Validating the automation of different measures of high temperature tolerance of small terrestrial insects.

Accurately phenotyping numerous test subjects is essential for most experimental research. Collecting such data can be tedious or time-consuming, and it can be biased or limited using manual observations. The thermal tolerance of small ectotherms is a good example of this type of phenotypic data, and it is widely used to investigate thermal adaptation, acclimation capacity and climate change resilience of small ectotherms. Here, we present the results of automatically generated thermal tolerance data using motion-tracking software on video recordings. The automatization was applied to two different heat tolerance assays, in two Drosophila species and used temperature acclimation to create variation in thermal tolerances. We find similar effect sizes of acclimation and hardening responses between manual and automated approaches, but different absolute tolerance estimates. This discrepancy likely reflects both technical differences in the assay conditions as well as the measured end-points of the assays. We conclude that both methods generate biological meaningful results, which reflect different aspects of the thermal biology, find no evidence of inflated variance in the manually scored assays, but find that automation can increase throughput several times without compromising quality. Further we show that the method can be applied to a wide range of arthropod taxa. We suggest that this automated method is a useful example of high throughput phenotyping. Further, we suggest this approach might be applied to other tedious laboratory traits, such as desiccation or starvation tolerance, with similar benefits to throughput but caution that the interpretation and potential comparison to results using different methodology rely on thorough validation of the assay and the involved biological mechanism.

Drawing the line: Linear or non-linear reaction norms in response to adult acclimation on lower thermal limits.

Estimates of lower thermal limits are widely used to infer sensitivity to climate variability, local adaptation and adaptive acclimation responses in ectotherms. These inferences build on the ecological relevance of the tolerance estimates and assume that estimates can be extrapolated to relevant conditions. Methodological effects for upper thermal limits have been extensively investigated, with different ramping rates and acclimation regimes giving rise to varying, and even disparate, conclusions. However, methodological effects have received much less attention for lower thermal limits. In this study, we explicitly test whether methodology could affect estimates of lower thermal limits in interaction with acclimation temperature and thermal variability, by acclimating adult Drosophila melanogaster to different constant and fluctuating temperature regimes and generating reaction norms at different ramping rates. We find that ramping rates have no significant effect on the lower thermal limits. Constant temperature acclimation resulted in non-linear reaction norms, while the introduction of thermal variability during adult life result in linear reaction norms. Thus, applying ecologically relevant conditions (here thermal variability) potentially impacts the results and conclusions of insect low temperature tolerance and acclimation capacity.

Temperature preference across life stages and acclimation temperatures investigated in four species of Drosophila.

Ectotherms can use microclimatic variation and behavioral thermoregulation to cope with unfavorable environmental temperatures. However, relatively little is known about how and if thermoregulatory behavior is used across life stages in small ectothermic insects. Here we investigate differences between three specialized Drosophila species from temperate, tropical or desert habitats and one cosmopolitan species by estimating the preferred temperature (Tpref) and the breadth (Tbreadth) of the distribution of adults, adult egg-laying, and larvae in thermal gradients. We also assess the plasticity of thermal preference following developmental acclimation to three constant temperatures. For egg-laying and larvae, we observe significant species differences in preferred temperature but this is not predicted by thermal ecology of the species. We corroborated this with previous studies of other Drosophila species and found that Tpref for egg laying and larvae have no relationship with annual mean temperature of the species' natural habitat. While adults have the greatest mobility, they show the greater variation in preference compared to juveniles contradicting common assumptions. We found evidence of developmental thermal acclimation in adult egg-laying preferred temperature, Tpref increasing with acclimation temperature, and in the breadth of the temperature preference distributions, Tbreadth decreasing with increasing acclimation temperature. Together, these data provide a high resolution and comprehensive look at temperature preferences across life stages and in response to acclimation. Results suggest that thermal preference, particularly in the early life stages, is relatively conserved among species and unrelated to temperature at species origin. Measuring thermal preference, in addition to thermal performance, is essential for understanding how species have adapted/will adapt to their thermal environment.

Evolution and plasticity of thermal performance: an analysis of variation in thermal tolerance and fitness in 22 Drosophila species.

The thermal biology of ectotherms is often used to infer species' responses to changes in temperature. It is often proposed that temperate species are more cold-tolerant, less heat-tolerant, more plastic, have broader thermal performance curves (TPCs) and lower optimal temperatures when compared to tropical species. However, relatively little empirical work has provided support for this using large interspecific studies. In the present study, we measure thermal tolerance limits and thermal performance in 22 species of Drosophila that developed under common conditions. Specifically, we measure thermal tolerance (CTmin and CTmax) as well as the fitness components viability, developmental speed and fecundity at seven temperatures to construct TPCs for each of these species. For 10 of the species, we also measure thermal tolerance and thermal performance following developmental acclimation to three additional temperatures. Using these data, we test several fundamental hypotheses about the evolution and plasticity of heat and cold resistance and thermal performance. We find that cold tolerance (CTmin) varied between the species according to the environmental temperature in the habitat from which they originated. These data support the idea that the evolution of cold tolerance has allowed species to persist in colder environments. However, contrary to expectation, we find that optimal temperature ( Topt) and the breadth of thermal performance ( Tbreadth) are similar in temperate, widespread and tropical species and we also find that the plasticity of TPCs was constrained. We suggest that the temperature range for optimal thermal performance is either fixed or under selection by the more similar temperatures that prevail during growing seasons. As a consequence, we find that Topt and Tbreadth are of limited value for predicting past, present and future distributions of species. This article is part of the theme issue 'Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen'.

Using museum specimens to track morphological shifts through climate change.

Museum specimens offer a largely untapped resource for detecting morphological shifts in response to climate change. However, morphological shifts can be obscured by shifts in phenology or distribution or sampling biases. Additionally, interpreting phenotypic shifts requires distinguishing whether they result from plastic or genetic changes. Previous studies using collections have documented consistent historical size changes, but the limited studies of other morphological traits have often failed to support, or even test, hypotheses. We explore the potential of collections by investigating shifts in the functionally significant coloration of a montane butterfly, Colias meadii, over the past 60 years within three North American geographical regions. We find declines in ventral wing melanism, which correspond to reduced absorption of solar radiation and thus reduced risk of overheating, in two regions. However, contrary to expected responses to climate warming, we find melanism increases in the most thoroughly sampled region. Relationships among temperature, phenology and morphology vary across years and complicate the distinction between plastic and genetic responses. Differences in these relationships may account for the differing morphological shifts among regions. Our findings highlight the promise of using museum specimens to test mechanistic hypotheses for shifts in functional traits, which is essential for deciphering interacting responses to climate change.This article is part of the theme issue 'Biological collections for understanding biodiversity in the Anthropocene'.

A critical test of Drosophila anaesthetics: Isoflurane and sevoflurane are benign alternatives to cold and CO2.

Anaesthesia is often a necessary step when studying insects like the model organism Drosophila melanogaster. Most studies of Drosophila and other insects that require anaesthesia use either cold exposure or carbon dioxide exposure to induce a narcotic state. These anaesthetic methods are known to disrupt physiology and behavior with increasing exposure, and thus ample recovery time is required prior to experimentation. Here, we examine whether two halogenated ethers commonly used in vertebrate anaesthesia, isoflurane and sevoflurane, may serve as alternative means of insect anaesthesia. Using D. melanogaster, we generated dose-response curves to identify exposure times for each anaesthetic (cold, CO2, isoflurane and sevoflurane) that allow for five-minutes of experimental manipulation of the animals after the anaesthetic was removed (i.e. 5min recovery doses). We then compared the effects of this practical dose on high temperature, low temperature, starvation, and desiccation tolerance, as well as locomotor activity and fecundity of female flies following recovery from anaesthesia. Cold, CO2 and isoflurane each had significant or near significant effects on the traits measured, but the specific effects of each anaesthetic differed, and effects on stress tolerance generally did not persist if the flies were given 48h to recover from anaesthesia. Sevoflurane had no measureable effect on any of the traits examined. Care must be taken when choosing an anaesthetic in Drosophila research, as the impacts of specific anaesthetics on stress tolerance, behavior and reproduction can widely differ. Sevoflurane may be a practical alternative to cold and CO2 anaesthesia in insects - particularly if flies are to be used for experiments shortly after anesthesia.

Experimental winter warming modifies thermal performance and primes acorn ants for warm weather.

The frequency of warm winter days is increasing under global climate change, but how organisms respond to warmer winters is not well understood. Most studies focus on growing season responses to warming. Locomotor performance is often highly sensitive to temperature, and can determine fitness outcomes through a variety of mechanisms including resource acquisition and predator escape. As a consequence, locomotor performance, and its impacts on fitness, may be strongly affected by winter warming in winter-active species. Here we use the acorn ant, Temnothorax curvispinosus, to explore how thermal performance (temperature-driven plasticity) in running speed is influenced by experimental winter warming of 3-5°C above ambient in a field setting. We used running speed as a measure of performance as it is a common locomotor trait that influences acquisition of nest sites and food in acorn ants. Experimental winter warming significantly altered thermal performance for running speed at high (26 and 36°C) but not low test temperatures (6 and 16°C). Although we saw little differentiation in thermal performance at cooler test temperatures, we saw a marked increase in running speed at the hotter test temperatures for ants that experienced warmer winters compared with those that experienced cooler winters. Our results provide evidence that overwintering temperatures can substantially influence organismal performance, and suggest that we cannot ignore overwintering effects when forecasting organismal responses to environmental changes in temperature.

Morphological and physiological determinants of local adaptation to climate in Rocky Mountain butterflies.

Flight is a central determinant of fitness in butterflies and other insects, but it is restricted to a limited range of body temperatures. To achieve these body temperatures, butterflies use a combination of morphological, behavioural and physiological mechanisms. Here, we used common garden (without direct solar radiation) and reciprocal transplant (full solar radiation) experiments in the field to determine the thermal sensitivity of flight initiation for two species of Colias butterflies along an elevation gradient in the southwestern Rocky Mountains. The mean body temperature for flight initiation in the field was lower (24-26°C) than indicated by previous studies (28-30°C) in these species. There were small but significant differences in thermal sensitivity of flight initiation between species; high-elevation Colias meadii initiated flight at a lower mean body temperature than lower-elevation Colias eriphyle. Morphological differences (in wing melanin and thoracic setae) drive body temperature differences between species and contributed strongly to differences in the time and probability of flight and air temperatures at flight initiation. Our results suggest that differences both in thermal sensitivity (15% contribution) and in morphology (85% contribution) contribute to the differences in flight initiation between the two species in the field. Understanding these differences, which influence flight performance and fitness, aids in forecasting responses to climate change.

Plasticity of upper thermal limits to acute and chronic temperature variation in Manduca sexta larvae.

In many ectotherms, exposure to high temperatures can improve subsequent tolerance to higher temperatures. However, the differential effects of single, repeated or continuous exposure to high temperatures are less clear. We measured the effects of single heat shocks and of diurnally fluctuating or constant rearing temperatures on the critical thermal maximum (CTmax) for final instar larvae of Manduca sexta Brief (2 h) heat shocks at temperatures of 35°C and above significantly increased CTmax relative to control temperatures (25°C). Increasing mean temperatures (from 25 to 30°C) or greater diurnal fluctuations (from constant to ±10°C) during larval development also significantly increased CTmax Combining these data showed that repeated or continuous temperature exposure during development improved heat tolerance beyond the effects of a single exposure to the same maximum temperature. These results suggest that both acute and chronic temperature exposure can result in adaptive plasticity of upper thermal limits.

Geographic divergence in upper thermal limits across insect life stages: does behavior matter?

Insects with complex life cycles vary in size, mobility, and thermal ecology across life stages. We examine how differences in the capacity for thermoregulatory behavior influence geographic differences in physiological heat tolerance among egg and adult Colias butterflies. Colias adults exhibit differences in morphology (wing melanin and thoracic setal length) along spatial gradients, whereas eggs are morphologically indistinguishable. Here we compare Colias eriphyle eggs and adults from two elevations and Colias meadii from a high elevation. Hatching success and egg development time of C. eriphyle eggs did not differ significantly with the elevation of origin. Egg survival declined in response to heat-shock temperatures above 38-40 °C and egg development time was shortest at intermediate heat-shock temperatures of 33-38 °C. Laboratory experiments with adults showed survival in response to heat shock was significantly greater for Colias from higher than from lower elevation sites. Common-garden experiments at the low-elevation field site showed that C. meadii adults initiated heat-avoidance and over-heating behaviors significantly earlier in the day than C. eriphyle. Our study demonstrates the importance of examining thermal tolerances across life stages. Our findings are inconsistent with the hypothesis that thermoregulatory behavior inhibits the geographic divergence of physiological traits in mobile stages, and suggest that sessile stages may evolve similar heat tolerances in different environments due to microclimatic variability or evolutionary constraints.

Evolutionary change in continuous reaction norms.

Understanding the evolution of reaction norms remains a major challenge in ecology and evolution. Investigating evolutionary divergence in reaction norm shapes between populations and closely related species is one approach to providing insights. Here we use a meta-analytic approach to compare divergence in reaction norms of closely related species or populations of animals and plants across types of traits and environments. We quantified mean-standardized differences in overall trait means (Offset) and reaction norm shape (including both Slope and Curvature). These analyses revealed that differences in shape (Slope and Curvature together) were generally greater than differences in Offset. Additionally, differences in Curvature were generally greater than differences in Slope. The type of taxon contrast (species vs. population), trait, organism, and the type and novelty of environments all contributed to the best-fitting models, especially for Offset, Curvature, and the total differences (Total) between reaction norms. Congeneric species had greater differences in reaction norms than populations, and novel environmental conditions increased the differences in reaction norms between populations or species. These results show that evolutionary divergence of curvature is common and should be considered an important aspect of plasticity, together with slope. Biological details about traits and environments, including cryptic variation expressed in novel environmental conditions, may be critical to understanding how reaction norms evolve in novel and rapidly changing environments.

Common garden experiments reveal uncommon responses across temperatures, locations, and species of ants.

Population changes and shifts in geographic range boundaries induced by climate change have been documented for many insect species. On the basis of such studies, ecological forecasting models predict that, in the absence of dispersal and resource barriers, many species will exhibit large shifts in abundance and geographic range in response to warming. However, species are composed of individual populations, which may be subject to different selection pressures and therefore may be differentially responsive to environmental change. Asystematic responses across populations and species to warming will alter ecological communities differently across space. Common garden experiments can provide a more mechanistic understanding of the causes of compositional and spatial variation in responses to warming. Such experiments are useful for determining if geographically separated populations and co-occurring species respond differently to warming, and they provide the opportunity to compare effects of warming on fitness (survivorship and reproduction). We exposed colonies of two common ant species in the eastern United States, Aphaenogaster rudis and Temnothorax curvispinosus, collected along a latitudinal gradient from Massachusetts to North Carolina, to growth chamber treatments that simulated current and projected temperatures in central Massachusetts and central North Carolina within the next century. Regardless of source location, colonies of A. rudis, a keystone seed disperser, experienced high mortality and low brood production in the warmest temperature treatment. Colonies of T. curvispinosus from cooler locations experienced increased mortality in the warmest rearing temperatures, but colonies from the warmest locales did not. Our results suggest that populations of some common species may exhibit uniform declines in response to warming across their geographic ranges, whereas other species will respond differently to warming in different parts of their geographic ranges. Our results suggest that differential responses of populations and species must be incorporated into projections of range shifts in a changing climate.

Complex life cycles and the responses of insects to climate change.

Many organisms have complex life cycles with distinct life stages that experience different environmental conditions. How does the complexity of life cycles affect the ecological and evolutionary responses of organisms to climate change? We address this question by exploring several recent case studies and synthetic analyses of insects. First, different life stages may inhabit different microhabitats, and may differ in their thermal sensitivities and other traits that are important for responses to climate. For example, the life stages of Manduca experience different patterns of thermal and hydric variability, and differ in tolerance to high temperatures. Second, life stages may differ in their mechanisms for adaptation to local climatic conditions. For example, in Colias, larvae in different geographic populations and species adapt to local climate via differences in optimal and maximal temperatures for feeding and growth, whereas adults adapt via differences in melanin of the wings and in other morphological traits. Third, we extend a recent analysis of the temperature-dependence of insect population growth to demonstrate how changes in temperature can differently impact juvenile survival and adult reproduction. In both temperate and tropical regions, high rates of adult reproduction in a given environment may not be realized if occasional, high temperatures prevent survival to maturity. This suggests that considering the differing responses of multiple life stages is essential to understand the ecological and evolutionary consequences of climate change.

Does including physiology improve species distribution model predictions of responses to recent climate change?

Thermal constraints on development are often invoked to predict insect distributions. These constraints tend to be characterized in species distribution models (SDMs) by calculating development time based on a constant lower development temperature (LDT). Here, we assessed whether species-specific estimates of LDT based on laboratory experiments can improve the ability of SDMs to predict the distribution shifts of six U.K. butterflies in response to recent climate warming. We find that species-specific and constant (5 degrees C) LDT degree-day models perform similarly at predicting distributions during the period of 1970-1982. However, when the models for the 1970-1982 period are projected to predict distributions in 1995-1999 and 2000-2004, species-specific LDT degree-day models modestly outperform constant LDT degree-day models. Our results suggest that, while including species-specific physiology in correlative models may enhance predictions of species' distribution responses to climate change, more detailed models may be needed to adequately account for interspecific physiological differences.

N-terminal atrial natriuretic peptide immunoreactivity in plasma of cats with hypertrophic cardiomyopathy.

Atrial natriuretic peptide (ANP) is an important regulator of fluid homeostasis and vascular tone. We sought to compare N-terminal ANP immunoreactivity (ANP-IR) in plasma from cats with and without hypertrophic cardiomyopathy (HCM). Secondarily, we evaluated relationships between ANP-IR and echocardiographical variables in cats with HCM and healthy cats. Venous blood samples were obtained from 17 cats with HCM and from 19 healthy cats. Plasma ANP-IR concentration was determined by an enzyme-linked immunoassay. Two cats with HCM had clinical evidence of congestive heart failure; the remainder had subclinical disease. Plasma ANP-IR concentration was higher in cats with HCM (3,808 +/- 1,406 fmol/L, mean +/- SD) than in control cats (3,079 +/- 1,233 fmol/L), but this difference was not statistically significant (P = .11; 95% confidence interval [CI] = -166 to 1,622). There was a significant, but modest correlation between plasma ANP-IR concentration and left ventricular posterior wall thickness (r = 0.42; P = .01). Additionally, plasma ANP-IR concentration was weakly correlated with left atrial size (r = 0.35; P = .03). A linear regression model was developed to further explore these relationships. Atrial size and wall thickness were included in the model; the 2 explanatory variables had an interactive effect on plasma ANP-IR concentration (R2 = 0.27; P = .02). There was no appreciable correlation between plasma ANP-IR concentration and any other echocardiographical variable. In a population that included cats with subclinical disease, those with HCM did not have significantly higher plasma ANP-IR concentration than did healthy cats. An exploratory multivariable regression analysis suggested a linear relationship between ANP-IR concentration and atrial size, wall thickness, and their interaction.

Two-dimensional echocardiographic assessment of the feline left atrium.

Atrial size determined by echocardiography provides a surrogate measure of the hemodynamic burden of cardiac disease. Linear atrial dimensions often are indexed to aortic diameter. Whereas quantitative variables obtained from healthy cats, using 2-dimensional echocardiography (2DE), have been reported, indices from 2DE, have not. Using 2DE and M-mode echocardiography, we calculated indices of left atrial size and a single index of atrial function, left atrial fractional shortening, in 17 healthy cats. Specifically, left atrial dimensions from short- and long-axis 2DE planes were indexed to aortic diameter and also to end-diastolic left ventricular dimension. Additionally, left atrial circumference and area were indexed to aortic circumference and area, respectively. The same variables were obtained from 20 cats with hypertrophic cardiomyopathy (HCM), so that agreement between 2DE indices and indices from M-mode echocardiography could be evaluated over a clinically relevant range of atrial sizes. Atrial dimensions and indices of atrial size from cats with HCM exceeded those of healthy cats. Left atrial dimension from 2D short-axis images indexed to aortic diameter generally was less than the analogous index obtained from M-mode (mean bias, [95% limits of agreement] -0.13, [-0.42, 0.17]). Left atrial dimension from 2D long-axis images indexed to aortic diameter generally was greater than the index obtained from M-mode (0.15, [-0.28, 0.58]). We conclude that ratios of left atrial size and aortic diameter, from 2DE and M-mode echocardiography, are not interchangeable. Normative data that may serve as reference intervals for 2DE assessment of atrial size are presented.

Comparison of Doppler-derived peak aortic velocities obtained from subcostal and apical transducer sites in healthy dogs.

The accuracy of Doppler-derived blood flow velocity depends on the angle of incidence between the ultrasound beam and the direction of flow. In dogs with subaortic stenosis, it is known that the subcostal transducer site provides higher left ventricular ejection velocities than does the apical site. Left ventricular ejection velocities obtained from the subcostal site in healthy dogs have not been reported; accordingly, we examined healthy, random-source dogs to test the hypothesis that aortic velocities obtained from the subcostal and apical transducer sites differ. In 38 unsedated dogs, high-pulsed repetition frequency (HPRF) and continuous-wave (CW) Doppler studies of the proximal aorta and left ventricular outflow tract were performed using two-dimensional echocardiographic guidance. The effects of Doppler modality, transducer site, and their interaction on peak aortic velocity were tested through the use of analysis of variance. P values less than 0.05 were considered to indicate significance. The mean (+/-SE) peak aortic velocities were as follows: subcostal site (HPRF) = 1.44 +/- 0.03; apical site (HPRF) = 1.39 +/- 0.03; subcostal site (CW) = 1.48 +/- 0.03; apical site (CW) = 1.39 +/- 0.03. Aortic velocities obtained from the subcostal site were significantly greater than those obtained from the cardiac apex (P = 0.0001). Lower and upper limits of 95% reference intervals for aortic velocities by method of measurement and transducer location are proposed. In a population of healthy dogs, peak aortic velocities obtained from the subcostal site exceeded those obtained from the cardiac apex but did so only to a marginal degree.