Laura M. Zimmerman

Understanding the vertebrate immune system: insights from the reptilian perspective

Summary Reptiles are ectothermic amniotes, providing the key link between ectothermic anamniotic fishes and amphibians, and endothermic amniotic birds and mammals. A greater understanding of reptilian immunity will provide important insights into the evolutionary history of vertebrate immunity as well as the growing field of eco-immunology. Like mammals, reptile immunity is complex and involves innate, cell-mediated and humoral compartments but, overall, there is considerably less known about immune function in reptiles. We review the current literature on each branch of the reptilian immune system, placing this information in context to other vertebrates. Further, we identify key areas that are prime for research as well as areas that are lagging because of lack of reagents in non-model systems.

Variation in the seasonal patterns of innate and adaptive immunity in the red-eared slider (Trachemys scripta)

SUMMARY The primary function of the immune system is to protect the organism from invading pathogens. In vertebrates, this has resulted in a multifaceted system comprised of both innate and adaptive components. The immune system of all jawed vertebrates is complex, but unlike the endothermic vertebrates, relatively little is known about the functioning of the ectothermic vertebrate immune system, especially the reptilian system. Because turtles are long-lived ectotherms, factors such as temperature and age may affect their immune response, but comprehensive studies are lacking. We investigated variation in immune responses of adult male and female red-eared sliders (Trachemys scripta) across the entire active season. We characterized seasonal variation in innate, cell-mediated and humoral components via bactericidal capacity of plasma, delayed-type hypersensitivity and total immunoglobulin levels, respectively. Results indicate that all immune measures varied significantly across the active season, but each measure had a different pattern of variation. Interestingly, temperature alone does not explain the observed seasonal variation. Immune measures did not vary between males and females, but immunoglobulin levels did vary with age. This study demonstrates the highly dynamic nature of the reptilian immune system, and provides information on how biotic and abiotic factors influence the immune system of a long-lived ectotherm.

Phagocytic B cells in a reptile

Evidence for a developmental relationship between B cells and macrophages has led to the hypothesis that B cells evolved from a phagocytic predecessor. The recent identification of phagocytic IgM+ cells in fishes and amphibians supports this hypothesis, but raises the question of when, evolutionarily, was phagocytic capacity lost in B cells? To address this, leucocytes were isolated from red-eared sliders, Trachemys scripta, incubated with fluorescent beads and analysed using flow cytometry and confocal microscopy. Results indicate that red-eared slider B cells are able to ingest foreign particles and suggest that ectothermic vertebrates may use phagocytic B cells as part of a robust innate immune response.

A vertebrate cytokine primer for eco-immunologists

Summary The field of eco-immunology seeks to address the causes and consequences of natural variation in immune responses. Recently, the development of more immunological resources for nonmodel systems along with the growing availability of genomic data has made it feasible to explore more complex immunological differences in nonmodel systems. Cytokines are small molecular weight proteins that are the major signalling molecules of the immune system. Studies in mice and humans have demonstrated the importance of tightly regulated cytokine signalling, and thus, cytokines may play a major, unexplored role in explaining natural variation in immune responses. In this review, we highlight cytokines that are likely to be important for eco-immunology studies and what is currently known about their presence or absence in the major vertebrate groups. We also explore what types of questions an eco-immunologist might ask involving cytokines and discuss potential ways to measure cytokines in nonmodel systems. Inclusion of cytokines in eco-immunology will advance the field as it will help us to better understand mechanisms behind both natural variation in immune responses and how different taxa respond to immune challenges.

Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle

SUMMARY Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxon’s immune strategy can influence how the immune system is affected by age.

No evidence that estrogens affect the development of the immune system in the red-eared slider turtle, Trachemys scripta.

Exposure to maternally derived substances during development can affect offspring phenotype. In ovo exposure to maternally derived steroids has been shown to influence traits such as growth and behavior in the offspring. The development of the immune system also can be altered by exposure to both androgens and glucocorticoids in a variety of species, but much less is known about the potential for estrogens to influence the development of this system. We examined the effect of estradiol on the development of both innate and adaptive immune components in the red-eared slider turtle (Trachemys scripta). A bacterial killing assay was used to assess innate immunity, a delayed-type hypersensitivity test for cellular immunity, and total immunoglobulin levels to measure the humoral immune response. We found no effect of in ovo estradiol treatment on any of our immune measures despite using doses that are known to influence other phenotypic parameters during development and varying the timing of dosing across development. Our results suggest that maternally derived estradiol does not affect the development of the immune system in T. scripta.

Red-Eared Slider Turtles Lack Response to Immunization with Keyhole Limpet Hemocyanin but Have High Levels of Natural Antibodies

While the specific humoral response of reptiles is slow and does not typically increase in titer or binding affinity upon secondary immunization, reptiles produce polyreactive natural antibodies (NAbs) that have low binding affinity and are produced in the absence of antigen stimulation. Given the poor specific response, NAbs may be an important protective resource in reptiles. In order to investigate the relative contributions of natural and specific antibodies, we immunized turtles with the novel antigen keyhole limpet hemocyanin (KLH). We did not detect an increase in antibody titers. However, preimmunization titers to KLH, as well as to a series of other novel antigens, were high in the turtles, indicating a strong NAb response. Interestingly, we found an age-associated increase in NAb titers in adults. Overall, our data suggest that reptiles may use NAbs as part of a strong innate immune response rather than relying on slower specific humoral responses.

Corticosterone Levels during the Nesting Process in Red-eared Sliders (Trachemys scripta)

Abstract Free-living vertebrates tend to respond to environmental challenges by increasing circulating glucocorticoid levels. Changes in glucocorticoids can modulate various aspects of physiology and behavior, which can have fitness benefits in the short term but may be detrimental to fitness if elevated levels are frequent, prolonged, or both. The transfer of glucocorticoids from females to offspring might also have fitness consequences, but there is less known about the potential effects of transgenerational glucocorticoid transfer. Our study examined corticosterone levels in female Red-eared Sliders (Trachemys scripta) during the nesting season because of the potential fitness consequences for both females and offspring. Whether caught in aquatic traps or caught during the nesting process, females had similar circulating corticosterone levels that were relatively low. However, when females were subjected to a restraint protocol, corticosterone rose. Therefore, corticosterone levels may be elevated during the nesting process, but the nesting process itself does not result in elevated corticosterone levels. Understanding how natural processes such as nesting influence corticosterone levels has important consequences for interpreting corticosterone levels as an index of fitness.