Testing androgen-induced immunosuppression: Environmental androgens as a model system for steroid-immune interaction.

It is well known that hormones influence and direct most facets of physiology; however, there is still contention regarding the directions of certain relationships, for example, between gonadal hormones and immunity. Among the many proposed relationships relating to gonadal-immune interactions, support for immunosuppressive effects of androgens remains prominent within physiological literature. Although ample study has been directed toward the immunosuppressive effects of androgens, considerable disagreement remains regarding their influence on immune function. In this study, we test the hypothesis that androgens inhibit immunocompetence in the American alligator (Alligator mississippiensis). Developing alligators were incubated at female-producing temperatures with a subset of individuals being exposed to 17-α-methyltestosterone (MT) before sexual determination. 17-α-methyltestosterone is a potent androgen, not aromatizable by crocodilians, that has been found to exert masculinizing effects in exposed crocodilian populations in vivo and in vitro. Additionally, a subset of animals was exposed to a novel antigen to quantify innate and acquired immune function. We recovered no significant differences in leukocyte ratios or proportions between groups and found no significant differences in innate immune function as measured by hemolysis-hemagglutination. However, we did find significant differences in acquired immune function, where masculinized individuals expressed greater antibody titers. Our findings reject the hypothesis that androgens suppress immune function; rather, androgens may be immunoenhancing to acquired humoral responses and neutral to innate humoral immunity in crocodilians.

Latitudinal gradients in sexual dimorphism: Alternative hypotheses for variation in male traits.

Biological patterns across latitudinal gradients elucidate a number of striking natural clines from which numerous processes can be further explored. The trade-off between reproduction and somatic maintenance and growth represents a suite of life-history traits with variable energy allocation and potential latitudinal patterns. Specifically, male sexually dimorphic traits in female choice systems represent one such reproductive investment constrained by resource acquisition and subsequent allocation. Latitudinal variation in sexual dimorphism has been suggested although the relationship between dimorphic traits and latitude are conflicting. Here, we test alternative hypotheses regarding this pattern using two broadly distributed vertebrates exhibiting sexually dimorphic traits. We hypothesized that the exaggeration of dimorphic traits correlates with latitude, with males having exaggerated sexually dimorphic traits at either higher or lower latitudes. Results indicate that male sexually dimorphic traits are exaggerated at lower latitudes while relative gonopodium size in Poecilia latipinna was larger at higher latitudes. This pattern may be a result of lower latitude populations experiencing greater population densities and longer access to resources that could manifest in females more intensively selecting for higher quality males in lower latitudes. Experimental work should address this pattern and investigate mechanistic processes.

Testing the febrile response of snakes inoculated with Ophidiomyces ophidiicola, the causative agent of snake fungal disease.

Snake Fungal Disease (SFD) negatively impacts wild snake populations in the eastern United States and Europe. Ophidiomyces ophidiicola causes SFD and manifests clinically by the formation of heterophilic granulomas around the mouth and eyes, weight loss, impaired vision, and sometimes death. Field observations have documented early seasonal basking behaviors in severely infected snakes, potentially suggesting induction of a behavioral febrile response to combat the mycosis. This study tested the hypothesis that snakes inoculated with Ophidiomyces ophidiicola would seek elevated basking temperatures to control body temperature and behaviorally induce a febrile response. Eastern ribbon snakes (Thamnophis saurita, n = 29) were experimentally or sham inoculated with O. ophidiicola. Seven days after inoculation, snakes were tested on a thermal gradient and the internal body temperature and substrate temperature of each snake was recorded over time. Quantitative PCR was used when snakes arrived, during pre-inoculation, and post-inoculation to test snakes for the presence of O. ophidiicola. Some snakes arrived with O. ophidiicola and were subsequently inoculated, allowing for an assessment of secondary exposure effects. Snake thermoregulatory behavior was compared between 1) O. ophidiicola inoculated vs. sham inoculated treatments, 2) infected vs. disease negative groups, and 3) disease naïve vs. pre-exposed immune response categories. Neither internal nor substrate temperatures differed among initially prescribed, and qPCR recovered disease states, although infected snakes tended to reach a preferred body temperature faster than disease negative snakes. Snakes experiencing their first exposure (disease naïve) sought higher substrate temperatures than snakes experiencing their second exposure (pre-exposed). Here, we recover no evidence for behaviorally induced fever in snakes with SFD but do elucidate a febrile immune response associated with secondary exposure.

Physiological responses of gopher tortoises (Gopherus polyphemus) to trapping.

With a growing number of species of conservation concern, understanding the physiological effects of routine sampling of vertebrate species remains a priority to maintain the welfare status of wildlife and ensure such activities are not counter to conservation goals. The gopher tortoise (Gopherus polyphemus) is a species of conservation concern throughout its range and is among the most frequently trapped turtles globally (for both research and conservation activities). Several studies have found equivocal results on the effects of trapping and handling on the glucocorticoid stress response. In this study, we tested how multiple physiological biomarkers (i.e. plasma lactate, corticosterone (cort), heterophil:lymphocyte ratio (HLR) and bactericidal ability (BA)) respond to four different combinations of trapping conditions in comparison to baseline reference sampling. We found that trapping and handling of gopher tortoises yielded a rapid rise in plasma lactate concentration followed by elevations of cort and stress-associated immune changes. In visibly distressed animals that were in traps for fewer than 2 hours, lactate, cort, HLR and BA were all elevated, and generally more so than animals that remained calm in traps for a similar amount of time. Animals that had been trapped and then held for a 3-hour restraint showed similar degrees of physiological alteration as those that showed outward signs of distress. This study demonstrates that trapping may yield physiological disturbances in gopher tortoises, although the intensity of this response is highly variable between individuals and the duration of such alterations remains unknown. This research emphasizes the need for continued work to refine trapping and handling processes in an effort to minimize impacts on individuals and populations.

Sex-based trade-offs in the innate and acquired immune systems of Sternotherus minor.

Longevity patterns in most vertebrates suggest that females benefit most from maintenance investment. A reversed longevity pattern in loggerhead musk turtles (Sternotherus minor) allowed us to test trade-offs between maintenance and survivorship. We tested the hypothesis that the sex with greater longevity has greater maintenance than the sex with shorter longevity. We also compared the following parameters between sexes: Bactericidal ability (BA) and heterophil:lymphocyte ratios (HLR). Baseline blood samples were collected from turtles in the field; a subset of turtles was returned to a laboratory for experiments of acquired immune responses to sheep red blood cells (SRBC). We found no support for the original hypothesis of reversal in sex-dependent immune trade-offs (difference between sex SRBC titers: p = .102; interaction between treatment and sex: p = .177; difference between treatments: p < .001; effect of sex on BA: p = .830; effect of sex on HLR: p = .717). However, we did find support for sex-dependent differences in immunity in the relationship between HLR and body condition (BCI) (effect of BCI on HLR: p = .015). In field conditions, we found that males with higher body condition indices express stressed phenotypes more than males with lower body condition indices (p = .002). However, females expressed similar stress loads across all body conditions (p = .900). Testosterone concentrations were assayed in free-living turtles and were not related to any of the immune parameters. Our results suggest that the immune systems play an important role in balancing sex-specific responses to different selective pressures in S. minor.

Upper respiratory tract disease and associated diagnostic tests of mycoplasmosis in Alabama populations of Gopher tortoises, Gopherus polyphemus.

Upper respiratory tract disease (URTD) in North American tortoises (Gopherus) has been the focus of numerous laboratory and field investigations, yet the prevalence and importance of this disease remains unclear across many tortoise populations. Furthermore, much research has been focused on understanding diagnostic biomarkers of two known agents of URTD, Mycoplasma agassizii and Mycoplasma testudineum, yet the reliability and importance of these diagnostic biomarkers across populations is unclear. Gopher Tortoises (Gopherus polyphemus) have experienced significant declines and are currently protected range wide. Geographically, Alabama represents an important connection for Gopher Tortoise populations between the core and periphery of this species' distribution. Herein, we systematically sampled 197 Gopher Tortoises for URTD across seven sites in south-central and south-eastern Alabama. Plasma samples were assayed for antibodies to M. agassizii and M. testudineum; nasal lavage samples were assayed for the presence of viable pathogens as well as pathogen DNA. Lastly, animals were scored for the presence of external symptoms and nasal scarring consistent with URTD. External symptoms of URTD were present in G. polyphemus in all sites sampled in Alabama. There was no relationship between active symptoms of URTD and Mycoplasma antibodies, however the presence of URTD nasal scarring was positively related to M. agassizii antibodies (P = 0.032). For a single site that was sampled in three sequential years, seroprevalence to M. agassizii significantly varied among years (P < 0.0001). Mycoplasma agassizii DNA was isolated from four of the seven sites using quantitative PCR, yet none of the samples were culture positive for either of the pathogens. An analysis of disease status and condition indicated that there was a significant, positive relationship between the severity of URTD symptoms and relative body mass (P < 0.05). This study highlights the need for continued monitoring of disease in wild populations. Specifically, focus must be placed on identifying other likely pathogens and relevant biomarkers that may be important drivers of URTD in North American tortoises. Special consideration should be given to environmental contexts that may render wild populations more susceptible to disease.

Rapid thermal immune acclimation in common musk turtles (Sternotherus odoratus).

As infectious diseases in ectothermic vertebrates increasingly threaten wild populations, understanding how host immune systems are affected by the environment is key to understanding the process of infection. In this study, we investigated how temperature change and simulated bacterial infection (via lipopolysaccharide [LPS] injection) interacted to regulate innate immunity, as measured by bactericidal ability (BA), phagocytosis rate, and heterophil:lymphocyte ratio (HLR) in common musk turtles (Sternotherus odoratus). We found that LPS stimulated an acute immune response, as measured by an increase in BA, phagocytosis rate, and HLR. When exposed to a 5 or 10°C temperature change for 48 hr, turtles rapidly acclimated to the new temperature by adjusting their immune output. This acclimation was compensatory as seen by elevated rates of immune output in colder animals and decreased rates of immune output in warmer animals. These results indicate that while temperature change may be a constraint on some animals, S. odoratus have the ability to rapidly adjust immunity to match environmental thermal demand. This rapid ability to adjust immunity may be related to the broad geographic distribution of musk turtles. Future research should focus on how immune acclimation in ectotherms varies both intraspecifically and interspecifically across regional scales and geographic distributions.

Emendation and new species of Hapalorhynchus Stunkard, 1922 (Digenea: Schistosomatoidea) from musk turtles (Kinosternidae: Sternotherus) in Alabama and Florida rivers.

Hapalorhynchus Stunkard, 1922 is emended based on morphological study of existing museum specimens (type and voucher specimens) and newly-collected specimens infecting musk turtles (Testudines: Kinosternidae: Sternotherus spp.) from rivers in Alabama and Florida (USA). Hapalorhynchus conecuhensis n. sp. is described from an innominate musk turtle, Sternotherus cf. minor, (type host) from Blue Spring (31°5'27.64″N, 86°30'53.21″W; Pensacola Bay Basin, Alabama) and the loggerhead musk turtle, Sternotherus minor (Agassiz, 1857) from the Wacissa River (30°20'24.73″N, 83°59'27.56″W; Apalachee Bay Basin, Florida). It differs from congeners by lacking a body constriction at level of the ventral sucker, paired anterior caeca, and a transverse ovary as well as by having a small ventral sucker, proportionally short posterior caeca, nearly equally-sized anterior and posterior testes, a small cirrus sac, and a uterus extending dorsal to the ovary and the anterior testis. Specimens of Hapalorhynchus reelfooti Byrd, 1939 infected loggerhead musk turtles, stripe-necked musk turtles (Sternotherus peltifer Smith and Glass, 1947), Eastern musk turtles (Sternotherus odoratus [Latreille in Sonnini and Latreille, 1801]), and S. cf. minor. Those of Hapalorhynchus cf. stunkardi infected S. minor and S. odoratus. Sternothorus minor, S. peltifer, and S. cf. minor plus S. minor and S. odoratus are new host records for H. reelfooti and H. cf. stunkardi, respectively. This is the first report of an infected musk turtle from the Coosa and Tallapoosa Rivers (Mobile-Tensaw River Basin), Pensacola Bay Basin, or Apalachee Bay Basin. Sequence analysis of the large subunit rDNA (28S) showed a strongly-supported clade for Hapalorhynchus.

More than Fever: Thermoregulatory Responses to Immunological Stimulation and Consequences of Thermoregulatory Strategy on Innate Immunity in Gopher Tortoises (Gopherus polyphemus).

Organisms possess a range of thermoregulatory strategies that may vary in response to sickness, thereby driving important life-history consequences. Because the immune system is vital to maintaining organism function, understanding the suite of immune responses to infection indicates basic costs and benefits of physiological strategies. Here, we assessed consequences of thermoregulation and seasonality on immune function in both immunologically stimulated and nonstimulated gopher tortoises (Gopherus polyphemus). An ectothermic vertebrate was used as an experimental model because the effects of thermoregulation on immunity remain understudied and are of increasing importance in light of anthropogenic alterations to thermal environments. We found that G. polyphemus increased body temperature (Tb) at 1 h after injection with lipopolysaccharide (LPS) when compared with saline controls (P = 0.04), consistent with behavioral fever. LPS increased plasma bactericidal ability (BA; P = 0.006), reduced plasma iron concentration (P = 0.041), and increased heterophil∶lymphocyte ratios (P < 0.001). In nonstimulated animals, thermoregulatory strategy had a strong effect on innate immunity, which demonstrated that individuals have the ability to facultatively adjust immune function when infection burden is low; this relationship was not present in LPS-injected animals, which suggested that animals stimulated with LPS maximize bactericidal ability independently of temperature. Seasonal acclimation state did not influence responses to LPS, although baseline plasma iron was significantly lower in animals acclimated to winter. These results support that a trade-off exists between immunity and other conflicting physiological interests. Moreover, these results clearly demonstrate the ability of individuals to modulate immune function as a direct result of thermoregulatory decisions.

Lag of Immunity Across Seasonal Acclimation States in Gopher Tortoises (Gopherus Polyphemus).

Disease outbreaks are of increasing importance to ectothermic vertebrates as one of numerous results of global change. Anthropogenic climate change is predicted to increase climatic instability, thereby altering natural thermal environments. In this study, we evaluated the direct effects of rapid temperature change on immunity in Gopher Tortoises (Gopherus polyphemus). Specifically, we tested the lag hypothesis, which predicts significant misalignment of optimal and realized immunity when temperature rapidly changes. We assayed constitutive innate immunity, B-cell humoral responses, and heterophil: lymphocyte ratios in response to rapid temperature changes corresponding to realistic changes in body temperature between winter and summer. We found that during summer, rapid temperature reduction caused a series of changes in immunity, including reduced bactericidal ability (P = 0.002), reduced humoral response (P < 0.0001), and increased heterophil:lymphocyte ratios (P < 0.0001). During winter, we found that a temperature increase provided no benefit to immunity. Specifically, there was no increase in bactericidal ability as was predicted by the lag hypothesis. In winter, humoral responses were significantly reduced as a result of rapid warming (P = 0.011) and the rapid warming caused a significant reduction in heterophil:lymphocyte ratios (P < 0.0001). Independent of temperature, we found a significant acclimation effect of winter relative to summer conditions in humoral response (P < 0.001), which showed an overall increase in this parameter during winter. Our findings demonstrate that rapid temperature change, regardless of its direction, is a constraint on immunity in ectothermic vertebrates.

Seasonal Acclimation of Constitutive Immunity in Gopher Tortoises Gopherus polyphemus.

Studies have suggested a role for natural seasonal change to drive patterns of disease, especially within ectothermic vertebrates. In light of recent climate change, it is important to understand baseline disease resistance in a seasonal context to further understand the role that changes in seasonal weather patterns may have in increasing disease frequency. Herein we found support for the seasonal acclimation hypothesis in Gopherus polyphemus (gopher tortoise), which indicated that natural seasonal variation causes differences in baseline immune function across seasonal acclimation states. We found that an innate immune parameter, bactericidal ability (BA), was significantly elevated in the summer (P < 0.00001). Circulating leukocyte profiles varied significantly among seasons, with heterophils and monocytes increased (P = 0.00019 and P = 0.0001, respectively) and lymphocytes decreased (P < 0.00001) during winter. We assayed baseline glucocorticoid concentration (e.g., corticosterone [CORT]) across seasons and sampling conditions to test whether CORT drove the seasonal pattern in immunological acclimation. CORT was significantly lowest during winter and in animals temporarily maintained in seminatural conditions. These changes in CORT occurred independently of the immunological adjustments, suggesting that the seasonal pattern of immunity was not mediated by CORT secretion. The reduction in lymphocytes and BA and also BA during winter suggest that seasonal acclimation is likely a restraint on energetic output when temperature is low and physiological performance is thermally constrained. While these parameters were reduced in winter, the increase in heterophils and monocytes may indicate a compensatory immune adjustment to increase the number of innate phagocytic cells.