High sugar diet alters immune function and the gut microbiome in juvenile green iguanas (Iguana iguana).

The present work aimed to study whether a high sugar diet can alter immune responses and the gut microbiome in green iguanas. Thirty-six iguanas were split into four treatment groups using a 2×2 design. Iguanas received either a sugar-supplemented diet or a control diet, and either a lipopolysaccharide (LPS) injection or a phosphate-buffered saline (PBS) injection. Iguanas were given their respective diet treatment through the entire study (∼3 months) and received a primary immune challenge 1 and 2 months into the experiment. Blood samples and cloacal swabs were taken at various points in the experiment and used to measure changes in the immune system (bacterial killing ability, lysis and agglutination scores, LPS-specific IgY concentrations), and alterations in the gut microbiome. We found that a sugar diet reduces bacterial killing ability following an LPS challenge, and sugar and the immune challenge temporarily alters gut microbiome composition while reducing alpha diversity. Although sugar did not directly reduce lysis and agglutination following the immune challenge, the change in these scores over a 24-h period following an immune challenge was more drastic (it decreased) relative to the control diet group. Moreover, sugar increased constitutive agglutination outside of the immune challenges (i.e. pre-challenge levels). In this study, we provide evidence that a high sugar diet affects the immune system of green iguanas (in a disruptive manner) and alters the gut microbiome.

Sex-specific effects of immune challenges on green anole lizard metabolism.

Immune responses can increase survival, but they can also incur a variety of costs that may lead to phenotypic trade-offs. The nature of trade-offs between immune activity and other components of the phenotype can vary and depend on the type and magnitude of immune challenge, as well as the energetic costs of simultaneously expressing other traits. There may also be sex-specific differences in both immune activity and trade-offs, particularly with regard to energy expenditure that might differ between males and females during the breeding season. Females are generally expected to invest less in nonspecific immune responses compared to males due to differences in the allocation of resources to reproduction, which may lead to sex differences in the metabolic costs of immunity. We tested for sex-specific differences in metabolic costs of different types of immune challenges in Anolis carolinensis lizards, including lipopolysaccharide (LPS) injection and wounding. We also tested for differences in immune prioritization by measuring bacterial killing ability (BKA). We predicted males would show a greater increase in metabolism after immune challenges, with combined immune challenges eliciting the greatest response. Furthermore, we predicted that metabolic costs would result in decreased BKA. LPS injection increased the resting metabolic rate (RMR) of males but not females. Wounding did not affect RMR of either sex. However, there was an inverse relationship between BKA and wound healing in LPS-injected lizards, suggesting dynamic tradeoffs among metabolism and components of the immune system.

Invading nonnative frogs use different microhabitats and change physiology along an elevation gradient.

The coqui frog (Eleutherodactylus coqui) was introduced to the island of Hawai'i in the 1980s, and has spread across much of the island. There is concern they will invade higher elevation areas where negative impacts on native species are expected. It is not known if coqui change behavior and baseline physiology in ways that allow them to invade higher elevations. We investigated where coqui are found across the island and whether that includes recent invasion into higher elevations. We also investigated whether elevation is related to coqui's microhabitat use, including substrate use and height off the forest floor, and physiological metrics, including plasma osmolality, oxidative status, glucose, free glycerol, and triglycerides, that might be associated with invading higher elevations. We found coqui have increased the area they occupy along roads from 31% to 50% and have moved into more high-elevation locations (16% vs. 1%) compared to where they were found 14 years ago. We also found frogs at high elevation on different substrates and closer to the forest floor than frogs at lower elevations-perhaps in response to air temperatures which tended to be warmer close to the forest floor. We observed that blood glucose and triglycerides increase in frogs with elevation. An increase in glucose is likely an acclimation response to cold temperatures while triglycerides may also help frogs cope with the energetic demands of suboptimal temperatures. Finally, we found that female coqui have higher plasma osmolality, reactive oxygen metabolites (dROMs), free glycerol, and triglycerides than males. Our study suggests coqui behavior and physiology in Hawai'i may be influenced by elevation in ways that allow them to cope with lower temperatures and invade higher elevations.

Invasive frogs show persistent physiological differences to elevation and acclimate to colder temperatures.

The coqui frog (Eleutherodactylus coqui) was introduced to the island of Hawai'i in the 1980s and has spread across much of the island. Concern remains that this frog will continue to expand its range and invade higher elevation habitats where much of the island's endemic species are found. We determined whether coqui thermal tolerance and physiology change along Hawai'i's elevational gradients. We measured physiological responses using a short-term experiment to determine baseline tolerance and physiology by elevation, and a long-term experiment to determine the coqui's ability to acclimate to different temperatures. We collected frogs from low, medium, and high elevations. After both the short and long-term experiments, we measured critical thermal minimum (CTmin), blood glucose, oxidative stress, and corticosterone levels. CTmin was lower in high elevation frogs than low elevation frogs after the short acclimation experiment, signifying that they acclimate to local conditions. After the extended acclimation, CTmin was lower in frogs acclimated to cold temperatures compared to warm-acclimated frogs and no longer varied by elevation. Blood glucose levels were positively correlated with elevation even after the extended acclimation, suggesting glucose may also be related to lower temperatures. Oxidative stress was higher in females than males, and corticosterone was not significantly related to any predictor variables. The extended acclimation experiment showed that coquis can adjust their thermal tolerance to different temperatures over a 3-week period, suggesting the expansion of coqui into higher elevation habitats may still be possible, and they may not be as restricted by cold temperatures as previously thought.

Egg viability and egg mass underlie immune tradeoffs and differences between urban and rural lizard egg yolk physiology.

Urbanization can cause innumerable abiotic and biotic changes that have the potential to influence the ecology, behavior, and physiology of native resident organisms. Relative to their rural conspecifics, urban Side-blotched Lizard (Uta stansburiana) populations in southern Utah have lower survival prospects and maximize reproductive investment via producing larger eggs and larger clutch sizes. While egg size is an important predictor of offspring quality, physiological factors within the egg yolk are reflective of the maternal environment and can alter offspring traits, especially during energetically costly processes, such as reproduction or immunity. Therefore, maternal effects may represent an adaptive mechanism by which urban-dwelling species can persist within a variable landscape. In this study, we assess urban and rural differences in egg yolk bacterial killing ability (BKA), corticosterone (CORT), oxidative status (d-ROMs), and energy metabolites (free glycerol and triglycerides), and their association with female immune status and egg quality. Within a laboratory setting, we immune challenged urban lizards via lipopolysaccharide injection (LPS) to test whether physiological changes associated with immune system activity impacted egg yolk investment. We found urban females had higher mite loads than rural females, however mite burden was related to yolk BKA in rural eggs, but not urban eggs. While yolk BKA differed between urban and rural sites, egg mass and egg viability (fertilized vs. unfertilized) were strong predictors of yolk physiology and may imply tradeoffs exist between maintenance and reproduction. LPS treatment caused a decrease in egg yolk d-ROMs relative to the control treatments, supporting results from previous research. Finally, urban lizards laid a higher proportion of unfertilized eggs, which differed in egg yolk BKA, CORT, and triglycerides in comparison to fertilized eggs. Because rural lizards laid only viable eggs during this study, these results suggest that reduced egg viability is a potential cost of living in an urban environment. Furthermore, these results help us better understand potential downstream impacts of urbanization on offspring survival, fitness, and overall population health.

Blood chemistry and biliverdin differ according to reproduction and tourism in a free-living lizard.

Changes in the physiological health of species are an essential indicator of changing conditions and environmental challenges. Reponses to environmental challenges can often induce stress, influence physiology, and change metabolism in organisms. Here we tested blood chemistry parameters indicative of stress and metabolic activity using an i-STAT point-of-care blood analyzer in seven populations of free-ranging rock iguanas exposed to varying levels of tourism and supplemental feeding. We found significant differences in blood chemistry (glucose, oxygen, carbon dioxide, hematocrit, hemoglobin, calcium, potassium, and biliverdin levels) among populations exposed to varying levels of tourism, and some variation between sexes and reproductive states. However, different variables are not directly related to one another, suggesting that the causal physiological pathways driving tourism-induced differences are influenced by mechanisms that are not detected by common analyses of blood chemistry. Future work should investigate upstream regulators of these factors affected by tourism. Regardless, these blood metrics are known to be both stress sensitive and related to metabolic activity, suggesting that exposure to tourism and associated supplemental feeding by tourists are generally driven by stress-related changes in blood chemistry, biliverdin, and metabolism.

Short-term impact of a wildfire on the homeostasis of Tropidurus oreadicus lizards.

Wildfires cause significant changes in natural habitats and can impact lizard populations. Through changes in the thermal environment, reduced prey availability, and increased exposure to parasite vectors, wildfires affect lizard physiology, immunity, and health. We sampled 56 Tropidurus oreadicus lizards from Cerrado savannas of Brazil living in two adjacent sites: one burned 14 days before the study, and the other unburned for 6 years. We logged the air temperatures of those sites throughout fieldwork. We assessed the short-term possible homeostatic imbalances caused by the fires via measuring body mass, circulating levels of corticosterone (CORT), leukocytes profile changes in heterophile-lymphocyte ratios (HLRs), innate immunity using the bacterial killing assay (BKA), and the diagnosis of hemoparasites using molecular techniques. The air temperature was significantly higher in the burned site. There was no difference in lizard body mass between the two sites, suggesting that prey availability was not affected by the wildfire. While parasite presence was seemingly not affected by fire, the timing of initial parasite infection for animals in the study was unknown, so we also evaluated parasitism as an independent variable relative to the other metrics. Our results showed that parasitic infections lead to reduced bactericidal capacity and body mass in lizards, suggesting clinical disease and depletion of innate immune resources. Moreover, we observed increased HLR with fire and parasitic infections and a strong negative correlation with BKA. These findings suggest that the increased environmental temperature following wildfires may lead to increased CORT and decreased BKA.

Complex tourism and season interactions contribute to disparate physiologies in an endangered rock iguana.

To promote survival and fitness, organisms use a suite of physiological systems to respond to both predictable and unpredictable changes in the environment. These physiological responses are also influenced by changes in life history state. The continued activation of physiological systems stemming from persistent environmental perturbations enable animals to cope with these challenges but may over time lead to significant effects on the health of wildlife. In the present study, we tested how varying environmental perturbations driven by tourism and associated supplemental feeding affects the energetics, corticosterone and immunity of six discrete populations of the northern Bahamian rock iguana (Cyclura cychlura inornata and Cyclura cychlura figginsi). We studied populations within and outside the reproductive season and quantified tourist numbers during sample collection. Specifically, we measured clutch size, body condition, plasma energy metabolites, reactive oxygen species, baseline corticosterone concentrations and immune function of male and female iguanas from each population to address whether (i) disparate physiologies are emerging across a gradient of tourism and feeding, (ii) both subspecies respond similarly and (iii) responses vary with season/reproductive condition. We found significant effects of tourism level, season and their interaction on the physiology of both C. c. inornata and C. c. figginsi, supporting the idea that tourism is leading to the divergence of phenotypes. Specifically, we found elevated plasma energy metabolites, oxidative stress and a measure of innate immunity (bactericidal ability), but reduced corticosterone concentrations with increasing tourism in both subspecies of rock iguanas. These physiological metrics differ according to the level of tourism in both subspecies and persist across seasons despite variation with natural seasonal and reproductive changes. These findings suggest that anthropogenic disturbance results in disparate physiologies in northern Bahamian rock iguanas.

Glucose tolerance of iguanas is affected by high-sugar diets in the lab and supplemental feeding by ecotourists in the wild.

There is great interspecific variation in the nutritional composition of natural diets, and the varied nutritional content is physiologically tolerated because of evolutionarily based balances between diet composition and processing ability. However, as a result of landscape change and human exposure, unnatural diets are becoming widespread among wildlife without the necessary time for evolutionary matching between the diet and its processing. We tested how a controlled, unnatural high glucose diet affects glucose tolerance using captive green iguanas, and we performed similar glucose tolerance tests on wild Northern Bahamian rock iguanas that are either frequently fed grapes by tourists or experience no such supplementation. We evaluated both short and longer-term blood glucose responses and corticosterone (CORT) concentrations as changes have been associated with altered diets. Experimental glucose supplementation in the laboratory and tourist feeding in the wild both significantly affected glucose metabolism. When iguanas received a glucose-rich diet, we found greater acute increases in blood glucose following a glucose challenge. Relative to unfed iguanas, tourist-fed iguanas had significantly lower baseline CORT, higher baseline blood glucose, and slower returns to baseline glucose levels following a glucose challenge. Therefore, unnatural consumption of high amounts of glucose alters glucose metabolism in laboratory iguanas with short-term glucose treatment and free-living iguanas exposed to long-term feeding by tourists. Based on these results and the increasing prevalence of anthropogenically altered wildlife diets, the consequences of dietary changes on glucose metabolism should be further investigated across species, as such changes in glucose metabolism have health consequences in humans (e.g. diabetes).

Climate effects on nesting phenology in Nebraska turtles.

A frequent response of organisms to climate change is altering the timing of reproduction, and advancement of reproductive timing has been a common reaction to warming temperatures in temperate regions. We tested whether this pattern applied to two common North American turtle species over the past three decades in Nebraska, USA. The timing of nesting (either first date or average date) of the Common Snapping Turtle (Chelydra serpentina) was negatively correlated with mean December maximum temperatures of the preceding year and mean May minimum and maximum temperatures in the nesting year and positively correlated with precipitation in July of the previous year. Increased temperatures during the late winter and spring likely permit earlier emergence from hibernation, increased metabolic rates and feeding opportunities, and accelerated vitellogenesis, ovulation, and egg shelling, all of which could drive earlier nesting. However, for the Painted Turtle (Chrysemys picta), the timing of nesting was positively correlated with mean minimum temperatures in September, October, December of the previous year, February of the nesting year, and April precipitation. These results suggest warmer fall, and winter temperature may impose an increased metabolic cost to painted turtles that impedes fall vitellogenesis, and April rains may slow the completion of vitellogenesis through decreased basking opportunities. For both species, nest deposition was highly correlated with body size, and larger females nested earlier in the season. Although average annual ambient temperatures have increased over the last four decades of our overall fieldwork at our study site, spring temperatures have not yet increased, and hence, nesting phenology has not advanced at our site for Chelydra. While Chrysemys exhibited a weak trend toward later nesting, this response was likely due to increased recruitment of smaller females into the population due to nest protection and predator control (Procyon lotor) in the early 2000s. Should climate change result in an increase in spring temperatures, nesting phenology would presumably respond accordingly, conditional on body size variation within these populations.