class I loci in this species. The low classical class I genetic diversity is likely the result of repeated bottleneck events, which arose as a result of the cane toad's complex history of introductions as a biocontrol agent and its subsequent invasion across Australia.

Effect of a Punica granatum enriched diet on immunocompetence in Rhinella marina.

Direct ingestion of plant materials has been shown to have immunomodulatory effects on a variety of herbivores. Studies have also shown that compounds ingested indirectly by predators through prey items can affect the general physiology of the ingesting organism. Relatively little data exists, however, concerning the modulation of a predator's immune system via compounds obtained indirectly through prey. In this study, we sought to determine if the immune-stimulating properties of Punica granatum (pomegranate) could be conveyed from a prey organism, Acheta domestica, to a predator, Rhinella marina, through diet specialization. Experimental crickets were fed a diet of agar supplemented with 10 mg/mL of lyophilized, powdered, whole pomegranate while control crickets were fed unadulterated agar. Experimental toads consumed a diet consisting of crickets fed the pomegranate-enriched diet, while control toads consumed a diet consisting of crickets fed the standard agar diet. Blood samples were taken weekly and leukocyte profiles and neutrophil phagocytic activity were determined for all toads over an 8-week period. Complement activity was measured at 6 weeks. Toads fed the pomegranate-enriched diet showed altered leukocyte profiles as evidenced by an increase in circulating eosinophil number and a decrease in the number of circulating lymphocytes, monocytes, and basophils as compared to controls, indicating an immunomodulatory effect of the pomegranate-enhanced diet. These results suggest that pomegranate-derived immunomodulatory compounds can be transferred from prey to predator, and suggests that the flora in the environment where insectivores forage could have a significant effect on the physiology of the animal.

Corticosterone-immune interactions during captive stress in invading Australian cane toads (Rhinella marina).

Vertebrates cope with physiological challenges using two major mechanisms: the immune system and the hypothalamic pituitary-adrenal axis (e.g., the glucocorticoid stress response). Because the two systems are tightly integrated, we need simultaneous studies of both systems, in a range of species, to understand how vertebrates respond to novel challenges. To clarify how glucocorticoids modulate the amphibian immune system, we measured three immune parameters and plasma corticosterone (CORT), before and after inflicting a stressor (capture and captive confinement) on introduced cane toads (Rhinella marina) near their invasion front in Australia. Stress increased CORT levels, decreased complement lysis capacity, increased leukocyte oxidative burst, and did not change heterologous erythrocyte agglutination. The strength of the CORT response was positively correlated with leukocyte oxidative burst, and morphological features associated with invasiveness in cane toads (relative leg length) were correlated with stress responsiveness. No immune parameter that we measured was affected by a toad's infection by a parasitic nematode (Rhabdias pseudosphaerocephala), but the CORT response was muted in infected versus uninfected toads. These results illustrate the complex immune-stress interactions in wild populations of a non-traditional model vertebrate species, and describe immune adaptations of an important invasive species.

Behavioral responses to immune-system activation in an anuran (the cane toad, Bufo marinus): field and laboratory studies.

The challenges posed by parasites and pathogens evoke behavioral as well as physiological responses. Such behavioral responses are poorly understood for most ectothermic species, including anuran amphibians. We quantified effects of simulated infection (via injection of bacterial lipopolysaccharide [LPS]) on feeding, activity, and thermoregulation of cane toads Bufo marinus within their invasive range in tropical Australia. LPS injection reduced feeding rates in laboratory trials. For toads in outdoor enclosures, LPS injection reduced activity and shifted body temperature profiles. Although previous research has attributed such thermal shifts to behavioral fever (elevated body temperatures may help fight infection), our laboratory studies suggest instead that LPS-injected toads stopped moving. In a thermal gradient, LPS-injected toads thus stayed close to whichever end of the gradient (hot or cold) they were first introduced; the introduction site (rather than behavioral thermoregulation) thus determined body temperature regimes. Shifts in thermal profiles of LPS-injected toads in outdoor enclosures also were a secondary consequence of inactivity. Thus, the primary behavioral effects of an immune response in cane toads are reduced rates of activity and feeding. Thermoregulatory modifications also occur but only as a secondary consequence of inactivity.

Effects of ozone exposure on nonspecific phagocytic capacity of pulmonary macrophages from an amphibian, Bufo marinus.

Pulmonary macrophages are an important component of immune defense against inhaled foreign particles and microorganisms. In humans and other mammals, exposure to moderate amounts of ozone (O3) can inhibit functional capacities of alveolar macrophages. In many wilderness areas downwind of urban centers, ozone levels frequently exceed national standards. We report results of 4-h inhalation exposures to 0.8 parts per million O3 on pulmonary macrophage viability and phagocytosis capacity in marine toads, Bufo marinus. At 1 and 24 h after ozone exposure, macrophages had reduced in vitro capacity to phagocytize fluorescent polystyrene microspheres. By 48 h postexposure, there were no differences in these macrophage functions between ozone- and air-exposed toads. Macrophage yield did not differ among exposure groups nor did exposure to elevated temperatures (30 degrees C) for up to 48 h affect recovery of macrophages. However, compared with the millions of macrophages per milliliter recovered in mammals by similar procedures, pulmonary macrophage yield was typically in the range of 50 to 200 x 10(3) per milliliter extracted fluid. These results are the first to report effects of an air pollutant on amphibian immune system function and suggest a possible role of oxidant air pollutants in regional declines of amphibian populations.

Role of environmental pollutants on immune functions, parasitic infections and limb malformations in marine toads and whistling frogs from Bermuda.

Soil, water, and amphibian tissues collected between 1995 and 1999 from 15 study sites in Bermuda were analysed for pesticides and heavy metals. The most abundant pesticide residue in soil was p,p'-dichlorodiphenyldichloroethylene (DDE) which was found at all sites in concentrations ranging from 0.003 to 4.023 p.p.m. No pesticide residues were found in water. DDE was also recovered from the livers and fat bodies of marine toads (Bufo marinus) and whistling frogs (Eleutherodactylus johnstonei). Analyses of food sources consumed by these anuran species revealed residue levels of p, p'-DDE ranging from 0.05 to 0.217 p.p.m. Other soil residues included dichlorodiphenyltrichloroethane (DDT) at eight study sites, Dicofol(kelthane) at eight sites, dieldrin at five sites, and polychlorinated biphenyls (PCBs) as Arochlor 1254 and Arochlor 1260 at seven sites. Analyses of toad livers revealed significant concentrations of cadmium, chromium, copper and zinc. Livers of Bermuda toads exhibited altered hepatocytic morphology and an increased number of melanomacrophages and possible granulomas, while spleens showed a marked decrease in white pulp. Spleen cells from Bufo marinus collected at one site having high levels of cadmium exhibited a decreased B cell response to lipopolysaccharide. The incidence of trematode infection in Bufo marinus increased from 53.8% in 1995 to 90% in 1999. Deformity rates in the limbs of subadult and adult toads ranged between 15 and 25%. Examination of 1,995 newly-metamorphosed toads revealed deformity rates as high as 47%. The current comprehensive study suggests that environmental pollutants may account for immunosuppression, increased susceptibility to infections, limb malformations and possible decline in amphibian populations from Bermuda.

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus).

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.

Cyclicity and memory in the humoral immune response of the marine toad Bufo marinus.

Bufo marinus toads immunized with a single dose of 100 mug of polymerized flagellin (POL) produce a longlasting cyclic humoral response at 22 degrees C in which the serum antibody titers rise and fall within periods of 2-3 weeks. This seems to be regulated by the catabolism of immunoglobulins, since passive 125I-labeled IgM in the serum has a half-life of 17 days, corresponding to the cyclicity of the titers of active antibodies. In toads injected with between 10 ng and 10 mug antigen the serum antibody titer peaks between 4 and 7 weeks, depending on the antigen dose, and declines by the 9th week. At this time a second stimulation with an equal dose of POL induces a secondary response during which higher titers of antibodies rise faster than in the primary response. A dose of 10 mug POL stimulates an optimal primary response and a second equal dose, given when the serum antibodies are disappearing, induces a secondary response enhanced in time but not in antibody titers.

Regulation of the immune response. II. Qualitative and quantitative differences between thymus- and bone marrow-derived lymphocytes in the recognition of antigen.

The specificity of antigen recognition by thymus-derived helper cells (T cells) and antibody was examined in mice, heterologous erythrocyte antigens from sheep (SRBC), goat (GRBC), burro (BRBC), chicken (CRBC), and toad (TRBC) being used. Antibody specificity was tested by a number of functional assays: hemagglutination, hemolysis, and immune suppression. The specificity of T cells was determined by titrating their ability to help the in vitro antitrinitrophenol (TNP) responses of mouse spleen cultures immunized with the hapten coupled to the various test erythrocytes as carrier. Anti-SRBC antibody cross-reacted with GRBC, but not with BRBC, CRBC, or TRBC. In contrast, SRBC-primed helper T cells cross-reacted with both GRBC and BRBC, but not with CRBC or TRBC, indicating a difference in the specificity of antigen recognition between the cellular and the humoral immune responses.