Adaptive Immunity in Reptiles: Conventional Components but Unconventional Strategies.

Recent studies have established that the innate immune system of reptiles is broad and robust, but the question remains: What role does the reptilian adaptive immune system play? Conventionally, adaptive immunity is described as involving T and B lymphocytes that display variable receptors, is highly specific, improves over the course of the response, and produces a memory response. While reptiles do have B and T lymphocytes that utilize variable receptors, their adaptive response is relatively non-specific, generates a prolonged antibody response, and does not produce a typical memory response. This alternative adaptive strategy may allow reptiles to produce a broad adaptive response that complements a strong innate system. Further studies into reptile adaptive immunity cannot only clarify outstanding questions on the reptilian immune system but can shed light on a number of important immunological concepts, including the evolution of the immune system and adaptive immune responses that take place outside of germinal centers.

The reptilian perspective on vertebrate immunity: 10†years of progress.

Ten years ago, 'Understanding the vertebrate immune system: insights from the reptilian perspective' was published. At the time, our understanding of the reptilian immune system lagged behind that of birds, mammals, fish and amphibians. Since then, great progress has been made in elucidating the mechanisms of reptilian immunity. Here, I review recent discoveries associated with the recognition of pathogens, effector mechanisms and memory responses in reptiles. Moreover, I put forward key questions to drive the next 10 years of research, including how reptiles are able to balance robust innate mechanisms with avoiding self-damage, how B cells and antibodies are used in immune defense and whether innate mechanisms can display the hallmarks of memory. Finally, I briefly discuss the links between our mechanistic understanding of the reptilian immune system and the field of eco-immunology. Overall, the field of reptile immunology is poised to contribute greatly to our understanding of vertebrate immunity in the next 10 years.

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.

Mucosal antibody quantity but not avidity predicts likelihood of Salmonella infection in red-eared slider turtles (Trachemys scripta).

Natural antibodies (NAbs) are polyreactive, have low avidity, and are a product of B-1 cells. Evidence suggests that NAbs may play a key role in immune defense in turtles, as increased total mucosal antibodies are associated with a decreased number of extracellular intestinal parasites. However, it is unknown if this trend extends to other types of pathogens and if avidity of the NAb to the pathogen is a factor in protection. We examined the relationship between a common intracellular bacteria in turtles-Salmonella-and NAbs. Plasma and mucosal samples were taken from red-eared slider turtles. We measured levels and avidity of antibodies that bound to lipopolysaccharide (LPS), a component of Salmonella cell wall. We examined the relationship between these measures and the ability of plasma to kill Salmonella as well as infection status. Higher mucosal antibody levels were significantly associated with a decrease in likelihood of infection with Salmonella; however, plasma antibody levels were not. There was a trend for bactericidal ability of the plasma to be positively correlated with plasma antibody levels bound to LPS, but not mucosal antibody levels. Avidity was not significantly related to either killing capacity or likelihood of infection suggesting that only increased quantity and not better binding is responsible for the decreased likelihood of infection. These findings suggest that NAb regulation was sufficient to isolate the infection to the gastrointestinal tract of the turtles, allowing it to be cleared with the mucus layer. Our results add further evidence that turtles use a general, nonspecific NAb response to combat pathogens.

Effects of imidacloprid on Rana catesbeiana immune and nervous system.

Imidacloprid (IMD), a neonicotinoid, is generally considered to be of low toxicity in vertebrates. However, the inhibition of acetylcholine (ACh) receptors can have a profound effect on both the immune and nervous system due to the anti-inflammatory effects of ACh. Vertebrates, such as amphibians, might be affected by IMD because they breed in wetlands where the concentration of IMD is high. In our study, we experimentally exposed Rana catesbeiana tadpoles to environmentally relevant IMD and then quantified the ACh and antibody to non-replicating antigens. We hypothesized that IMD exposure would result in higher AChE and antibody levels. We completed a factorial experiment in which tadpoles were divided into four groups, two of which were exposed to 100 ng/L of IMD. After five weeks, two groups were injected with the novel antigen keyhole limpet hemocyanin (KLH) and two injected with a control. Three weeks later, tadpoles were euthanized and blood samples collected. At 100 ng/L, IMD exposure did not cause a significant difference in AChE levels or KLH-specific IgY antibodies. However, tadpoles injected with KLH had slightly higher levels of AChE. In addition, we saw a trend in total IgM with higher levels in tadpoles exposed to IMD. While we found no effect of IMD at 100 ng/L on antibody response to a novel, non-replicating antigen nor on ACh production, further research is needed to determine if higher concentrations of IMD or parasite infection can influence development of R. catesbeiana.

Relationships between parasitic infection and natural antibodies, age, and sex in a long-lived vertebrate.

Reptilian immune systems are believed to rely more heavily on the innate response than the adaptive response. Past research on reptilian immune systems has indicated that natural antibodies (NAbs) play an important role in fighting antigens as the first responder and initiating the innate immune response. However, there is a gap in research on NAbs in reptiles as there is little data on how they may influence parasite prevalence in reptiles. Furthermore, no studies have examined NAbs at mucosal sites, which represent an important point of entry for parasites. We investigated NAbs in male and female red-eared slider turtles (Trachemys scripta) in order to begin to fill this gap in our understanding. Plasma and mucosal samples were collected from T. scripta to determine total immunoglobulin levels in relation to intestinal parasite load, which was determined from fecal samples. We also determined antibody levels to various antigens to examine if NAbs are present in mucosal samples and how levels compare to plasma NAbs. In female turtles, parasite intensity decreased with increasing host age and mucosal antibody levels. We also found that the levels of antibodies that bind to a novel antigen and an antigen they are likely to have encountered were significantly related in both plasma and mucosal samples, indicating a NAb response. This study contributes to our understanding of how reptiles may rely on a broad innate response that allows them to resist potential pathogens while potentially avoiding some negative consequences of immunosenescence.

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

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.

Sex-biased terminal investment in offspring induced by maternal immune challenge in the house wren (Troglodytes aedon).

The reproductive costs associated with the upregulation of immunity have been well-documented and constitute a fundamental trade-off between reproduction and self-maintenance. However, recent experimental work suggests that parents may increase their reproductive effort following immunostimulation as a form of terminal parental investment as prospects for future reproduction decline. We tested the trade-off and terminal investment hypotheses in a wild population of house wrens (Troglodytes aedon) by challenging the immune system of breeding females with lipopolysaccharide, a potent but non-lethal antigen. Immunized females showed no evidence of reproductive costs; instead, they produced offspring of higher phenotypic quality, but in a sex-specific manner. Relative to control offspring, sons of immunized females had increased body mass and their sisters exhibited higher cutaneous immune responsiveness to phytohaemagglutinin injection, constituting an adaptive strategy of sex-biased allocation by immune-challenged females to enhance the reproductive value of their offspring. Thus, our results are consistent with the terminal investment hypothesis, and suggest that maternal immunization can induce pronounced transgenerational effects on offspring phenotypes.

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

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.