Stefanny Christie Monteiro Titon

Amphibians face threats from emerging bacterial, viral, and fungal pathogens, and their innate immune response plays a key role in early defense. The liver is a central immune organ that produces acute-phase proteins (APPs) and complement system proteins, which are essential for early pathogen neutralization and a positive disease outcome. Here, we investigated the temporal dynamics of hepatic gene expression for APPs [C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen-like protein 1 (FGL1)] and complement components (C1s, C3, C4, C5, C9) in Rhinella diptycha toads injected with lipopolysaccharide (LPS) or saline. Using RT-qPCR at four time points post-injection (1h, 3h, 6h, and 18h), we detected a significant effect of LPS on C1s expression and a treatment x time interaction for C4, C5, and C9, indicating time-dependent activation of different complement proteins. In contrast, we found no evidence for LPS-induced changes in APPs expression. However, several APPs were positively correlated with complement components, suggesting coordinated regulation even in the absence of differential expression. Our findings provide a temporal analysis of hepatic acute-phase responses to LPS in Rhinella diptycha toads, unraveling a complex network of complement system activation. By showing that complement components, rather than APPs, dominate the early hepatic response to a bacterial antigen in a widespread Neotropical toad species, our study provides a temporal and mechanistic baseline for future investigations of amphibian innate immunity and underscores the need for future work to determine how these early pathways respond to different pathogens and contribute to variation in disease outcomes among amphibians.

Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3 h over 24 h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22 h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10 h) or at night (22 h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retinal light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group.

Amphibian populations have been declining worldwide, with global climate changes and infectious diseases being among the primary causes of this scenario. Infectious diseases are among the primary drivers of amphibian declines, including ranavirosis and chytridiomycosis, which have gained more attention lately. While some amphibian populations are led to extinction, others are disease-resistant. Although the host's immune system plays a major role in disease resistance, little is known about the immune mechanisms underlying amphibian disease resistance and host–pathogen interactions. As ectotherms, amphibians are directly subjected to changes in temperature and rainfall, which modulate stress-related physiology, including immunity and pathogen physiology associated with diseases. In this sense, the contexts of stress, disease and ecoimmunology are essential for a better understanding of amphibian immunity. This issue brings details about the ontogeny of the amphibian immune system, including crucial aspects of innate and adaptive immunity and how ontogeny can influence amphibian disease resistance. In addition, the papers in the issue demonstrate an integrated view of the amphibian immune system associated with the influence of stress on immune–endocrine interactions. The collective body of research presented herein can provide valuable insights into the mechanisms underlying disease outcomes in natural populations, particularly in the context of changing environmental conditions. These findings may ultimately enhance our ability to forecast effective conservation strategies for amphibian populations. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’.

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’.

Glucocorticoid (GC) release is triggered by adverse stimuli that activate the hypothalamus-pituitary-adrenal/interrenal axis. Glucocorticoids may enhance or suppress immune functions depending on the level of elevation. In this study, we investigated the effects of transient and chronic increase of corticosterone (CORT) on the wound healing of the American bullfrog. Frogs were submitted to a daily transdermal hormonal application that acutely elevated CORT plasma levels, or vehicle as a control. Other frogs were surgically implanted with a silastic tube filled with CORT that resulted in chronic elevation of CORT plasma levels or received empty implants as a control. A dermal biopsy was performed to create a wound and was photographed every 3 days. Individuals treated with transdermal CORT started healing faster than their control 32 days after the biopsy. Frogs that received CORT implants tended to heal slower than control subjects. Plasma bacterial killing ability was not affected by treatment, which reinforces the constitutive nature of this innate immune trait. By the end of the experiment, frogs from the acute CORT treatment had smaller wounds compared with those receiving the CORT-filled implants, highlighting the differential effects of acute (immunoenhancing) and chronic (immunosuppressive) elevation of CORT plasma levels. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’.

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.

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.