Effects of elevated corticosterone on humoral innate and antibody-mediated immunity in southern leopard frog (Lithobates sphenocephalus) tadpoles.

As a free-living larval stage of a vertebrate, tadpoles are good subjects for the study of the development of physiological systems and the study of evolutionarily conserved, context-dependent responses to variable environments. While the basic components of innate and adaptive immune defenses in tadpoles are known, the impact of glucocorticoids on immune defenses in tadpoles is not well-studied. We completed four experiments to assess effects of elevation of corticosterone on humoral innate defenses and antibody-mediated immunity in southern leopard frog tadpoles (Lithobates sphenocephalus). To test humoral innate defense within the tadpoles exposed to short-term and long-term elevation of glucocorticoids, we exposed tadpoles to exogenous corticosterone for different lengths of time in each experiment (0-84 days). We used bacterial killing assays to assess humoral innate immune defense. To test antibody-mediated immune responses, we again exposed tadpoles to exogenous corticosterone, while also exposing them to Aeromonas hydrophila. We used A. hydrophila ELISA comparing IgM and IgY responses among groups. Plasma from corticosterone-dosed tadpoles killed more A. hydrophila than control tadpoles each following a short-term (14 day) and long-term (56 day) exposure to exogenous corticosterone. Conversely, corticosterone-dosed tadpoles had significantly lower IgM and IgY against A. hydrophila after 12 weeks. Our fourth experiment revealed that the lower IgY response is a product of weaker, delayed isotype switching compared with controls. These results show that elevated corticosterone has differential effects on innate and acquired immunity in larval southern leopard frogs, consistent with patterns in more derived vertebrates and in adult frogs.

Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity.

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 µg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 µg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.

Immediate and lag effects of pesticide exposure on parasite resistance in larval amphibians.

Across host-parasite systems, there is evidence that pesticide exposure increases parasite loads and mortality following infection. However, whether these effects are driven by reductions in host resistance to infection or slower rates of parasite clearance is often unclear. Using controlled laboratory experiments, we examined the ability of larval northern leopard frogs (Lithobates pipiens) and American toads (Anaxyrus americanus) to resist and clear trematode (Echinoparyphium sp.) infections following exposure to the insecticide carbaryl. Northern leopard frogs exposed to 1 mg L-1 of carbaryl had 61% higher parasite loads compared with unexposed individuals, while there was no immediate effect of carbaryl on parasite encystment in American toads. However, when tadpoles were exposed to carbaryl and moved to freshwater for 14 days before the parasite challenge, we recovered 37 and 63% more parasites from carbaryl-exposed northern leopard frogs and American toads, respectively, compared with the control. No effects on clearance were found for either species. Collectively, our results suggest that pesticide exposure can reduce the ability of amphibians to resist parasite infections and that these effects can persist weeks following exposure. It is critical for researchers to incorporate species interactions into toxicity studies to improve our understanding of how contaminants affect ecological communities.

Dietary antioxidants enhance immunocompetence in larval amphibians.

Dietary antioxidants have been shown to confer a variety of benefits through their ability to counter oxidative stress, including increased immunocompetence and reduced susceptibility to both infectious and non-infectious diseases. However, little is known about the effects of dietary antioxidants on immune function in larval amphibians, a group experiencing worldwide declines driven by factors that likely involve altered immunocompetence. We investigated the effects of dietary antioxidants (quercetin, vitamin E, and ß-carotene) on two components of the immune system, as well as development and growth. Lithobates pipiens tadpoles fed diets with supplemental ß-carotene or vitamin E exhibited an enhanced swelling response as measured with a phytohemagglutinin assay (PHA), but there was no induced antibody response. Effects were often dose-dependent, with higher antioxidant levels generally conferring stronger swelling that possibly corresponds to the innate immune response. Our results indicate that the antioxidant content of the larval amphibian diets not only had a detectable effect on their immune response capability, but also promoted tadpole growth (mass gain), although developmental stage was not affected. Given that many environmental perturbations may cause oxidative stress or reduce immunocompetence, it is critical to understand how nutrition may counter these effects.

Immunomodulation in post-metamorphic northern leopard frogs, Lithobates pipiens, following larval exposure to polybrominated diphenyl ether.

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis.

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis.

One issue of great concern for the scientific community is the continuing loss of diverse amphibian species on a global scale. Amphibian populations around the world are experiencing serious losses due to the chytrid fungus, Batrachochytrium dendrobatidis. This pathogen colonizes the skin, leading to the disruption of ionic balance and eventual cardiac arrest. In many species, antimicrobial peptides secreted into the mucus are thought to contribute to protection against colonization by skin pathogens. Although it is generally thought that antimicrobial peptides are an important component of innate immune defenses against B. dendrobatidis, much of the current evidence relies on correlations between effective antimicrobial peptide defenses and species survival. There have been few studies to directly demonstrate that antimicrobial peptides play a role. Using the northern leopard frog, Rana pipiens, we show here that injection of noradrenaline (norepinephrine) brings about a long-term depletion of skin peptides (initial concentrations do not recover until after day 56). When peptide stores recovered, the renewed peptides were similar in composition to the initial peptides as determined by MALDI-TOF mass spectrometry and in activity against B. dendrobatidis as determined by growth inhibition assays. Newly metamorphosed froglets depleted of their peptide stores and exposed to B. dendrobatidis died more rapidly than B. dendrobatidis-exposed froglets with their peptides intact. Thus, antimicrobial peptides in the skin mucus appear to provide some resistance to B. dendrobatidis infections, and it is important for biologists to recognize that this defense is especially important for newly metamorphosed frogs in which the adaptive immune system is still immature.

Effects of agricultural pesticides on the health of Rana pipiens frogs sampled from the field.

There is evidence that over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations due to disease outbreaks. Multiple causes have been suggested to explain this increase in disease incidence. Among these, climate changes, environmental pollution and reduced water quality are gaining attention. Indeed, some chemicals of environmental concerns are known to alter the immune system. It is possible that exposure to these pollutants could alter the immune system of anurans and render them more susceptible to different pathogens. In this study, we sampled Rana pipiens in five different sites near St. Lawrence River (Quebec, Canada) during the months of July and September in 2001. Two of these sites were protected areas, in which low levels of pesticides were detected, while the remaining three sites were located in areas with intensive corn and soybeans cultivations. Our results demonstrated that frogs living in agricultural regions are smaller in size and weight than frogs living in areas with lower levels of pesticides at both sampling times. Moreover, we have observed a significant decrease in the number of splenocytes (cellularity) and the phagocytic activity in frogs sampled in impacted sites. Taken together, these results suggest that frogs living in agricultural regions might be more vulnerable to infections and diseases through their smaller size and alteration of their immune system. Our results also contribute to the overall discussion on factors involved in amphibian declines.

The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians.

BACKGROUND: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them. OBJECTIVE: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States. METHODS: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 µg/L). We also conducted two dose-response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone. RESULTS: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 µg/L killed 100% of R. sphenocephala, and 0.0164 µg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 µg/L). CONCLUSIONS: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

A revised leopard frog phylogeny allows a more detailed examination of adaptive evolution at ranatuerin-2 antimicrobial peptide loci.

Ranatuerins are antimicrobial peptides of the innate immune system found in ranid frogs. We previously presented evidence that a positive selective sweep had fixed a single allele at the Ranatuerin2 locus in the northern leopard frog (Rana pipiens). In this paper, we further investigate the evolutionary history of ranatuerins as follows. First, we sequenced Ranatuerin2 in additional individuals of R. pipiens and related frog species and compared diversity and divergence at these sequences with that at four putatively neutrally evolving loci. Second, we asked whether the evolutionary patterns observed at Ranatuerin2 were typical for ranatuerin loci by sequencing our samples at a paralogous locus, Ranatuerin2b, and performing the same neutrality tests. Ranatuerin2b also showed strong and significant evidence of at least one selective sweep. Third, we used the neutral loci to independently resolve conflicting hypotheses about phylogenetic relationships among our study species. Both the neutral loci and the ranatuerin loci supported an older phylogeny inferred from allozyme data and strongly rejected a more recent phylogeny inferred from mitochondrial DNA. Finally, in order to test whether the sweep was driven by the evolution of substantially new peptide function, we used the phylogeny to reconstruct the hypothetical Ranatuerin2 peptide that existed before the sweep. We synthesized this peptide and tested its activity and that of the extant peptide against six bacterial pathogens of frogs. We observed antibacterial activity but found no significant functional differences between the two peptides.

Agrochemicals increase trematode infections in a declining amphibian species.

Global amphibian declines have often been attributed to disease, but ignorance of the relative importance and mode of action of potential drivers of infection has made it difficult to develop effective remediation. In a field study, here we show that the widely used herbicide, atrazine, was the best predictor (out of more than 240 plausible candidates) of the abundance of larval trematodes (parasitic flatworms) in the declining northern leopard frog Rana pipiens. The effects of atrazine were consistent across trematode taxa. The combination of atrazine and phosphate--principal agrochemicals in global corn and sorghum production--accounted for 74% of the variation in the abundance of these often debilitating larval trematodes (atrazine alone accounted for 51%). Analysis of field data supported a causal mechanism whereby both agrochemicals increase exposure and susceptibility to larval trematodes by augmenting snail intermediate hosts and suppressing amphibian immunity. A mesocosm experiment demonstrated that, relative to control tanks, atrazine tanks had immunosuppressed tadpoles, had significantly more attached algae and snails, and had tadpoles with elevated trematode loads, further supporting a causal relationship between atrazine and elevated trematode infections in amphibians. These results raise concerns about the role of atrazine and phosphate in amphibian declines, and illustrate the value of quantifying the relative importance of several possible drivers of disease risk while determining the mechanisms by which they facilitate disease emergence.

Acid exposure is an immune disruptor in adult Rana pipiens.

Acidic environments are physiological stressors for amphibians. The objective of the present study was to document the effect of an acidic environment on innate immune system function under controlled experimental conditions in Rana pipiens. We developed an in vivo assay, by injecting a suspension of 1-microm fluorescent beads in fluid thioglycollate, to induce peritonitis. The number of peritoneal exudate leukocytes and their phagocytic activity did not increase with thioglycollate injection when frogs were exposed to pH 5.5 compared to when frogs were exposed to pH 7.0. An environment of pH 5.5 disrupted the inflammatory response of frogs compared to an environment of pH 7.0; at pH 5.5, more nonphagocytic leukocytes and fewer highly phagocytic leukocytes were found compared to those in frogs exposed to pH 7.0. Frogs stimulated by thioglycollate injection and exposed to pH 5.5 had a 50% increase in cells that did not exhibit phagocytosis and a 4- to 10-fold reduction in the number of highly phagocytic cells. This is evidence that acid exposure functions as an immune disruptor in adult R. pipiens under laboratory conditions.

Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens.

Over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations. Multiple causes have been suggested to explain this decline. Among these, environmental pollution is gaining attention. Indeed, some chemicals of environmental concern are known to alter the immune system. Given that amphibians are frequently exposed to agricultural pesticides, it is possible that these pollutants alter their immune system and render them more susceptible to different pathogens. In this study, we exposed two frog species, Xenopus laevis and Rana pipiens, for a short period of time to a mixture of pesticides (atrazine, metribuzine, endosulfan, lindane, aldicarb and dieldrin) representative in terms of composition and concentrations to what it is found in the environment of the southwest region of the province of Quebec. The pesticides were known to be present in surface water of many tributaries of the St. Lawrence River (Quebec, Canada). Our results demonstrate that the mixture of pesticides could alter the cellularity and phagocytic activity of X. laevis and the lymphocyte proliferation of R. pipiens. Taken together, these results indicate that agricultural pesticides can alter some aspects of the immune response in frogs and could contribute to their global decline by rendering them more susceptible to certain infections.

Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection.

In the past 30 years, many amphibian species have suffered population declines throughout the world. Mass mortality have been frequently reported, and in several instances, infectious diseases appear to be the cause of death. The role that contaminants could play in these die-offs through immunotoxic effects has been poorly investigated. In this study, juvenile leopard frogs (Rana pipiens) were exposed for 21 d to a mixture of six pesticides (atrazine, metribuzin, aldicarb, endosulfane, lindane, and dieldrin) and subsequently challenged with a parasitic nematode, Rhabdias ranae. Exposure to the mixture at environmentally realistic concentrations significantly reduced lymphocyte proliferation. Three weeks after the end of the exposure, lymphocyte proliferation had recovered and was stimulated in frogs challenged with parasites with the exception of those previously exposed to the highest concentration. No pesticide effects on phagocytosis and splenocyte numbers were detectable at the end of the exposure period, but these two parameters were diminished 21 d after the infection challenge in frogs previously exposed to the highest levels of pesticides. In these animals, the prevalence of lung infection by R. ranae also tended to be higher. These results suggest that agricultural pesticides can alter the immune response of frogs and affect their ability to deal with parasitic infection.

ATPase and MHC class II molecules co-expression in Rana pipiens dendritic cells.

Mammalian Langerhans cells are antigen-presenting cells located in different epithelia. These cells have a characteristic ultrastructural pattern, present a plasmatic membrane ATPase activity and constitutively express class II molecules of the major histocompatibility complex. ATPase-positive dendritic cells that are morphologically similar to Langerhans cells have also been found in amphibian epidermis. In order to demonstrate that ATPase-positive dendritic cells of amphibian epidermis express class II molecules and are present in other stratified epithelia, histochemical and immunohistochemical as well as ultrastructural analysis were performed. ATPase-positive dendritic cells and class II-positive dendritic cells were observed in epidermis, nictitant membrane and cornea. In epidermis the number of ATPase-positive dendritic cells was 656+/-186/mm2 while class II-positive dendritic cells was 119+/-45/mm2. Some ATPase-positive dendritic cells showed co-expression of class II molecules. These results suggest the existence of dendritic cell subsets in amphibians as is clearly demonstrated in mammals.

Two ancient allelic lineages at the single classical class I locus in the Xenopus MHC.

Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and alpha2 peptide-binding domains, and there are highly diverse alleles (defined by the alpha1 peptide-binding domain) in each lineage. Surprisingly, the alpha3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.

Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold.

The effect of exposure to low temperatures (5 degrees C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 degrees C compared to that of animals kept at 22 degrees C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 degrees C for the same length of time. Mean serum complement activity after 1 month at 5 degrees C was significantly reduced in comparison to animals held at 22 degrees C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 degrees C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 degrees C) in frogs returned to 22 degrees C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 degrees C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 degrees C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 degrees C failed to cause death or observable symptoms of disease in frogs that were returned to 22 degrees C.

Hibernation alters the frog's immune system.

The lymphomyeloid organs and blood leukocyte populations of the leopard frog, Rana pipiens, undergo conspicuous changes during hibernation at 4 degrees C. Within the blood, spleen, thymus, jugular bodies, and bone marrow there was a progressive loss of hemopoietic populations resulting in a marked lymphocyte depletion. Termination of the 135-day hibernation period resulted in the restoration of all hemopoietic elements in the blood and lymphomyeloid organs within 30 days. Frogs subjected to experimental hibernation and immunized showed weakened immune responses when brought from the hibernaculum. Plaque-forming cells (PFC) were lower in spleen, jugular bodies, and bone marrow, and serum antibody titers were also lower. Although the kinetics of the primary responses were essentially the same, the secondary responses differed suggesting major rearrangements with respect to the numbers of cells and their function in secreting antibody. The apparent lymphocyte aplasia may contribute to the absence of immunological responsiveness during periods of hibernation.

Cellular and serum immune response to dinitrophenol in adult Rana pipiens.

A rabbit anti-Rana anti-DNP antiserum was used in order to estimate changes in frequency of bone marrow (BM) cells containing cytoplasmic anti-DNP antibodies (cId+) for eight weeks following a single injection. We found that (i) cId+ cells increased from 0.2 to 4.3 percent of total mononuclear cells (MNC's) during the first 8 weeks, (ii) BM granulocytes increased in frequency up to week 2, then gradually decreased to week 8, and (iii) serum anti-DNP antibody levels, evaluated by ELISA, increased to week 8. Our observations (i) support those which suggest that Rana BM resembles mammaliam BM as a source of antibody-producing cells, (ii) indicate that specific immune responses in BM may result in a 20-fold increase in the frequency of antigen-related plasma cells among total MNC's, and (iii) suggest that BM B cell clonal expansion, maturation and secretion may result in an increase in specific antibody levels in serum.