Baseline and stress-induced changes in plasma bacterial killing ability against gram-negative bacteria are partially mediated by the complement system in Rhinella diptycha toads.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

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.

Dehydration followed by restraint sustains high circulating corticosterone and improves immunity in toads.

Amphibians are suffering population declines due to a variety of factors such as increased ultraviolet radiation, climate change, habitat loss, pathogens, and pollution, or a combination of these. Such changes are associated with a reduction in the availability of water, exposing these animals to a greater risk of desiccation. In this context, understanding how dehydration can modulate the hypothalamic-pituitary-interrenal axis (HPI) and the immune response is an imperative question to predict how stressors can affect amphibian species. We investigated whether dehydration promotes long-lasting effects on toads' ability to respond to a consecutive stressor (restraint) even if the toads are allowed to rehydrate, as well as its effects on the immune function. We also tested the hypothesis that the toads showing more severe dehydration would exhibit lower responsiveness to restraint challenge, even if the animals were allowed to rehydrate. Individuals of R. ornata were dehydrated mildly and severely. Thereafter, they were submitted to a restraint stress challenge for 1 and 24 h. Our results show that dehydration increased hematocrit and CORT in R. ornata toads. The restraint induced an acute stress response in fully hydrated toads (increased CORT and neutrophil: lymphocyte ratio). Otherwise, restraint in moistened cloth bags allowed full rehydration in previously dehydrated toads and did not induce an additional increase in CORT, but those toads sustained elevated CORT up to 24 h of restraint. Also, these animals showed increased neutrophil: lymphocyte ratio and the phagocytic activity of blood cells, even when they rehydrated during restraint. These results point to a continuous activation of the HPA during dehydration and subsequent restraint, even when they recovered from the dehydration state. Also, acute stressors seem to promote immune cell redistribution and augmentation of immune cellular function in R. ornata toads.

Baseline and stress-induced steroid plasma levels and immune function vary annually and are associated with vocal activity in male toads (Rhinella icterica).

Theoretical models predict that elevated androgen and glucocorticoid levels in males during the reproductive season promote immunosuppression. However, some studies report decreased stress response during this season. This study investigated annual variation in plasma corticosterone and testosterone levels, plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) in free-living male toads (Rhinella icterica). Toads were sampled in the field (baseline) and 1 h-post restraint over five months, and we considered the occurrence of vocal activity. Baseline corticosterone, testosterone, and BKA showed higher values during the reproductive period, specifically in calling male toads. The NLR was similar throughout the year, but higher values were observed in calling toads. Moreover, baseline NLR and BKA were positively correlated with both testosterone and corticosterone, suggesting higher steroid levels during reproduction are associated with enhanced cellular and humoral immunity. Despite fluctuation of baseline values, post-restraint corticosterone levels remained uniform over the year, indicating that toads reached similar maximum values throughout the year. Testosterone levels decreased following restraint before one specific reproductive period but increased in response to restraint during and after this period. Meanwhile, BKA decreased due to restraint only after the reproductive period, indicating immune protection and resilience to immunosuppression by stressors associated with steroid hormones during reproduction. Our results show that baseline and stress-induced hormonal and immune regulation varies throughout the year and are associated with vocal activity in R. icterica males, indicating a possible compromise between steroids and immune function in anuran males.

Testosterone immunomodulation in free-living and captive Rhinella icterica male toads.

Testosterone (T) regulates immune function, with both immunostimulatory and immunosuppressive effects on several vertebrates. We investigated the covariation between plasma T and corticosterone (CORT) levels and immunity (plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR)) in free-living Rhinella icterica male toads inside and outside the reproductive season. We found an overall positive correlation between steroids and immune traits, with toads during the reproductive season displaying increased T, CORT and BKA. We also investigated the T transdermal application effects on T, CORT, phagocytosis of blood cells, BKA and NLR in captive toads. Toads were treated with T (1, 10 or 100 µg) or vehicle (sesame oil) for eight consecutive days. Animals were bled on the first and eighth days of treatment. Increased plasma T was observed on the first and last day of T-treatment, while increased BKA was observed following all T doses on the last day, with a positive correlation between T and BKA. Plasma CORT, NLR and phagocytosis increased on the last day for all T-treated and vehicle groups. Overall, we demonstrated a positive covariation between T and immune traits in the field and T-induced augmented BKA in captive toads, indicating a T immunoenhancing effect in R. icterica males. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Contaminated meal intake (Aeromonas hydrophila) does not elicit hormonal or immune modulation in bullfrogs (Lithobates catesbeianus).

The gastrointestinal tract (GIT) is colonized by resident microbiota but contact with foreign microbiota during feeding can impair GIT functions. During meal digestion, several vertebrates modulate the systemic immune function and immunoregulatory hormones concentration. However, in ectothermic animals, it is not known if this hormonal and immune modulation during the postprandial period is affected by the presence of pathogenic microbiota in the food. This study aimed to investigate the effects of contaminated meal ingestion on hormonal and innate immune responses in bullfrogs (Lithobates catesbeianus). Bullfrogs were divided into three treatments: fed three times with sterilized fish feed (control group), fed twice with sterilized fish feed and once with fish feed containing live bacteria (Aeromonas hydrophila, 109 UFC/mL), and fed three times with fish feed containing live bacteria. Blood and GIT tissues were collected 24 h after treatments to measure plasma and tissue corticosterone levels, NL ratio, and plasma bacterial killing ability. The ingestion of contaminated meal did not affect the hormonal and immune parameters. In conclusion, ingestion of contaminated food was not capable of intensifying the hypothalamic-pituitary-interrenal axis activation and the consequent hormonal and immune responses observed after feeding in bullfrogs. However, our results suggest that the ingestion of three contaminated meals tended to decrease stomach corticosterone levels (nonstatistically significant), possibly contributing to preventing the transmigration of the bacteria to organs outside the GIT.

Heat-Induced Hatching of Red-Eyed Treefrog Embryos: Hydration and Clutch Structure Increase Behavioral Thermal Tolerance.

Climate change is increasing both environmental temperatures and droughts. Many ectotherms respond behaviorally to heat, thereby avoiding damage from extreme temperatures. Within species, thermal tolerance varies with factors such as hydration as well as ontogenetic stage. Many tropical anurans lay terrestrial eggs, relying on environmental moisture for embryonic development. These eggs are vulnerable to dehydration, and embryos of some species can hatch prematurely to escape from drying eggs. Warmer temperatures can accelerate development and thus hatching, but excess heat can kill embryos. Thus, we hypothesize that embryos may show a behavioral thermal tolerance limit, hatching prematurely to avoid potentially lethal warming. If so, because warming and drying are often associated, we hypothesize this limit, measurable as a voluntary thermal maximum, may depend on hydration. We manipulated the hydration of the terrestrial eggs of Agalychnis callidryas, in intact clutches and egg-groups isolated from clutch jelly, then warmed them to assess if embryos hatch early as a behavioral response to high temperatures and whether their thermal tolerance varies with hydration or surrounding structure. We discovered that heating induces hatching; these embryos show a behavioral escape-hatching response that enables them to avoid potentially lethal warming. Hydrated eggs and clutches lost more water and warmed more slowly than dehydrated ones, indicating that hydration buffers embryos from environmental warming via evaporative cooling. Embryos in hydrated clutches tolerated greater warming before hatching and suffered higher mortality, suggesting their behavioral Thermal Safety Margin is small. In contrast, lower thermal tolerance protected dry embryos, and those isolated from clutch jelly, from lethal warming. Heat-induced hatching offers a convenient behavioral assay for the thermal tolerance of terrestrial anuran embryos and the interactive effects of warming and dehydration at an early life stage. This work expands the set of threats against which embryos use hatching in self-defense, creating new opportunities for comparative studies of thermal tolerance as well as integrative studies of self-defense mechanisms at the egg stage.

El cambio climático está aumentando tanto las temperaturas ambientales como las sequías. Muchos ectotermos responden conductualmente al calor, evitando así los daños por temperaturas extremas. Dentro de las especies, la tolerancia térmica varía con factores como la hidratación y la ontogenia. Muchos anuros tropicales depositan huevos terrestres que dependen de la humedad ambiental para el desarrollo embrionario. Estos huevos son vulnerables a la deshidratación y los embriones de algunas especies pueden eclosionar prematuramente para escapar de la desecación de los huevos. Las temperaturas más cálidas pueden acelerar el desarrollo y, por lo tanto, la eclosión, pero el exceso de calor puede matar a los embriones. Por lo tanto, planteamos la hipótesis de que los embriones pueden mostrar un límite de tolerancia térmica conductual, eclosionando prematuramente para evitar un calentamiento potencialmente letal. Si es así, debido a que el calentamiento y la desecación a menudo están asociados, planteamos la hipótesis de que este límite, medible como una tolerancia térmica voluntaria, puede depender de la hidratación. Manipulamos la hidratación de los huevos terrestres de Agalychnis callidryas, en posturas intactas y grupos de huevos aislados de la gelatina de la postura. Luego, los calentamos para evaluar si los embriones eclosionan temprano como respuesta conductual a las altas temperaturas y si su tolerancia térmica varía con la hidratación o la estructura circundante. Descubrimos que el calentamiento induce la eclosión. Estos embriones muestran una respuesta conductual de eclosión de escape que les permite evitar un calentamiento potencialmente letal. Los huevos y las posturas hidratadas perdieron más agua y se calentaron más lentamente que los deshidratados, lo que indica que la hidratación protege a los embriones del calentamiento ambiental a través del enfriamiento por evaporación. Los embriones en posturas hidratadas toleraron un mayor calentamiento antes de la eclosión y sufrieron una mayor mortalidad, lo que sugiere que su margen de seguridad térmica conductual es pequeño. En contraste, la tolerancia térmica más baja protegió a los embriones deshidratados, y a los aislados de la gelatina, del calentamiento letal. La eclosión inducida por calor ofrece un ensayo de comportamiento conveniente para la tolerancia térmica de los embriones terrestres de anuros y los efectos integrados del calentamiento y la deshidratación en una etapa temprana de la vida. Este estudio amplía el conjunto de amenazas contra las cuales los embriones usan la eclosión en defensa propia, creando nuevas oportunidades para estudios comparativos de tolerancia térmica, así como estudios integradores de mecanismos de autodefensa en etapa de huevo.

As mudanças climáticas estão aumentando as temperaturas ambientais e as secas. Muitos ectotermos respondem comportamentalmente ao calor, evitando danos devido a temperaturas extremas. Dentro das espécies, a tolerância térmica varia com fatores como a hidratação e a ontogenia. Muitos anuros tropicais depositam ovos terrestres que dependem da umidade ambiental para o desenvolvimento embrionário. Esses ovos são vulneráveis à desidratação, e os embriões de algumas espécies podem eclodir prematuramente para escapar da dessecação dos ovos. Temperaturas mais altas podem acelerar o desenvolvimento e, portanto, a eclosão, mas o excesso de calor pode matar os embriões. Portanto, propomos a hipótese de que os embriões podem mostrar um limite de tolerância térmica comportamental, eclodindo prematuramente para evitar o aquecimento potencialmente letal. Nesse caso, como o aquecimento e a dessecação são frequentemente associados, propomos a hipótese de que esse limite, mensurável como uma tolerância térmica voluntária, pode depender da hidratação. Manipulamos a hidratação dos ovos terrestres de Agalychnis callidryas, em desovas completas e grupos de ovos isolados da geleia da desova. Depois, aquecemos os ovos para avaliar se os embriões eclodem cedo como uma resposta comportamental a altas temperaturas, e se sua tolerância térmica varia com a hidratação ou a estrutura circundante. Descobrimos que o aquecimento induz a eclosão. Esses embriões mostram uma resposta comportamental de eclosão que lhes permite evitar o aquecimento potencialmente letal. Ovos e desovas hidratadas perderam mais água e se aqueceram mais lentamente que os desidratados, indicando que a hidratação protege os embriões do aquecimento ambiental através do resfriamento por evaporação. Os embriões em desovas hidratadas toleraram maior aquecimento antes da eclosão e sofreram maior mortalidade, sugerindo que sua margem de segurança térmica comportamental é menor. Por outro lado, a menor tolerância térmica protegeu os embriões desidratados e os isolados da geleia, do aquecimento letal. A eclosão induzida pelo calor oferece um teste comportamental conveniente para a estimativa da tolerância térmica de embriões de anuros terrestres e dos efeitos integrados do aquecimento e da desidratação em um estágio inicial da vida. Este estudo expande o conjunto de ameaças contra as quais os embriões usam a eclosão como defesa, criando novas oportunidades para estudos comparativos de tolerância térmica, além de estudos integrativos dos mecanismos de autodefesa no estágio de ovo.

The Impacts of Transdermal Application of Corticosterone on Toad (Rhinella icterica) Immunity.

Recent studies have shown that acute physiological increases in endogenous glucocorticoid levels have immunostimulatory effects. Although post-acute stress immunosuppressive effects have also been described, the difference between enhancing and suppressing the immune response seems mediated by the stressor's duration, intensity, and the immune component under analysis. To elicit physiologically relevant corticosterone levels that can be found in Rhinella icterica toads after stressful events (e.g., restraint or captivity) and understand how acute increased glucocorticoid levels of different intensities affect corticosterone and testosterone plasma levels and immune parameters (in vitro plasma bacterial killing ability, neutrophil-to-lymphocyte ratio, and in vivo phagocytosis of peritoneal leukocytes), we submitted toads to the transdermal application of two corticosterone doses (1 and 10 µg). Corticosterone transdermal application increased corticosterone plasma levels with different intensities: 3 times for 1 µg and fourteen times for 10 µg, compared to the vehicle, and the neutrophil-to-lymphocyte ratio increased regardless of the corticosterone dose. However, there was no effect on testosterone levels and bacterial killing ability. Interestingly, both corticosterone doses promoted immunosuppression, decreasing peritoneal leukocytes' phagocytosis activity by 60% for toads receiving the dose of 1µg and 40% for those receiving 10 µg. Our results show the complexity of the relationship between increased corticosterone levels and immunomodulation. The different corticosterone doses promoted increases of distinct magnitudes in corticosterone plasma levels, with the less intense increase in corticosterone levels generating greater cell-mediated immunosuppression. Future studies using different corticosterone doses to achieve and compare physiological vs. pharmacological hormone levels are imperative to understanding these interrelationships between corticosterone and immune response.

Temperature Extreme Events Decrease Endocrine and Immune Reactive Scope in Bullfrogs (Lithobates catesbeianus).

Currently, effects of increased atmospheric temperature, in the context of ongoing climate change, have been investigated in multiple organisms and levels of biological organization. While there has been a focus on the impacts of increased mean temperature, an emergent and equally important point is the consequences of recurrent exposure to extreme temperature events, simulating heat waves. This study investigated the effects of serial exposure to high temperatures on immune and endocrine variables before and after exposure to an acute secondary stressor in bullfrogs (Lithobates catesbeianus). Adult males were divided into three groups and subjected to three thermal regimes: control (c; constant 22°C); experimental 1 (E1; kept at 22°C and exposed to 4 days of 30°C every 16 days); and experimental 2 (E2; kept at 22°C and exposed to 4 days of 30°C every 6 days). Blood samples were collected on the last day of key extreme heat events. Two weeks after the last extreme heat event, animals were subjected to restraint stress (1 h) and sampled again. Blood samples were used to determine neutrophil: lymphocyte ratio, plasma bacterial killing ability, as well as, corticosterone and plasma testosterone levels. Overall, we found exposure to extreme heat events did not affect immune and endocrine variables over time. Meanwhile, the previous exposure to extreme heat events modulated the responsiveness to restraint. The amplitude of increased corticosterone plasma levels and neutrophil: lymphocyte ratio in response to restraint decreased with the number of previous exposures to extreme heat events. These results suggest that exposure to extreme climatic events has hidden effects on bullfrog's stress response, expressed as diminished reactive scope to a novel stressor. This represents a highly deleterious facet of climate change since diminished responsiveness prevents proper coping with wildlife challenges.

Day Versus Night Melatonin and Corticosterone Modulation by LPS in Distinct Tissues of Toads (Rhinella Icterica).

Pathogen-associated molecular patterns modulate melatonin (MEL) production in the pineal and extra-pineal sites and corticosterone (CORT) synthesis in the adrenal/interrenal and other tissues. Both MEL and CORT play essential and complex immunomodulatory roles, controlling the inflammatory response. Given that most of what we know about these interactions is derived from mammalian studies, discovering how MEL and CORT are modulated following an immune challenge in anurans would increase understanding of how conserved these immune-endocrine interactions are in vertebrates. Herein, we investigated the modulation of MEL and CORT in plasma vs. local tissues of toads (Rhinella icterica) in response to an immune challenge with lipopolysaccharide (LPS; 2 mg/kg) at day and night. Blood samples were taken 2 hours after injection (noon and midnight), and individuals were killed for tissue collection (bone marrow, lungs, liver, and intestine). MEL and CORT were determined in plasma and tissue homogenates. LPS treatment increased MEL concentration in bone marrow during the day. Intestine MEL levels were higher at night than during the day, particularly in LPS-injected toads. Bone marrow and lungs showed the highest MEL levels among tissues. Plasma MEL levels were not affected by either the treatment or the phase. Plasma CORT levels increased in LPS-treated individuals, with an accentuated increase at night. Otherwise, CORT concentration in the tissues was not affected by LPS exposure. Modulation of MEL levels in bone marrow suggests this tissue may participate in the toad's inflammatory response assembly. Moreover, MEL and CORT levels were different in tissues, pointing to an independent modulation of hormonal concentration. Our results suggest an important role of immune challenge in modulating MEL and CORT, bringing essential insights into the hormone-immune interactions during anuran's inflammatory response.

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.

Daily and LPS-induced variation of endocrine mediators in cururu toads (Rhinella icterica).

Increased plasma glucocorticoids (corticosterone - CORT, in amphibians) and melatonin (MEL) are associated with the daily activity phase and with environmental darkness, respectively. Besides, CORT and MEL also play pivotal immunomodulatory roles in several vertebrates. Herein we described the daily profile of plasma MEL and CORT for Rhinella icterica toads in captivity. Thereafter, we investigated the effects of lipopolysaccharide (LPS)-induced systemic inflammation on the production of CORT and MEL in the R. icterica. Captive toads showed CORT and MEL diurnal variation typical of nocturnal species, with increased values for CORT at ZT12 (18 h) and MEL peak at ZT18 (24 h). LPS-induced hormonal changes included increased plasma CORT and decreased ocular and plasma MEL when compared to those from toads treated with saline 2 h post-injection. Our results demonstrated the presence of a diurnal CORT and MEL variation in toads. We also showed the crosstalk between CORT and MEL during the toad's systemic inflammation in response to an immune challenge with LPS. Additionally, our results demonstrated that anuran eyes' MEL production might be regulated during the inflammatory processes.

Skin and poison glands in toads (Rhinella) and their role in defence and water balance

Toads are considered poisonous animals since they have a passive mode of defence relying on cutaneous poison glands, differently from venomous animals who can inject venom in predators/aggressors or prey. Toads of Rhinella marina group are generally large and have a broad distribution in South America, inhabiting a wide range of environments. In this paper, we studied the toads Rhinella icterica from the Atlantic rainforest, and Rhinella jimi from the Brazilian Semiarid Caatinga, analysing aspects of natural history and comparing their skin morphology, the presence of macroglands, their resistance to water loss and rates of water uptake. In periods of extreme drought, R. jimi uses rock cracks as refuges, exposing only the head and regions of accumulation of poison glands. The skin of R. jimi showed higher number of poison glands with hydrophilic content than R. icterica. R. jimi also had a thicker skin, which can be related to its superior resistance to water loss. It also showed high rates of rehydration in association with a peculiar behaviour using the limbs to spread water onto highly glandular skin areas, suggesting that poison glands may also act in water balance in addition to chemical defence.

Short-term stressors and corticosterone effects on immunity in male toads (Rhinella icterica): A neuroimmune-endocrine approach.

In the last decades, it is growing the idea that stress-induced immunomodulation is bimodal: with acute stress associated with enhancing effects while chronic stress with suppressive effects. However, the immune-endocrine interactions and its implications are often overlooked in ectotherms. We investigated the impact of corticosterone (CORT) treatment and short-term stressors on CORT plasma levels and the immunity of male toads (Rhinella icterica), using three distinct protocols: restraint, immune challenge (with lipopolysaccharide, LPS), and CORT transdermal application (TA). Our results showed increased CORT and neutrophil: lymphocyte ratio (NLR) regardless of the stress input (restraint, LPS challenge) or CORT TA. In the meantime, the bacterial killing ability (BKA) was not affected by any treatment, suggesting this immune parameter might be a more constitutive and robust response. Interestingly, the cellular immune response showed distinct patterns. Increased phagocytosis of blood leukocytes and phytohemagglutinin edema followed LPS and CORT TA (15 â€‹µg), respectively. In contrast, the phagocytosis of peritoneal leukocytes decreased after CORT TA (1 and 10 â€‹µg), indicating that short-term increases in CORT levels might impair local immune function. Such differences in cellular immunity might also be associated with CORT doses or the interaction between CORT and other immune mediators, such as melatonin, testosterone, and cytokines. Overall, our results highlight the immune-enhancing effects of the acute stress response and CORT TA, and the complexity of the immune-endocrine interaction in anurans. It also highlights the relevance of investigating distinct contexts for CORT increase arising from different situations, as well as diverse immune components for a better understanding of the stress-induced immunomodulation.

LPS-induced immunomodulation and hormonal variation over time in toads.

The inflammatory response is a complex process that relies on interactions among multiple endocrine and immune modulators. Studies incorporating time-related and integrative endocrine and immune responses to an immune challenge might shed light on the characterization of the phases of the inflammatory response in anurans. The present study investigated time-related changes (1, 3, 6, and 18 h post-challenge) in plasma corticosterone (CORT), melatonin (MEL) and testosterone (T) levels, phagocytosis percentage (PP), plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) following a lipopolysaccharide (LPS) immune challenge in Rhinella diptycha toads. Our results showed the response to LPS injection was characterized by increased CORT, PP, BKA, and NLR, with a concomitant decrease in plasma MEL and T. Increased CORT was more pronounced at 6 and 18 h, while increased NLR was observed only 18 h post-LPS injection. Meanwhile, plasma MEL and T decreased independently of the time post-LPS injection. Additionally, toads in better body condition showed higher BKA and PP in the LPS-treated group, regardless of the time postinjection. Our results show that toads (R. diptycha) were sensitive to the LPS challenge, mounting an inflammatory response, which started quickly (after 1 h) and developed over time and was influenced by body condition. These results demonstrate a time-related hormonal and immune variation as a consistent pattern of activation of the immune system, as well as of hypothalamic-pituitary-adrenal/interrenal and immune-pineal axes following an immune challenge more deeply studied in mammals, suggesting the evolutionary conservation of the regulatory mechanisms for tetrapod vertebrates.

Eutrophication effects on fatty acid profiles of seston and omnivorous fish in tropical reservoirs.

It has been postulated that eutrophication causes replacement of n3 highly unsaturated fatty acids (n3 HUFA) rich taxa, such as Bacillariophyta, Cryptophyta and Dinophyta, with taxa poor in these fatty acids (FA), such as Chlorophyta and Cyanobacteria. Such a change in community composition at the basis of the food web may alter the FA composition of consumer tissues. Here, we investigated the effects of eutrophication on phytoplankton composition and FA profiles of seston and muscle of two omnivorous fish species (Astyanax fasciatus and Astyanax altiparanae) from reservoirs of different trophic status in Southeast Brazil. The phytoplankton composition and seston FA profiles reflected the degree of eutrophication at most of the studied sites. Three of the five most eutrophic sites were dominated by cyanobacteria and had the highest saturated fatty acid (SFA) and lowest polyunsaturated fatty acid (PUFA) relative contents among all sites. In contrast, the remaining two sites presented a higher phytoplankton diversity and higher relative contribution of sestonic PUFAs with 18 carbons (C18) and HUFAs than less eutrophic systems. However, there were no clear effects of sestonic FA profiles on the FA profiles of muscle of both fish species. A higher percentage of n3 HUFAs was found in the fish samples from a hypereutrophic and cyanobacteria dominated reservoir than in those from sites with a more diverse phytoplankton community in which fish mainly showed higher percentages of C18 PUFA. These results suggest a lack of a direct relationship between the degree of eutrophication and the percentage of n3 HUFAs in both fish species, which can be caused by specific characteristics of the reservoirs that may modulate eutrophication effects. Therefore, consumer FA biochemistry seemed to be dictated by their ability to select, accumulate, and modify dietary FAs, rather than by the eutrophication degree of the studied tropical reservoirs.

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.

Hormonal daily variation co-varies with immunity in captive male bullfrogs (Lithobates catesbeianus).

Almost all physiological processes within the organism, including immune parameters and hormones, follow a circadian rhythm. These daily fluctuations are often observed in free-living organisms; however, little is known regarding hormonal and immune daily variations in anurans, particularly under laboratory conditions. This study aimed to investigate the hormonal and immune daily variation in captive-bred Bullfrogs (Lithobates catesbeianus) under constant conditions (21 °C and 12:12 LD cycle). Our results showed a daily variation for plasma corticosterone (CORT), testosterone (T), and melatonin (MEL), as well as for blood leukocyte profile, phagocytic activity, and plasma bacterial killing ability (BKA). Hormonal profile and immune activity were higher at the dark when compared with the light phase; however, monocytes and lymphocytes followed the opposite pattern. Moreover, CORT was positively correlated with phagocytosis percentage of blood cells, BKA, and monocytes, while MEL and T showed a positive correlation with PP. Our results demonstrate the daily covariation of different immune variables and immunomodulatory hormones. These 24 h-day variations and covariation certainly have broad implications and need to be considered for better understanding anuran physiology both in the context of laboratory and field studies.

Effects of dehydration on thermoregulatory behavior and thermal tolerance limits of Rana catesbeiana ().

Predicting the effects of high environmental temperatures and drought on populations requires understanding how these conditions will influence the thermoregulatory behavior and thermal tolerance of organisms. Ectotherms show proportional (fine-tuned) and all-or-none (abrupt) responses to avoid overheating. Scattered evidence suggests that dehydration alters these behavioral responses and thermal tolerance, but these effects have not been evaluated in an integrative manner. We examined the effects of hydration level on the behavioral thermoregulation and behavioral and physiological thermal limits of the "bullfrog" (Rana catesbeiana), a well-studied and important invasive species. To examine the effects of dehydration on proportional responses, we compared the Preferred Body Temperatures (PBT) of frogs with restricted and unrestricted access to water. To assess the effect of dehydration on all-or-none responses, we measured and compared the Voluntary Thermal Maximum (VTMax) at different hydration levels (100%, 90%, 80% of body weight at complete hydration). Finally, to understand the effect of dehydration on physiological thermal tolerance, we measured the Critical Thermal Maximum (CTMax) of frogs at matched hydration levels. PBT, VTMax, and CTMax all decreased in response to higher dehydration levels. However, bullfrogs changed their PBT more than their VTMax or CTMax in response to dehydration. Moreover, some severely dehydrated individuals did not exhibit a VTMax response. We discuss the implications of our results in the context of plasticity of thermoregulatory responses and thermal limits, and its potential application to mechanistic modeling.