Incubation Temperature Affects Duckling Body Size and Food Consumption Despite No Effect on Associated Feeding Behaviors.

Developmental conditions can have consequences for offspring fitness. For example, small changes (<1°C) in average avian incubation temperature have large effects on important post-hatch offspring phenotypes, including growth rate, thermoregulation, and behavior. Furthermore, average incubation temperatures differ among eggs within the same nest, to the extent (i.e., >1°C) that differences in offspring phenotypes within broods should result. A potential consequence of within-nest incubation temperature variation is inequality in behaviors that could cause differences in resource acquisition within broods. To investigate this, we incubated wood duck (Aix sponsa) eggs at one of two ecologically-relevant incubation temperatures (35°C or 36°C), formed mixed-incubation temperature broods after ducklings hatched, and conducted trials to measure duckling behaviors associated with acquisition of heat (one trial) or food (three trials). Contrary to our predictions, we found no effect of incubation temperature on duckling behaviors (e.g., time spent occupying heat source, frequency of feeding bouts). However, we found evidence that ducklings incubated at the higher temperature consumed more food during the 1-h feeding trials, and grew faster in body mass and structural size (culmen and tarsus) throughout the study, than those incubated at the lower temperature. Apparent food consumption during the trials was positively related to culmen length, suggesting that differences in food consumption may be driven by structural size. This could result in positive feedback, which would amplify size differences between offspring incubated at different temperatures. Thus, our study identifies incubation temperature as a mechanism by which fitness-related phenotypic differences can be generated and even amplified within avian broods.

TRANSLATED BY JAZEL OULED-CHEIKH BONAN: Título: La temperatura de incubación afecta el tamaño corporal y el consumo de alimentos de los patos juveniles a pesar de no tener ningún efecto sobre los comportamientos alimentarios asociadosResumen: Las condiciones del desarrollo pueden tener consecuencias para la eficacia biológica de la descendencia. Por ejemplo, en aves, pequeños cambios (<1°C) en la temperatura promedio de incubación tienen grandes efectos sobre los fenotipos importantes de la descendencia después de la eclosión, incluida la tasa de crecimiento, la termorregulación y el comportamiento. Además, las temperaturas promedio de incubación difieren entre los huevos dentro del mismo nido, de forma que deberían dar lugar a diferencias en los fenotipos de descendencia dentro de la nidada. Una posible consecuencia de la variación de la temperatura de incubación dentro del nido es la diferencia en los comportamientos que podrían causar contrastes en la adquisición de recursos entre polluelos. Para investigar esto, incubamos huevos de pato joyuyo (Aix sponsa) a diferentes temperaturas de incubación ecológicamente relevantes (35 o 36°C). A continuación, después de la eclosión de los polluelos, formamos grupos mixtos con respecto a la temperatura de incubación y realizamos ensayos para medir los comportamientos de los polluelos asociados con la adquisición de calor (un ensayo) o comida (tres ensayos). Contrariamente a nuestras predicciones, no encontramos ningún efecto de la temperatura de incubación en los comportamientos de los polluelos (p. Ej., tiempo dedicado a ocupar la fuente de calor, frecuencia de las peleas relacionadas con la comida). Sin embargo, encontramos evidencia de que los polluelos incubados a la temperatura más alta consumieron más alimentos durante las pruebas de alimentación de 1 hora, y crecieron más rápido en masa corporal y tamaño estructural (culmen y tarso) durante todo el estudio, que los incubados a la temperatura más baja. El consumo aparente de alimento durante los ensayos se relacionó positivamente con la longitud del culmen, lo que sugiere que las diferencias en el consumo de alimentos pueden deberse al tamaño estructural. Esto podría dar como resultado una retroalimentación positiva, lo que amplificaría las diferencias de tamaño entre las crías incubadas a diferentes temperaturas. Por lo tanto, nuestro estudio identifica la temperatura de incubación como un mecanismo por el cual se pueden generar diferencias fenotípicas relacionadas con la eficacia biológica dentro de las nidadas e incluso amplificarlas.Palabras clave: Aix sponsa, entorno de desarrollo temprano, consumo de alimentos, crecimiento, efecto parental, interacciones fraternales.

TRANSLATED BY MATHIAS DEZETTER AND FRÉDÉRIC ANGELIER: Titre: La température d'incubation affecte la taille corporelle et la consommation de nourriture des canetons malgré l'absence d'effet sur les comportements alimentaires associésRésumé: Les conditions de développement peuvent avoir des conséquences sur la valeur sélective de la progéniture. Par exemple, chez les oiseaux, de faibles changements de la température d'incubation moyenne (<1°C) ont des effets forts sur des paramètres phénotypiques importants de la progéniture après l'éclosion, notamment sur le taux de croissance, la thermorégulation et le comportement. De plus, les températures d'incubation moyennes diffèrent généralement entre les œufs d'une même nichée, ce qui devrait également engendrer des différences phénotypiques au sein de la couvée. De telles variations de la température d'incubation au sein des nids pourraient conduire à des différences comportementales, notamment en ce qui concerne l'acquisition des ressources des jeunes éclos. Nous avons incubé des œufs de canard carolin (Aix sponsa) à deux températures contrastées, pertinentes écologiquement (35 ou 36°C). Après l'éclosion, les canetons incubés aux deux températures ont été regroupés dans des lots mixtes et des tests comportementaux ont été menés: un test d'accès à la chaleur, et trois tests d'accès à la nourriture. Contrairement à nos prédictions, nous n'avons trouvé aucun effet de la température d'incubation sur les comportements des canetons (i.e., le temps passé à occuper la source de chaleur, la fréquence d'alimentation). Cependant, les canetons incubés à une température plus élevée consomment plus de nourriture pendant les tests d'alimentation d'une heure, et grandissent plus rapidement (masse corporelle et taille structurelle (culmen et tarse)) que ceux incubés à température plus basse. La consommation alimentaire mesurée au cours des tests était positivement liée à la longueur du culmen, ce qui suggère que les différences de consommation alimentaire peuvent être à l'origine des différences de taille observées. Cela pourrait entraîner un effet sur la croissance, qui amplifierait les différences de taille entre les progénitures incubées à des températures contrastées. Notre étude démontre que la température d'incubation peut générer des différences phénotypiques au sein des couvées d'oiseaux, et même les amplifier.Mots-clés: Aix sponsa, environnement de développement précoce, consommation alimentaire, croissance, effet parental, interactions fraternelles.

TRANSLATED BY RODRIGO S B GAVIRA: Título: A temperatura de incubação afeta o tamanho corpóreo e o consumo de alimento em filhotes de pato-carolino, apesar de nenhum efeito sobre os comportamentos alimentares associadosResumo: As condições de desenvolvimento durante a incubação podem ter consequências para a aptidão da prole. Por exemplo, pequenas alterações na temperatura média (< 1°C) de incubação das aves têm grandes efeitos sobre fenótipos importantes da prole pós-eclosão, incluindo taxa de crescimento, termorregulação e comportamento. Além disso, as temperaturas médias de incubação diferem entre os ovos no mesmo ninho, podendo igualmente resultar em alterações fenotípicas sobre a prole. Tal variação de temperatura dentro do ninho durante o período de incubação acarreta diferenças comportamentais entre os filhotes, sobretudo quanto à obtenção de recursos pela ninhada. Para investigar tais efeitos, ovos de patos-carolinos (Aix sponsa) foram incubados em duas temperaturas ecologicamente relevantes (35 ou 36°C). Após a eclosão, os filhotes incubados em ambas temperaturas foram reagrupados em lotes mistos, e medidas de comportamento foram realizadas: um teste quanto à aquisição de calor e três testes associados à obtenção de alimento. Ao contrário de nossas predições, não encontramos efeito da temperatura de incubação no comportamento dos patinhos (e.g., tempo gasto ocupando fonte de calor, frequência de registro de alimentação). No entanto, os filhotes de patos incubados em temperaturas mais altas ingeriram mais alimento durante os testes de alimentação de uma hora, e cresceram mais rapidamente em massa corporal e tamanho estrutural (cúlmen e tarso) do que aqueles incubados em temperaturas mais baixas. Ademais, o consumo alimentar foi positivamente relacionado ao comprimento do cúlmen, sugerindo que as diferenças na ingestão de alimento podem ser causadas pelo tamanho estrutural. Isso resultaria em um feedback positivo, o que amplificaria as diferenças de tamanho entre os filhotes incubados em diferentes temperaturas. Desta forma, nosso estudo identifica a temperatura de incubação em aves como mecanismo capaz de gerar, e até mesmo ampliar, diferenças fenotípicas das ninhadas.Palavras-chave: Aix sponsa, ambiente inicial de desenvolvimento, consumo de alimentos, crescimento, efeito parental, interações entre irmãos.

Are thyroid hormones mediators of incubation temperature-induced phenotypes in birds?

Incubation temperature influences a suite of traits in avian offspring. However, the mechanisms underlying expression of these phenotypes are unknown. Given the importance of thyroid hormones in orchestrating developmental processes, we hypothesized that they may act as an upstream mechanism mediating the effects of temperature on hatchling phenotypic traits such as growth and thermoregulation. We found that plasma T3, but not T4 concentrations, differed among newly hatched wood ducks (Aix sponsa) from different embryonic incubation temperatures. T4 at hatching correlated with time spent hatching, and T3 correlated with hatchling body condition, tarsus length, time spent hatching and incubation period. In addition, the T3 : T4 ratio differed among incubation temperatures at hatch. Our findings are consistent with the hypothesis that incubation temperature modulates plasma thyroid hormones which in turn influences multiple aspects of duckling phenotype.

Thermal challenge severity differentially influences wound healing in wood duck (Aix sponsa) ducklings.

Environmental conditions during early development can profoundly influence an individual's phenotype. Development requires simultaneous maturation and orchestration of multiple physiological systems creating the potential for interaction among key systems and requiring substantial resources. We investigated the influence of thermoregulation on immunocompetence in Wood Duck ducklings (Aix sponsa). At both 1 and 2 days post hatch (dph) we evaluated ducklings' abilities to thermoregulate during a thermal challenge at one of four temperatures (36 [thermoneutral controls], 20, 10, or 5°C). At 3 dph, ducklings received a superficial wound, which was monitored until full recovery to quantify wound healing ability, an ecologically relevant, integrative measure of immune function. We demonstrated that duckling body temperature decreased with increasing thermal challenge severity, thermoregulatory ability increased with age, and thermoregulation had temperature-dependent effects on the immune system. Specifically, a more severe thermal challenge (5°C) resulted in decreased immune performance when compared to a mild challenge (20°C). We conclude that early thermoregulatory experiences are influential in shaping immune responses early in development. Furthermore, our results emphasize that future studies of environmental stressors need to consider multiple physiological endpoints since interaction among systems can result in competing physiological demands.

Incubation conditions are more important in determining early thermoregulatory ability than posthatch resource conditions in a precocial bird.

Recent research in birds suggests that investing in incubation is one mechanism by which parents can enhance the phenotype of their offspring. Posthatch environmental conditions can also shape an individual's phenotype, and it is thus possible for pre- and posthatch conditions to have interactive effects on an individual's phenotype. In this study, we examined the individual and interactive effects of prehatch incubation temperature and posthatch food availability on growth, food consumption, and thermoregulatory ability in wood duck (Aix sponsa) ducklings. Eggs were incubated at one of three temperatures (35.0°, 35.9°, or 37.0°C), and then ducklings were reared on an either ad lib. or time-restricted diet for 12 d after hatching. We found that food availability influenced duckling growth, with the slowest growth occurring in ducklings fed the restricted diet. Incubation temperature also interacted with food conditions to influence duckling growth: ducklings fed ad lib. from the lowest incubation temperature grew slower than ducklings fed ad lib. from the higher incubation temperatures. Most importantly, we found that the improvement in a duckling's ability to maintain body temperature in the face of a thermal challenge was influenced by embryonic incubation temperature but not feeding conditions. Ducklings from the highest incubation temperature experienced the greatest improvement in thermoregulatory performance with age. Our findings suggest that the prehatch environment is more important than posthatch resource conditions in determining some physiological functions and underscores the important role that incubation temperature plays in determining offspring phenotype in birds.

Larval wood frog (Rana [=Lithobates] sylvatica) development and physiology following infection with the trematode parasite, Echinostoma trivolvis.

Parasites can potentially affect host energetics through a variety of mechanisms including diverting energy from host functions or eliciting energetically costly responses. In many systems energetic costs of parasite infection remain poorly defined. The widespread trematode Echinostoma trivolvis can cause mortality of and pathology in larval amphibians. However, physiological impacts of E. trivolvis infection have received limited attention. To evaluate the effects of E. trivolvis on larval amphibian survival, growth and development, we studied a wide range of infection intensity in wood frog, Rana (=Lithobates) sylvatica, tadpoles in laboratory experiments and outdoor mesocosms. To assess potential underlying physiological costs of infection, we measured tadpole energetics and phenotypic plasticity of the intestines as a compensatory mechanism to offset increased energy costs. Survival was high in all tadpoles, but the highest infections decreased the growth and slowed the development of tadpoles raised in mesocosms and the laboratory. However, infections failed to elicit detectable energetic costs or phenotypic changes in intestinal size. The lack of energetic costs observed in our study emphasizes the complex and often context-dependent nature of energetic costs of parasitism and suggests that other mechanisms, such as changes in host behavior, may contribute to sub-lethal effects on growth and development.

Making leaps in amphibian ecotoxicology: translating individual-level effects of contaminants to population viability.

Concern that environmental contaminants contribute to global amphibian population declines has prompted extensive experimental investigation, but individual-level experimental results have seldom been translated to population-level processes. We used our research on the effects of mercury (Hg) on American toads (Bufo americanus) as a model for bridging the gap between individual-level contaminant effects and amphibian population viability. We synthesized the results of previous field and laboratory studies examining effects of Hg throughout the life cycle of B. americanus and constructed a comprehensive demographic population model to evaluate the consequences of Hg exposure on population dynamics. Our model explicitly considered density-dependent larval survival, which is known to be an important driver of amphibian population dynamics, and incorporated two important factors that have seldom been considered in previous amphibian modeling studies: environmental stochasticity and sublethal effects. We demonstrated that decreases in embryonic survival and sublethal effects (e.g., reduced body size) that delay maturation have minor effects on population dynamics, whereas contaminant effects that reduce late-larval or post-metamorphic survival have important population-level consequences. We found that excessive Hg exposure through maternal transfer or larval diet, alone, had minor effects on B. americanus populations. Simultaneous maternal and dietary exposure resulted in reduced population size and a dramatic increase in extinction probability, but explicit prediction of population-level effects was dependent on the strength of larval density dependence. Our results suggest that environmental contaminants can influence amphibian population viability, but that highly integrative approaches are needed to translate individual-level effects to populations.

Coal fly ash basins as an attractive nuisance to birds: parental provisioning exposes nestlings to harmful trace elements.

Birds attracted to nest around coal ash settling basins may expose their young to contaminants by provisioning them with contaminated food. Diet and tissues of Common Grackle (Quiscalus quiscala) nestlings were analyzed for trace elements to determine if nestlings were accumulating elements via dietary exposure and if feather growth limits elemental accumulation in other tissues. Arsenic, cadmium, and selenium concentrations in ash basin diets were 5× higher than reference diets. Arsenic, cadmium, and selenium concentrations were elevated in feather, liver, and carcass, but only liver Se concentrations approached levels of concern. Approximately 15% of the total body burden of Se, As, and Cd was sequestered in feathers of older (>5 days) nestlings, whereas only 1% of the total body burden of Sr was sequestered in feathers. Feather concentrations of only three elements (As, Se, and Sr) were correlated with liver concentrations, indicating their value as non-lethal indicators of exposure.

Embryonic developmental patterns and energy expenditure are affected by incubation temperature in wood ducks (Aix sponsa).

Recent research in birds has demonstrated that incubation temperature influences a suite of traits important for hatchling development and survival. We explored a possible mechanism for the effects on hatchling quality by determining whether incubation temperature influences embryonic energy expenditure of wood ducks (Aix sponsa). Because avian embryos are ectothermic, we hypothesized that eggs incubated at higher temperatures would have greater energy expenditure at any given day of incubation. However, because eggs incubated at lower temperatures take longer to hatch than embryos incubated at higher temperatures, we hypothesized that the former would expend more energy during incubation. We incubated eggs at three temperatures (35.0°, 35.9°, and 37.0°C) that fall within the range of temperatures of naturally incubated wood duck nests. We then measured the respiration of embryos every 3 d during incubation, immediately after ducks externally pipped, and immediately after hatching. As predicted, embryos incubated at the highest temperature had the highest metabolic rates on most days of incubation, and they exhibited faster rates of development. Yet, because of greater energy expended during the hatching process, embryos incubated at the lowest temperature expended 20%-37% more energy during incubation than did embryos incubated at the higher temperatures. Slower developmental rates and greater embryonic energy expenditure of embryos incubated at the lowest temperature could contribute to their poor physiological performance as ducklings compared with ducklings that hatch from eggs incubated at higher temperatures.

Slight differences in incubation temperature affect early growth and stress endocrinology of wood duck (Aix sponsa) ducklings.

Early developmental experiences, such as incubation conditions, can have important consequences for post-hatching fitness in birds. Although the effects of incubation temperature on phenotype of avian hatchlings are poorly understood, recent research suggests that subtle changes in incubation conditions can influence hatchling characteristics, including body size and condition. We designed an experiment to explore the effects of incubation temperature on hatching success, survival to 9 days post hatch, growth and the hypothalamo-pituitary-adrenal (HPA) axis in wood ducks (Aix sponsa). Wood duck eggs were collected from nest boxes and experimentally incubated at three temperatures (35.0, 35.9 and 37.0 degrees C), each falling within the range of temperatures of naturally incubated wood duck nests. Survival and growth were monitored in ducklings fed ad libitum for 9 days post hatch. In addition, baseline and stress-induced plasma corticosterone concentrations were measured in 2 and 9 day old ducklings. Hatching success and survival to 9 days was greatest in ducks incubated at the intermediate temperature. Ducklings incubated at 35.9 degrees C and 37.0 degrees C had 43% higher growth rates than ducklings incubated at 35.0 degrees C. In addition, ducklings incubated at 35.0 degrees C had higher baseline (17-50%) and stress-induced (32-84%) corticosterone concentrations than ducklings incubated at 35.9 degrees C and 37.0 degrees C at 2 and 9 days post hatch. We also found a significant negative correlation between body size and plasma corticosterone concentrations (baseline and stress-induced) in 9 day old ducklings. To our knowledge, this is the first study to demonstrate that thermal conditions experienced during embryonic development can influence the HPA axis of young birds. Our results illustrate that subtle changes (<1.0 degrees C) in the incubation environment can have important consequences for physiological traits important to fitness.

Differential swimming performance of two natricine snakes exposed to a cholinesterase-inhibiting pesticide.

Environmental contaminants have direct effects on organisms at the molecular, cellular, and tissue levels, but the net results of these sub-organismal effects are only consequential to exposed populations if they alter organism-level traits that ultimately influence fitness (e.g., growth, locomotor performance, reproduction, and survival). Here, we explore the possibility that the swimming performance of neonate black swamp snakes (Seminatrix pygaea) and diamondback water snakes (Nerodia rhombifer) may be affected by exposure to carbaryl (2.5 and 5.0 mg/L). The highest concentration of carbaryl caused greater reductions in swim velocity in S. pygaea than in N. rhombifer. Most individuals recovered from the effects of carbaryl on swimming performance within 96 h, but recovery was significantly slower in S. pygaea than in N. rhombifer. We hypothesize that the sensitivity of S. pygaea may arise from its highly permeable integument compared to other natricines. Our findings suggest that performance can serve as an ecologically relevant response to contaminant exposure in reptiles and warrants further study.

The influence of circadian rhythms on pre- and post-prandial metabolism in the snake Lamprophis fuliginosus.

Measuring standard metabolic rate (SMR) and specific dynamic action (SDA) has yielded insight into patterns of energy expenditure in snakes, but less emphasis has been placed on identifying metabolic variation and associated energy cost of circadian rhythms. To estimate SMR, SDA, and identify metabolic variation associated with circadian cycles in nocturnally active African house snakes (Lamprophis fuliginosus), we measured oxygen consumption rates (VO2) at frequent intervals before and during digestion of meals equaling 10%, 20% and 30% of their body mass. Circadian rhythms in metabolism were perceptible in the VO2 data during fasting and after the initial stages of digestion. We estimated SMR of L. fuliginosus (mean mass=16.7+/-0.3 g) to be 0.68+/-0.02 (+/-SEM) mL O2/h at 25 degrees C. Twenty-four hours after eating, VO2 peaked at 3.2-5.3 times SMR. During digestion of meals equaling 10-30% of their body mass, the volume of oxygen consumed ranged from 109 to 119 mL O2 for SMR, whereas extra oxygen consumed for digestion and assimilation ranged from 68 to 256 mL O2 (equivalent to 14.5-17.0% of ingested energy). The oxygen consumed due to the rise in metabolism during the active phase of the daily cycle ranged from 55 to 66 mL O2 during digestion. Peak VO2, digestive scope, and SDA increased with increasing meal size. Comparisons of our estimates to estimates derived from methods used in previous investigations resulted in wide variance of metabolic variables (up to 39%), likely due to the influence of circadian rhythms and activity on the selection of baseline metabolism. We suggest frequent VO2 measurements over multiple days, coupled with mathematical methods that reduce the influence of undesired sources of VO2 variation (e.g., activity, circadian cycles) are needed to reliably assess SMR and SDA in animals exhibiting strong circadian cycles.

Responses of benthic fish exposed to contaminants in outdoor microcosms--examining the ecological relevance of previous laboratory toxicity tests.

Previous laboratory studies indicate that coal combustion wastes (a mixture composed of fly ash and other lower volume wastes such as bottom ash; hereafter collectively referred to as ash) adversely affect the health of benthic fish (Erimyzon sucetta; lake chubsucker), but fish in these studies were provided with ample uncontaminated food resources. Because aquatic disposal of ash can also adversely affect food resources for benthic fish, we hypothesized that changes in resources might exacerbate the effects of ash on fish observed in laboratory studies. We exposed juvenile E. sucetta in outdoor microcosms to water, sediment, and benthic resources from an ash-contaminated site or a reference site for 45 days and compared our findings to previous laboratory studies. Benthic invertebrate biomass was nearly three times greater in controls compared to ash microcosms. Total organic content of control sediment (41%) was also greater than in ash sediments (17%), suggesting that additional benthic resources may have also been limited in ash microcosms. Benthic invertebrates isolated from the ash microcosms had trace element concentrations (As, Cd, Co, Cr, Cs, Se, Sr, and V) up to 18 times higher than in weathered ash used in laboratory studies. The concentrations of trace elements accumulated by fish reflected the high dietary concentrations encountered in the ash microcosms and were associated with reduced growth (final mass = 0.07 g) and survival (25%) compared to controls (0.37 g and 67%, respectively). Accumulation of trace elements, as well as reductions in growth and survival, were more pronounced than in previous laboratory studies, suggesting that resource conditions may be important in mediating ash toxicity. Taken together, our studies suggest that ash discharge into aquatic systems is a more serious threat to the health of benthic fish than previously predicted based upon laboratory toxicity tests.

Respiratory and reproductive characteristics of eastern mosquitofish (Gambusia holbrooki) inhabiting a coal ash settling basin.

Coal fly ash and effluent from coal ash settling basins negatively affects metabolism and reproduction in a variety of organisms, including a number of fish species. Some species, most notably the eastern mosquitofish (Gambusia holbrooki), are known to maintain viable populations in areas contaminated by coal ash. While eastern mosquitofish are present in these systems, their degree of tolerance to coal ash has not been investigated using sublethal metrics of exposure. It is possible that eastern mosquitofish persist in habitats affected by coal ash, but experience significant costs such as changes in metabolism and fecundity. Thus, we investigated the effects of coal ash on standard metabolic rate and reproduction of eastern mosquitofish inhabiting a coal-ash contaminated settling basin. Standard metabolic rates of mosquitofish from the ash contaminated site and a reference site (mean O2 consumption = 0.286 mL/g x h +/- 0.007 and 0.291 +/- 0.008 mL/g x h, respectively) were not significantly different. Despite elevated contaminant concentrations in ash basin females (selenium, arsenic, copper, and cadmium) and their offspring (selenium), brood sizes and offspring viability did not differ between clutches collected from ash basin and reference site females. Our data provide further evidence of the high degree of tolerance of eastern mosquitofish to exposure to aquatic coal ash disposal generated by power plants. However, the basis for such tolerance to ash remains unclear. Further investigations are required to determine whether such tolerance is a result of species-specific characteristics or population characteristics due to local adaptation.

Standard and digestive metabolism in the banded water snake, Nerodia fasciata fasciata.

Estimating energy costs by respirometry is fundamental to many studies of the ecology, behavior and evolution of reptiles. However, traditional respirometry procedures seldom incorporate objective techniques for removal of outliers from estimates of metabolic parameters. We demonstrate how computer-automated respirometry equipment, which records many respiratory measurements over short intervals, can be coupled with mathematical procedures to produce robust estimates of pre- and post-prandial metabolism in banded water snakes (Nerodia fasciata fasciata). Standard metabolic rate of N. f. fasciata was estimated to be 1.21 ml O2/h (mass = 30.21 +/- 0.74 g) at 25 degrees C. After ingestion of a fish equaling 20% of their body mass, snakes exhibited a fivefold increase in metabolic rate with peak O2 consumption rate (VO2) reaching 6.5 ml O2/h. Total cost of digestion was 5.44 kJ, equivalent to approximately 21% of the energy in the meal. Repeated measurements of metabolism in the same individuals revealed that our methods yielded similar results, even when individuals exhibited different patterns of VO2 variation between respiratory trials. Our results underscore the importance of obtaining many VO2 measurements, coupled with objective removal of outlier values from estimates of metabolic rate, especially when metabolic values are to be interpreted in a comparative context.

Maternal transfer of contaminants to eggs in common grackles (Quiscalus quiscala) nesting on coal fly ash basins.

Coal combustion is a major source of trace elements to the environment. Aquatic disposal of wastes from this process can result in reproductive failure in many wildlife species, but little is known regarding impacts on avian fauna. Individual eggs were collected from common grackles (Quiscalus quiscala) nesting in association with coal fly ash settling basins and a reference site to determine if females from the contaminated site transfered trace elements to their eggs. Whole clutches were also collected from both sites to examine inter- and intra-clutch variability of maternally transferred contaminants. Selenium was the only trace element found in significantly higher concentrations in ash basin eggs (x = 5.88 +/- 0.44 microg/g DW) than in reference eggs (x = 2.69 +/- 0.13 microg/g DW). Selenium concentrations in eggs from the ash basins were above background levels, but did not exceed higher proposed Se toxicity thresholds. Inter- and intra-clutch variation was higher for ash basin clutches than reference clutches. The relationship between selenium concentrations and laying order (estimated by egg mass) was not statistically significant, but increased Se concentration in the second egg of most ash basin clutches followed by declining concentrations in subsequent eggs suggested that further examination of this pattern might be warranted.

Altered swimming performance of a benthic fish (Erimyzon sucetta) exposed to contaminated sediments.

Numerous field surveys suggest that coal combustion wastes (ash) adversely affect fish populations, but few controlled laboratory studies have evaluated the responses of individual fish to ash exposure. Available information suggests that sublethal effects of ash, including decreased growth rates and reduced fecundity, may be important manifestations of ash toxicity. Here, we hypothesized that ash may also alter swimming performance, which could ultimately have important implications for the autecology of affected species. To test this hypothesis, we measured sprint speed and critical swimming speed (U(crit)) of juvenile lake chubsuckers (Erimyzon sucetta) exposed to ash in the laboratory. Fish exposed to ash for 90-100 days accumulated significant concentrations of As, Se, Sr, and V; exhibited severe fin erosion; and had reduced sprint speed and U(crit). Compared to controls, sprint speed of ash-exposed fish was reduced by 30% at 5 cm and the percent reduction was further reduced to 104% at 20 cm. Critical swimming speed was approximately 50% lower in fish exposed to ash compared to controls. Additionally, the typical positive relationship between standard length and U(crit) was absent in fish exposed to ash. Because reductions in swimming performance could not be attributed to pollutant-induced differences in body condition or fin morphology, we hypothesize that physiological disruptions (e.g., increased energy demands, decreased oxygen uptake or transport) are responsible for the observed effects and warrant further attention.

Trace element speciation in largemouth bass ( Micropterus salmoides) from a fly ash settling basin by liquid chromatography-ICP-MS.

Analytical techniques used to examine the chemical speciation of multiple trace elements are important for the investigation of biological systems. Size exclusion chromatography (SEC) coupled to ICP-MS was used to investigate the speciation of Se, As, Cu, Cd and Zn in tissue extracts from a largemouth bass (Micropterus salmoides) collected from a coal fly ash basin and results were compared to a largemouth bass collected at a reference site. Using a Biosil SEC column, with an effective separation range of 100-7 KDa, Cu, Zn and Cd were shown to be bound to metallothionein (MT) in the liver, gill and, to a lesser extent, gonad tissue extract. In liver, muscle and gill of the ash basin bass, Se was predominantly present as low molecular weight species. Only in the gonad extract was the major fraction of Se associated with high molecular weight species. For the liver and gill extracts, further SEC-ICP-MS on a column with an effective separation range of 7000-500 Da was performed, but Se species still eluted near the total volume of the column suggesting a low molecular weight organic or inorganic species. Ion chromatography (IC)-ICP-MS using an AS7 column and HNO(3) gradient elution indicated that the Se and As species in the liver and gill extracts had similar retention times but these retention times did not correspond to retention times for As(III), As(V), dimethylarsenate, arsenobetaine, Se(IV), Se(VI), seleno-methionine, or seleno-cystine.

Effects of food ration on survival and sublethal responses of lake chubsuckers (Erimyzon sucetta) exposed to coal combustion wastes.

Study organisms in chronic toxicological bioassays are often provided with excessive resources to remove food limitations as a confounding experimental variable. Under more ecologically realistic situations, resources are often less abundant and such restrictions may alter the responses of organisms to environmental contaminants. Here, we investigated the interaction between resource level and sediment toxicity in the lake chubsucker, Erimyzon sucetta. For 78 days we fed fish one of three ration levels (1X, 2X, 4X; uncontaminated food) that was grazed directly from either clean sand or coal ash-contaminated sediments. Despite provision of uncontaminated food, fish exposed to the contaminated sediments accumulated significant whole body concentrations of As, Se, Sr, and V. Food ration affected the pattern of Se accumulation, with lowest concentrations accumulated by fish supplied with the lowest rations (1X). Paradoxically, fish in the 1X-ash treatment were most adversely effected by ash-exposure, despite having Se burdens much lower than fish in the 2X- and 4X-ash treatments. Fish in the 1X-ash treatment exhibited higher mortality, lower proportional growth, and increased incidence of fin erosion compared to fish provided with higher rations. Such results may, in part, be explained by the apparent inability of fish with reduced rations to maintain positive energy balance, as evidenced by their higher standard metabolic rates compared to control fish fed similar rations. Our results underscore the importance of considering resource quantity and nutritional factors in chronic bioassays in order to draw more ecologically realistic conclusions about contaminant effects.

Nondestructive indices of trace element exposure in squamate reptiles.

Compared with birds, mammals, fish, and even amphibians, very little is known about the effects of contaminants on reptiles. Recent evidence that many reptile populations may be declining has stimulated demand for toxicological studies of reptiles as well as development of nondestructive sampling techniques useful for assessing and monitoring contaminant exposure. The current study experimentally evaluated the utility of shed skins, tail clips, and blood samples as nondestructive indices of trace element exposure in banded water snakes, Nerodia fasciata. For 13.5 months, snakes were either fed fish from a coal ash-contaminated site or uncontaminated food from a reference site. Snakes fed contaminated prey accumulated As, Cd, Se, Sr, and V in various organs (i.e. liver, kidney, and/or gonads). Moreover, non-parametric discriminant function analysis revealed that snakes could be placed in two groups that reliably reflected their experimental diet based upon Se, Sr, and As concentrations in tail clips, blood, and/or shed skins. We suggest that nondestructive sampling techniques, particularly analyses of blood and tail clips, may be easily applied in evaluations of contaminant exposure in the field and laboratory and may prevent excessive destructive sampling of potentially threatened reptile species.

Resource allocation-based life histories: a conceptual basis for studies of ecological toxicology.

Whereas ecological assessments of contaminants are concerned with populations and higher levels of organization, most mechanistic work in toxicology is directed at effects on individuals and their parts. We propose that studies based on individuals can be useful in ecological analysis of polluted systems when based on the concepts of resource allocation-based life history analysis. At the heart of the resource allocation approach is the concept of operative environments of individuals (i.e., environmental factors influencing birth, death, or migration). Contaminants can have strong influences on operative environments, modifying resource allocation strategies that reflect changes in energy assimilation and demands. By examining contaminant-induced responses of individuals from the perspective of changing operative environments, individual-based changes and population dynamics can be addressed in an ecologically rigorous manner.