Experimental manipulation of microbiota reduces host thermal tolerance and fitness under heat stress in a vertebrate ectotherm.

Identifying factors that influence how ectothermic animals respond physiologically to changing temperatures is of high importance given current threats of global climate change. Host-associated microbial communities impact animal physiology and have been shown to influence host thermal tolerance in invertebrate systems. However, the role of commensal microbiota in the thermal tolerance of ectothermic vertebrates is unknown. Here we show that experimentally manipulating the tadpole microbiome through environmental water sterilization reduces the host's acute thermal tolerance to both heat and cold, alters the thermal sensitivity of locomotor performance, and reduces animal survival under prolonged heat stress. We show that these tadpoles have reduced activities of mitochondrial enzymes and altered metabolic rates compared with tadpoles colonized with unmanipulated microbiota, which could underlie differences in thermal phenotypes. These results demonstrate a strong link between the microbiota of an ectothermic vertebrate and the host's thermal tolerance, performance and fitness. It may therefore be important to consider host-associated microbial communities when predicting species' responses to climate change.

CURE in a Box: an Online CURE for Introductory Biology Majors That Incorporates Vision and Change.

Course-based undergraduate research experiences (CUREs) help fulfill the recommendation of Vision and Change for biology curricula to focus on key concepts and skills to better prepare students for careers in the sciences. However, the COVID-19 pandemic has forced many schools to move instruction online, highlighting the dearth of resources available for offering a CURE for introductory biology outside of the traditional laboratory-based setting. Here, we present a revision of our first-semester General Biology laboratory for a synchronous online setting where students perform a research-based wet lab component at home using an affordable preassembled kit while still meeting the CURE learning objectives. This template can be utilized by other colleges and universities that are looking to offer a safe, reliable, and affordable CURE in their introductory biology courses whether in a virtual or face-to-face environment.

Changes in the gut microbial community of the eastern newt (Notophthalmus viridescens) across its three distinct life stages.

Understanding the forces that shape vertebrate gut microbial community assembly and composition throughout development is a major focus of the microbiome field. Here, we utilize the complex life cycle of the eastern newt (Notophthalmus viridescens) as a natural wild model to compare the effects of host and environmental factors on gut microbiome development. We compared bacterial inventories of each of the newt's three physiologically distinct developmental stages to determine if each hosted a unique community, or if the two stages which share an aquatic habitat (larvae and adults) harbored more similar communities than those of the third stage, the terrestrial juvenile eft. Additionally, we assessed how the contribution of selective processes to gut microbial assembly changed through development. We found that structurally, each life stage harbored a distinct community, which may be attributable to host factors. Further, across development, we found that community assembly processes shifted from a predominance of neutral to selective forces. However, habitat may also be important in determining community membership and diversity due the uniqueness of eft communities based on these metrics. Our results are similar to those in other vertebrate taxa, suggesting that gut microbiota assembly processes may be conserved across diverse lineages.

Differential plasticity of membrane fatty acids in northern and southern populations of the eastern newt (Notophthalmus viridescens).

Seasonal changes in membrane composition and metabolic activity allow many temperate ectotherms to contend with changes in body temperature, but few studies have investigated whether the plasticity of these traits has diverged within a single species. Therefore, we studied the effects of thermal acclimation on the membrane fatty acid composition and the activities of cytochrome c oxidase (CCO) and citrate synthase (CS) in the skeletal muscle and liver of eastern newts from Maine and Florida. Newts were acclimated to either 6 °C or 28 °C for 12 weeks prior to experiments. Cold acclimation resulted in a lower saturated fatty acid (SFA) content in the muscle membranes of both populations. SFA content in liver was lower in cold compared to warm-acclimated newts from Florida, but acclimation did not affect SFA content in liver membranes of the Maine population. In liver, cold acclimation resulted in a higher monounsaturated fatty acid (MUFA) content in the Florida population and a higher polyunsaturated fatty acid (PUFA) content in the Maine population. Regardless of acclimation conditions, the muscle and liver membranes of the Maine population had higher SFA and PUFA contents compared to those of the Florida population. MUFA content of muscle and liver membranes was higher in the Florida population compared to the Maine population. The effect of acclimation on CCO and CS activity was tissue-specific. In muscle, CCO and CS activities were higher in cold compared to warm-acclimated newts in both populations, and CS and CCO activities were higher in the Maine compared to the Florida population. In liver, CCO and CS activity were unaffected by acclimation in the Florida population, but activity was lower in cold compared to warm-acclimated Maine newts. These results demonstrate that the phenotypic plasticity of these traits in response to seasonal change has diverged between northern and southern populations.

Thermal Acclimatization in Overwintering Tadpoles of the Green Frog, Lithobates clamitans (Latreille, 1801).

Seasonal acclimatization permits organisms to maintain function in the face of environmental change. Tadpoles of the green frog (Lithobates clamitans) overwinter as tadpoles in much of their range. Because they are active in winter, we hypothesized that green frog tadpoles would display acclimatization of metabolic and locomotor function. We collected tadpoles in Sewanee, Tennessee (35.2°N) in winter and summer. Tadpoles collected during each season were tested at both winter (8°C) and summer (26°C) temperatures. Winter tadpoles were able to maintain swimming performance at both temperatures, whereas swimming performance decreased at cold temperatures in summer tadpoles. There was no evidence for seasonal acclimatization of whole-animal metabolic rate. Although whole-animal metabolic acclimatization was not observed, the activities of cytochrome c oxidase, citrate synthase, and lactate dehydrogenase measured in skeletal muscle homogenates showed higher activity in winter-acclimatized tadpoles indicating compensation for temperature. Further, the composition of muscle membranes of winter tadpoles had less saturated and more monounsaturated fatty acids and a higher ω-3 balance, unsaturation index, and peroxidation index than summer tadpoles. These data indicate that reversible phenotypic plasticity of thermal physiology occurs in larval green frog tadpoles. They appear to compensate for colder temperatures to maintain burst-swimming velocity and the ability to escape predators without the cost of maintaining a constant, higher standard metabolic rate in the winter.

Does the thermal plasticity of metabolic enzymes underlie thermal compensation of locomotor performance in the eastern newt (Notophthalmus viridescens)?

Eastern newts (Notophthalmus viridescens) upregulate the metabolic capacity of skeletal muscle in winter to compensate for thermodynamic effects on metabolism. However, whether this compensation facilitates locomotor performance at low temperature is unknown. Therefore, our aim was to determine if thermal acclimation of metabolic enzymes in muscle benefits locomotion. Eastern newts from southern Ohio were acclimated to cold (5°C, 10:14 L:D) or warm (25°C, 14:10 L:D) conditions for 12 weeks. Following acclimation, we measured the locomotor performance (burst speed and time until exhaustion) and the activities of metabolic enzymes in skeletal muscle at 5-30°C. Creatine kinase (CK) activity in skeletal muscle was higher in cold compared to warm-acclimated newts, and cold-acclimated newts had a higher burst speed at low temperature compared to warm-acclimated newts. At low temperature, time until exhaustion was higher in cold compared to warm-acclimated newts, but the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) in muscle were lower in cold compared to warm-acclimated newts. Together, these results demonstrate that eastern newts compensate for the effects of low temperature on locomotor performance. Whereas thermal compensation of CK activity is correlated with burst locomotion at low temperature, aerobic enzymes in skeletal muscle (CS and CCO) are not linked to compensation of sustained locomotion.

The thermal plasticity of locomotor performance has diverged between northern and southern populations of the eastern newt (Notophthalmus viridescens).

Many temperate ectotherms undergo thermal acclimation to remain functional over a wide range of body temperatures, but few studies have investigated whether populations of a single species have evolved differences in the thermal plasticity of locomotor performance. Therefore, we asked whether the thermal plasticity of locomotor performance has diverged between northern and southern populations of eastern newts (Notophthalmus viridescens). We acclimated eastern newts from Florida and Maine to cold (6 °C) or warm (28 °C) conditions for 12 weeks. Following acclimation, we measured the burst speed of newts at 6, 11.5, 17, 22.5, 28, and 33.5 °C. We also measured the activities of creatine kinase (CK) and lactate dehydrogenase (LDH) in skeletal muscle of newts. The newts from Maine were better able to acclimate to low temperature compared to newts from Florida. Regardless of acclimation, the thermal sensitivity of burst speed was higher in the Florida compared to the Maine population. In general, newts from Maine performed better at low temperatures, whereas newts from Florida performed better at high temperatures. The activities of CK and LDH were lower in cold compared to warm-acclimated newts in the Florida population, but acclimation did not affect the activities of these enzymes in the Maine population. The activities of CK and LDH do not explain differences in the thermal plasticity of locomotor performance between populations. Our results demonstrate that the thermal sensitivity and plasticity of locomotor performance differ between northern and southern populations of eastern newts, suggesting that these traits readily adapt to the thermal environment.

Animal galloping and human hopping: an energetics and biomechanics laboratory exercise.

This laboratory exercise demonstrates fundamental principles of mammalian locomotion. It provides opportunities to interrogate aspects of locomotion from biomechanics to energetics to body size scaling. It has the added benefit of having results with robust signal to noise so that students will have success even if not "meticulous" in attention to detail. First, using respirometry, students measure the energetic cost of hopping at a "preferred" hop frequency. This is followed by hopping at an imposed frequency half of the preferred. By measuring the O2 uptake and work done with each hop, students calculate mechanical efficiency. Lessons learned from this laboratory include 1) that the metabolic cost per hop at half of the preferred frequency is nearly double the cost at the preferred frequency; 2) that when a person is forced to hop at half of their preferred frequency, the mechanical efficiency is nearly that predicted for muscle but is much higher at the preferred frequency; 3) that the preferred hop frequency is strongly body size dependent; and 4) that the hop frequency of a human is nearly identical to the galloping frequency predicted for a quadruped of our size. Together, these exercises demonstrate that humans store and recover elastic recoil potential energy when hopping but that energetic savings are highly frequency dependent. This stride frequency is dependent on body size such that frequency is likely chosen to maximize this function. Finally, by requiring students to make quantitative solutions using appropriate units and dimensions of the physical variables, these exercises sharpen analytic and quantitative skills.

Does low daily energy expenditure drive low metabolic capacity in the tropical robin, Turdus grayi?

Temperate and tropical birds possess divergent life history strategies. Physiological parameters including energy metabolism correlate with the life history such that tropical species with a slower 'pace of life' have lower resting and maximal metabolic rates than temperate congeners. To better understand the physiological mechanisms underlying these differences, we investigated the relationship of metabolic capacity, muscle oxidative capacity and activity patterns to variation in life history patterns in American robins (Turdus migratorius), while resident in central North America and Clay-colored robins (Turdus grayi) resident in Panama. We measured summit metabolism [Formula: see text] in birds from both tropical and temperate habitats and found that the temperate robins have a 60 % higher metabolic capacity. We also measured the field metabolic rate (FMR) of free-living birds using heart rate (HR) telemetry and found that temperate robins' daily energy expenditure was also 60 % higher. Thus, [Formula: see text] and FMR both reflect life history differences between the species. Further, both species operate at a nearly identical ~50 % of their thermogenic capacity throughout a given day. As a potential mechanism to explain differences in activity and metabolic capacity, we ask whether oxidative properties of flight muscle are altered in accordance with life history variation and found minimal differences in oxidative capacity of skeletal muscle. These data demonstrate a close relationship between thermogenic capacity and daily activity in free-living birds. Further, they suggest that the slow pace of life in tropical birds may be related to the maintenance of low activity rather than functional capacity of the muscle tissue.