Corticosterone treatment results in fat deposition and body mass maintenance without effects on feeding behaviour or immunity in female lizards (Tropidurus catalanensis).

Throughout life, animals must maintain homeostasis while coping with challenging events. The period after reproduction can be challenging for oviparous females to maintain homeostasis since they direct most of their energy stores to vitellogenesis, possibly increasing the vulnerability to stressors. Changes in glucocorticoids' (GC) secretion promote various behavioural and physiological adjustments daily and to restore balance after facing stressors. However, when GC are elevated for extended periods, which usually occurs in response to chronic exposure to stressors, they can affect feeding behaviour and suppress the immune function. We aim to elucidate the effects of chronic corticosterone (CORT) exposure on feeding behaviour, body condition and immune function in female lizards, Tropidurus catalanensis, in the post-reproductive period. Thirty animals were divided into three groups: 1. Control (no experimental procedure performed); 2. Empty Implant (animals implanted with empty silastic tube); and 3. CORT Implant (animals implanted with silastic tube filled with CORT, with a chronic continuous release for at least a week). CORT plasma levels feeding behaviour, body condition (body index [BI] and fat index [FI]), leukocyte count, and several immune function variables (bacterial killing ability [BKA], hemagglutination titer, phytohemagglutinin [PHA] immune challenge and leukocyte count) were evaluated. After implantation, CORT treated animals maintained stable body mass through the experiment, while Control and Empty Implant groups displayed weight loss. In the CORT treated animals, there was also a positive relation between BI and FI, and higher FI when compared to groups 1 and 2. No effects of CORT were observed on feeding behaviour nor on the immune function.

Immunoendocrinology and Ecoimmunology in Brazilian Anurans.

This paper reviews several aspects of immunoendocrinology and ecoimmunology in Brazilian species of anurans under investigation for more than a decade, including (1) patterns of annual covariation of circulating steroids, calling behavior and innate immunity, (2) endocrine and immune correlates of calling performance, (3) behavioral and physiological correlates of parasite load, (4) thermal sensitivity of immune function, and (5) endocrine and immunomodulation by experimental exposure to acute and chronic stressors, as well as to endocrine manipulations and simulated infections. Integrated results have shown an immunoprotective role of increased steroid plasma levels during reproductive activity in calling males. Moreover, a higher helminth parasite load is associated with changes in several behavioral and physiological traits under field conditions. We also found anuran innate immunity is generally characterized by eurythermy, with maximal performance observed in temperatures close to normal and fever thermal preferendum. Moreover, the aerobic scope of innate immune response is decreased at fever thermal preferendum. Experimental exposure to stressors results in increased corticosterone plasma levels and immune redistribution, with an impact on immune function depending on the duration of the stress exposure. Interestingly, the fate of immunomodulation by chronic stressors also depends in part on individual body condition. Acute treatment with corticosterone generally enhances immune function, while prolonged exposure results in immunosuppression. Still, the results of hormonal treatment are complex and depend on the dose, duration of treatment, and the immune variable considered. Finally, simulated infection results in complex modulation of the expression of cytokines, increased immune function, activation of the Hypothalamus-Pituitary-Interrenal axis, and decreased activity of the Hypothalamus-Pituitary-Gonadal axis, as well as reduced melatonin plasma levels, suggesting that anurans have a functional Immune-Pineal axis, homologous to that previously described for mammals. These integrated and complementary approaches have contributed to a better understanding of physiological mechanisms and processes, as well as ecological and evolutionary implications of anuran immunoendocrinology.

Elevated corticosterone levels are associated with increased immunocompetence in male toads, both when calling and under experimental conditions.

Circulating androgens can influence immune responses and sexual traits in male vertebrates. However, in the last 20 years, glucocorticoids have also been implicated as a possible source of variation in male sexual traits and immunocompetence. In this context, we studied the relations between male vocal mating display, immunity, androgens, and glucocorticoids in the explosive breeding toad Rhinella granulosa. In the field, males with high calling effort display either high- or low-corticosterone (CORT) plasma levels, but only males with both high calling effort and high CORT plasma levels showed high bacterial killing ability (BKA), suggesting that the acute CORT elevation can be immunostimulatory. CORT treatments increased BKA in laboratory experiments, confirming the functional relationship observed in the field. However, toads treated with a low dose of CORT increased BKA for 10 h after the treatment, while toads that received a high dose increased BKA for only 1 h after the treatment. These results indicate that different CORT doses can result in temporal differences in the immune response. We did not find any relationship between calling effort, immune response (BKA and PHA swelling response), and testosterone plasma levels in the field, or any effects of testosterone treatment on immunocompetence. Our results suggest a complex relationship between calling effort and immunity, mediated by CORT plasma levels.

Challenges of a novel range: Water balance, stress, and immunity in an invasive toad.

Species introduced by human activities can alter the normal functioning of ecosystems promoting negative impacts on native biodiversity, as they can rapidly expand their population size, demonstrating phenotypic plasticity and possible adaptive capacity to novel environments. Twenty years ago, the guttural toad, Sclerophrys gutturalis, was introduced to a peri-urban area of Cape Town, with cooler and drier climatic characteristics than its native source population, Durban, South Africa. Our goal was to understand the phenotypic changes, in terms of physiology and immunity, of populations in native and novel environments. We evaluated body index (BI), field hydration level, plasma corticosterone levels (CORT), proportion of neutrophils: lymphocytes (N: L), plasma bacterial killing ability (BKA), and hematocrit (HTC) in the field, and after standardized stressors (dehydration and movement restriction) in males from the native and invasive populations. Toads from the invasive population presented lower BI and tended to show a lower field hydration state, which is consistent with living in the drier environmental conditions of Cape Town. Additionally, invasive toads also showed higher BKA and N:L ratio under field conditions. After exposure to stressors, invasive animals presented higher BKA than the natives. Individuals from both populations showed increased CORT after dehydration, an intense stressor for these animals. The highest BKA and N:L ratio in the field and after submission to stressors in the laboratory shows that the invasive population has a phenotype that might increase their fitness, leading to adaptive responses in the novel environment and, thus, favoring successful dispersion and population increase.

Helminth Parasites of Three Anuran Species during Reproduction and Drought in the Brazilian Semiarid Caatinga Region.

We investigated the mean abundance of helminths and analyzed helminth composition and structure at the infracommunity and component community levels for 3 anuran species (Pleurodema diplolister, Rhinella jimi, and Rhinella granulosa) from the Caatingas, a semiarid Brazilian region characterized by accentuated seasonality and unpredictability of rains. Data were collected during the reproductive period and during drought, when P. diplolister estivated buried underground but R. jimi and R. granulosa remained foraging actively. We expected higher parasitological parameters during the reproductive period when compared to drought for these 3 anurans. We also expected higher parasite infection in the Rhinella species and higher similarity between their helminth parasite communities when compared to the estivating species, P. diplolister. Contrary to our hypothesis, the season was not related to parasite community structure. As predicted, the Rhinella toads shared more similar species composition of parasite communities. These similarities in the composition of the parasite community between Rhinella species could be due to similar temporal/spatial patterns of activity and phylogenetic proximity. Pleurodema diplolister hosted a more restricted helminth fauna, a result that might be associated with estivation restricting the temporal window available to acquire parasites. This study also presents new helminth fauna records for R. jimi and P. diplolister, and the first helminth fauna record of R. granulosa from the Caatingas in semiarid Brazil.

Biomarker-based assessment of the muscle maintenance and energy status of anurans from an extremely seasonal semi-arid environment, the Brazilian Caatinga.

Strongly seasonal environments pose challenges for performance and survival of animals, especially when resource abundance seasonally fluctuates. We investigated the seasonal variation of key metabolic biomarkers in the muscles of males from three species (Rhinella jimi, R. granulosa and Pleurodema diplolister) of anurans from the drastically seasonal Brazilian semi-arid area, Caatinga. We examined the expression of proteins regulating energy turnover (AMP-activated protein kinase [AMPK] and protein kinase B [AKT]), protein synthesis and homeostasis (total and phosphorylated eukaryotic initiation factor 2α [eIF2α and p-eIF2α] and chaperone proteins [HSP 60, 70, and 90]) in muscles predominantly related to reproduction and locomotion. Cytochrome c oxidase (COX) activity was also assessed as an index of the muscle aerobic capacity. The expression pattern of metabolic biomarkers indicates that the maintenance of muscular function is regulated in a species-specific manner during the drastic seasonal variation. Rhinella jimi and R. granulosa that remain active during the drought appear to maintain muscles through more energy expensive pathways including elevated protein synthesis, while the aestivating P. diplolister employs energy conservation strategy suppressing protein synthesis, decreasing chaperone expression and increasing expression of AMPK. Two (P. diplolister and R. granulosa) of the three studied species activate cell survival pathways during the drought likely to prevent muscle atrophy, and all three studied species maintain the muscle aerobic capacity throughout the year, despite the resource limitation. These strategies are important considering the unpredictability of the reproductive event and high demand on muscular activity during the reproductive season in these amphibians. SUMMARY STATEMENT: We studied seasonal variation of key metabolic biomarkers in the muscles of anurans that experience drastic variation in environmental conditions and differ in seasonal activity patterns.

Immunomodulation by testosterone and corticosterone in toads: Experimental evidences from transdermal application.

Androgens and glucocorticoids play important roles in vertebrate's reproduction and display complex immunomodulatory functions that may affect survival. In anurans, testosterone and corticosterone are correlated to sexual behavior, reproduction, and immune function. Male toads (Rhinella jimi) were treated with acute doses of testosterone (T) and corticosterone (CORT) and immune variables (plasma bacterial killing ability [BKA], swelling after phytohemagglutinin [PHA] injection and the time point of maximum PHA swelling response) were measured. Transdermal T and CORT application increased androgen (T-DHT) and CORT plasma levels after 1 h of treatment, respectively, without a dose-specific effect. Transdermal T treatment did not affect BKA or PHA swelling response. Individuals treated with transdermal CORT showed an earlier maximum PHA swelling response and a tendency of lower BKA 15 h after treatment. Our results indicated that an acute experimental increase of CORT plasma levels diminished time for inflammatory resolution and suppressed non-cellular innate response.

Behavioral, physiological and morphological correlates of parasite intensity in the wild Cururu toad (Rhinella icterica).

Large numbers of parasites are found in various organs of anuran amphibians, with parasite intensities thought to modulate the host's Darwinian fitness traits. Interaction between the anuran hosts and their multiple parasites should modulate the host's phenotypic characteristic, such as those associated with high energetic demand (such as calling effort and locomotor performance), energy balance (standard metabolic rate), and morphological plasticity (as indicated by organ masses). The present study investigated the impact of parasite intensities on the behavioral, physiological, and morphological traits of wild adult male Rhinella icterica (Anura: Bufonidae). We tested as to whether individuals with higher parasite intensities would present: 1) lower vocal calling effort in the field, as well as poorer locomotor performance and body-condition index; and 2) higher standard metabolic rates and internal organ masses. Measurements included: calling effort in the field; standard metabolic rate; locomotor performance; parasite intensity; internal organ masses (heart, liver, kidneys, intestines, stomach, lungs, hind limb muscle, and spleen); and the body-condition index. Results showed a negative association of parasite intensities with locomotor performance, and standard metabolic rate of R. icterica. A positive association between parasite intensities and relative organ masses (heart, intestines and kidneys) was also evident. Toads with higher pulmonary and intestinal parasites intensities also showed higher total parasite intensities.

Seasonal Patterns of Variation in Steroid Plasma Levels and Immune Parameters in Anurans from Brazilian Semiarid Area.

Elevated androgens and glucocorticoids displayed by males during the reproductive season have been proposed to mediate a possible trade-off between reproduction and immunocompetence. Anurans living in arid and semiarid environments display a strong seasonal reproduction, which could accentuate the variation in physiological, immunological, and behavioral parameters. We studied covariation between steroid plasma levels, morphometric variables associated with body condition and immunity, leukocyte profile, parasite load, and response to an immunological challenge across different phases of the annual life-history cycle of three anuran species from a Brazilian semiarid area. Our results showed a seasonal pattern of covariation among leukocyte parameters, kidney mass, and steroid plasma levels, with higher values measured during the reproductive season, particularly when males were sampled during calling activity. Moreover, these anurans showed a stronger response to an immunological challenge during the reproductive period. The immunosuppression during the dry period was particularly evident for the species that aestivate, indicating that the availability of energetic resources might be an important factor determining seasonal variation in inflammatory response. Intensity of the helminth infection was associated with eosinophil count but showed a more complex pattern with regard to androgens levels. These data emphasize that variations in the intensity of helminth infection might be more closely related to specific aspects of the immune response than to the general seasonal patterns of variation in steroid plasma levels, total circulating leukocytes, and inflammatory response.

Breeding under unpredictable conditions: Annual variation in gonadal maturation, energetic reserves and plasma levels of androgens and corticosterone in anurans from the Brazilian semi-arid.

Anurans living in arid and semi-arid habitats are subjected to unpredictable rain patterns. Consequently, they should be prepared to reproduce at the onset of rain events. We investigated the covariation between calling behavior, testicular maturation, abdominal fat body index (FBI), plasma levels of androgens (T-DHT) and corticosterone (CORT) of males from three species of anurans in the Brazilian semi-arid during the reproductive period and drought. One of these species aestivates during the drought, while the other two species remain foraging. Although the three species display different behavioral strategies during the dry period, they present the same general reproductive patterns. T-DHT levels on the plasma and germinative cyst diameters were higher during the reproductive and breeding period compared to the drought. Additionally, the germinative cysts had all cell stages including sperm bundles during the dry season, however, it was only during the breeding event that free spermatozoa were found in the cyst lumen. These results suggest that these species present the reproductive pattern typical of desert anurans, consisting of opportunistic breeders that reproduce when triggered by a rain stimulus. Rhinella jimi and Pleurodema diplolister had higher CORT when males were calling. Moreover, Rhinella granulosa and P. diplolister showed lower FBI during breeding event, when males were calling. The high levels of CORT and lower FBI during reproductive period are associated, indicating that CORT modulates the recruitment of energy stores to prepare and maintain reproduction, particularly the expensive calling effort.

Helminth parasites of Hypsiboas prasinus (Anura: Hylidae) from two Atlantic forest fragments, São Paulo State, Brazil.

Seventy-seven males of Hypsiboas prasinus from 2 Atlantic forest fragments in the municipalities of Botucatu and Jundiaí, São Paulo State, Brazil, were examined for endoparasites. The frogs were captured in summer (January until March) and winter (July/August) of 2008 and 2009. Thirty-three males (75%) from Botucatu were infected with Rhabdias cf. fuelleborni , cosmocerciid nematodes, and Cylindrotaenia americana . Twenty-five tree frogs (78.5%) from Jundiaí were infected by Rhabdias cf. fuelleborni , Physaloptera sp., and cosmocerciid nematodes. Only cosmocerciid nematodes presented a statistically significance difference in prevalence (z  =  4.345; P < 0.001) and mean abundance (t  =  562.0; P < 0.001) between Botucatu and Jundiaí during the winter. Also, the cosmocerciids exhibited higher mean abundance (t  =  196.0; P  =  0.034) in winter when compared with summer at the Jundiaí site. Moreover, to our knowledge, this is the first report of C. americana in the Brazilian Hylidae. This study presents 4 new records of nematodes in H. prasinus .