Graham R. Scott

Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss)

SUMMARY Alarm substance is a chemical signal released from fish skin epithelial cells after a predator causes skin damage. When other prey fish detect alarm substance by olfaction, they perform stereotypical predator-avoidance behaviours to decrease predation risk. The objective of this study was to explore the effect of sublethal cadmium (Cd) exposure on the behavioural and physiological responses of juvenile rainbow trout (Oncorhynchus mykiss) to alarm substance. Waterborne exposure to 2 μg Cd l–1 for 7 days eliminated normal antipredator behaviours exhibited in response to alarm substance, whereas exposures of shorter duration or lower concentration had no effect on normal behaviour. Furthermore, dietary exposure to 3 μg Cd g–1 in the food for 7 days, which produced the same whole-body Cd accumulation as waterborne exposure to 2 μg l–1, did not alter normal behaviour, indicating that an effect specific to waterborne exposure alone (i.e. Cd accumulation in the olfactory system) results in behavioural alteration. Whole-body phosphor screen autoradiography of fish exposed to 109Cd demonstrated that Cd deposition in the olfactory system (rosette, nerve and bulb) during waterborne exposure was greater than in all other organs of accumulation except the gill. However, Cd could not be detected in the brain. A short-term elevation in plasma cortisol occurred in response to alarm substance under control conditions. Cd exposures of 2 μg l–1 waterborne and 3 μg g–1 dietary for 7 days both inhibited this plasma cortisol elevation but did not alter baseline cortisol levels. Our results suggest that exposure to waterborne Cd at environmentally realistic levels (2 μg l–1) can disrupt the normal behavioural and physiological responses of fish to alarm substance and can thereby alter predator-avoidance strategies, with potential impacts on aquatic fish communities.

Phenotypic plasticity and genetic adaptation to high-altitude hypoxia in vertebrates

Summary High-altitude environments provide ideal testing grounds for investigations of mechanism and process in physiological adaptation. In vertebrates, much of our understanding of the acclimatization response to high-altitude hypoxia derives from studies of animal species that are native to lowland environments. Such studies can indicate whether phenotypic plasticity will generally facilitate or impede adaptation to high altitude. Here, we review general mechanisms of physiological acclimatization and genetic adaptation to high-altitude hypoxia in birds and mammals. We evaluate whether the acclimatization response to environmental hypoxia can be regarded generally as a mechanism of adaptive phenotypic plasticity, or whether it might sometimes represent a misdirected response that acts as a hindrance to genetic adaptation. In cases in which the acclimatization response to hypoxia is maladaptive, selection will favor an attenuation of the induced phenotypic change. This can result in a form of cryptic adaptive evolution in which phenotypic similarity between high- and low-altitude populations is attributable to directional selection on genetically based trait variation that offsets environmentally induced changes. The blunted erythropoietic and pulmonary vasoconstriction responses to hypoxia in Tibetan humans and numerous high-altitude birds and mammals provide possible examples of this phenomenon. When lowland animals colonize high-altitude environments, adaptive phenotypic plasticity can mitigate the costs of selection, thereby enhancing prospects for population establishment and persistence. By contrast, maladaptive plasticity has the opposite effect. Thus, insights into the acclimatization response of lowland animals to high-altitude hypoxia can provide a basis for predicting how altitudinal range limits might shift in response to climate change.

Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport

SUMMARY We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl–, whereas the opercular epithelium actively absorbs Cl– but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3–cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase α1a, Na+,K+,2Cl–cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.

Temperature during embryonic development has persistent effects on thermal acclimation capacity in zebrafish

Global warming is intensifying interest in the mechanisms enabling ectothermic animals to adjust physiological performance and cope with temperature change. Here we show that embryonic temperature can have dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature (TE = 27 °C) or near the extremes for normal development (TE = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance (Ucrit), but each TE group had reduced thermal sensitivity at its respective TE. In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C TE groups compared with 27 °C TE controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fiber type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C TE groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodeling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some ectothermic organisms cope with the more variable temperatures that are expected under future climate-change scenarios.

Molecular Evolution of Cytochrome c Oxidase Underlies High-Altitude Adaptation in the Bar-Headed Goose

Bar-headed geese (Anser indicus) fly at up to 9,000 m elevation during their migration over the Himalayas, sustaining high metabolic rates in the severe hypoxia at these altitudes. We investigated the evolution of cardiac energy metabolism and O(2) transport in this species to better understand the molecular and physiological mechanisms of high-altitude adaptation. Compared with low-altitude geese (pink-footed geese and barnacle geese), bar-headed geese had larger lungs and higher capillary densities in the left ventricle of the heart, both of which should improve O(2) diffusion during hypoxia. Although myoglobin abundance and the activities of many metabolic enzymes (carnitine palmitoyltransferase, citrate synthase, 3-hydroxyacyl-coA dehydrogenase, lactate dehydrogenase, and pyruvate kinase) showed only minor variation between species, bar-headed geese had a striking alteration in the kinetics of cytochrome c oxidase (COX), the heteromeric enzyme that catalyzes O(2) reduction in oxidative phosphorylation. This was reflected by a lower maximum catalytic activity and a higher affinity for reduced cytochrome c. There were small differences between species in messenger RNA and protein expression of COX subunits 3 and 4, but these were inconsistent with the divergence in enzyme kinetics. However, the COX3 gene of bar-headed geese contained a nonsynonymous substitution at a site that is otherwise conserved across vertebrates and resulted in a major functional change of amino acid class (Trp-116 → Arg). This mutation was predicted by structural modeling to alter the interaction between COX3 and COX1. Adaptations in mitochondrial enzyme kinetics and O(2) transport capacity may therefore contribute to the exceptional ability of bar-headed geese to fly high.

Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation

SUMMARY We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10 g l-1) to freshwater. Differences in Na+ regulation between each population might partially account for this difference in tolerance, because plasma Na+ was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na+/K+-ATPase mRNA expression and activity in their gills to a greater extent 1-14 days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na+ at 14 days. All observed differences in Na+ regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl- regulation also existed between populations. Plasma Cl- was maintained in northern fish, but in southerns, plasma Cl- decreased rapidly and remained low for the duration of the experiment. Correspondingly, net Cl- loss from southern fish remained high after transfer, while northerns eliminated Cl- loss altogether. Elevated Cl- loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability (indicated by extrarenal clearance rate of PEG-4000) and apical crypt density in the gills (detected using scanning electron microscopy) were both higher than in northern fish. These large differences in the regulation of Cl- balance probably contributed to the marked differences in mortality after freshwater transfer. Glomerular filtration rate and urination frequency were also lower in southerns. Taken together, these data suggest that northern killifish are better adapted to freshwater environments and that minimizing Cl- imbalance appears to be the key physiological difference accounting for their greater freshwater tolerance.

The trans-Himalayan flights of bar-headed geese (Anser indicus)

Birds that fly over mountain barriers must be capable of meeting the increased energetic cost of climbing in low-density air, even though less oxygen may be available to support their metabolism. This challenge is magnified by the reduction in maximum sustained climbing rates in large birds. Bar-headed geese (Anser indicus) make one of the highest and most iconic transmountain migrations in the world. We show that those populations of geese that winter at sea level in India are capable of passing over the Himalayas in 1 d, typically climbing between 4,000 and 6,000 m in 7–8 h. Surprisingly, these birds do not rely on the assistance of upslope tailwinds that usually occur during the day and can support minimum climb rates of 0.8–2.2 km·h−1, even in the relative stillness of the night. They appear to strategically avoid higher speed winds during the afternoon, thus maximizing safety and control during flight. It would seem, therefore, that bar-headed geese are capable of sustained climbing flight over the passes of the Himalaya under their own aerobic power.

Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer.

SUMMARY We have explored the molecular and physiological responses of the euryhaline killifish Fundulus heteroclitus to transfer from brackish water (10% seawater) to 100% seawater for 12 h, 3 days or 7 days. Plasma [Na+] and [Cl–] were unchanged after transfer, and plasma cortisol underwent a transient increase. Na+/K+-ATPase activity increased 1.5-fold in the gills and opercular epithelium at 7 days (significant in gills only), responses that were preceded by three- to fourfold increases in Na+/K+-ATPase α1a mRNA expression. Expression of Na+/K+/2Cl– cotransporter 1, cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel, Na+/H+-exchanger 3 (significant in opercular epithelium only) and carbonic anhydrase II mRNA also increased two- to fourfold after transfer. Drinking rate increased over twofold after 12 h and remained elevated for at least 7 days. Surprisingly, net rates of water and ion absorption measured in vitro across isolated intestines decreased ∼50%, possibly due to reduced salt demands from the diet in seawater, but water absorption capacity still exceeded the drinking rate. Changes in bulk water absorption were well correlated with net ion absorption, and indicated that slightly hyperosmotic solutions (⩾298 mmol l–1) were transported. There were no reductions in unidirectional influx of Na+ from luminal to serosal fluid or intestinal Na+/K+-ATPase activity after transfer. Overall, our results indicate that gill and opercular epithelia function similarly at a molecular level in seawater, in contrast to their divergent function in freshwater, and reveal unexpected changes in intestinal function. As such they provide further insight into the mechanisms of euryhalinity in killifish.

Cadmium affects the social behaviour of rainbow trout, Oncorhynchus mykiss.

The present study investigated both the effects of cadmium on the social interactions of rainbow trout and the differential accumulation of waterborne cadmium among social ranks of fish. Fish exposed to waterborne cadmium concentrations of 2 microg l(-1) for 24 h, followed by a 1, 2 or 3 day depuration period in clean water, had a decreased ability to compete with non-exposed fish. However, the competitive ability of exposed fish given a 5 day depuration period was not significantly impaired. Cadmium accumulated in the olfactory apparatus of fish exposed to waterborne cadmium for 24 h and decreased significantly only after 5 days depuration in clean water. Among groups of ten fish held in stream tanks, where all fish were exposed to cadmium, there were significant effects on social behaviour and growth rate. Dominance hierarchies formed faster among fish exposed to cadmium than among control fish, and overall growth rates were higher in the cadmium treatment. In groups of ten fish, social status also affected tissue accumulation of cadmium during waterborne exposure, with dominant fish accumulating more cadmium at the gill. In conclusion, exposure to low levels of cadmium, affects the social behaviour of fish, in part due to accumulation in the olfactory apparatus, and dominant fish accumulate more gill cadmium than subordinates during chronic waterborne exposure.

Tribute to R. G. Boutilier: the effect of size on the physiological and behavioural responses of oscar, Astronotus ocellatus, to hypoxia.

SUMMARY The physiological and behavioural responses of two size groups of oscar (Astronotus ocellatus) to hypoxia were studied. The physiological responses were tested by measuring ṀO2 during decreasing environmental oxygen tensions. Larger oscars were better able to maintain oxygen consumption during a decrease in PO2, regulating routine ṀO2 to a significantly lower PO2 threshold (50 mmHg) than smaller oscars (70 mmHg). Previous studies have also demonstrated a longer survival time of large oscars exposed to extreme hypoxia, coupled with a greater anaerobic enzymatic capability. Large oscars began aquatic surface respiration (ASR) at the oxygen tension at which the first significant decrease in ṀO2 was seen (50 mmHg). Interestingly, smaller oscars postponed ASR to around 22 mmHg, well beyond the PO2 at which they switched from oxyregulation to oxyconformation. Additionally, when given the choice between an hypoxic environment containing aquatic macrophyte shelter and an open normoxic environment, small fish showed a greater preference for the hypoxic environment. Thus shelter from predators appears particularly important for juveniles, who may accept a greater physiological compromise in exchange for safety. In response to hypoxia without available shelter, larger fish reduced their level of activity (with the exception of aggressive encounters) to aid metabolic suppression whereas smaller oscars increased their activity, with the potential benefit of finding oxygen-rich areas.