Extreme High-Elevation Mammal Surveys Reveal Unexpectedly High Upper Range Limits of Andean Mice.

AbstractIn the world's highest mountain ranges, uncertainty about the upper elevational range limits of alpine animals represents a critical knowledge gap regarding the environmental limits of life and presents a problem for detecting range shifts in response to climate change. Here we report results of mountaineering mammal surveys in the Central Andes, which led to the discovery of multiple species of mice living at extreme elevations that far surpass previously assumed range limits for mammals. We livetrapped small mammals from ecologically diverse sites spanning >6,700 m of vertical relief, from the desert coast of northern Chile to the summits of the highest volcanoes in the Andes. We used molecular sequence data and whole-genome sequence data to confirm the identities of species that represent new elevational records and to test hypotheses regarding species limits. These discoveries contribute to a new appreciation of the environmental limits of vertebrate life.

Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis.

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Carbon dioxide and bicarbonate accumulation in caiman erythrocytes during diving.

The ability of crocodilian haemoglobins to bind HCO3 - has been appreciated for more than half a century, but the functional implication of this is exceptional mechanism has not previously been assessed in vivo Therefore, the goal of the present study was to address the hypothesis that CO2 primarily binds to Hb, rather than being accumulated in plasma as in other vertebrates, during diving in caimans. Here, we demonstrate that CO2 primarily accumulates within the erythrocyte during diving and that most of the accumulated CO2 is bound to haemoglobin. Furthermore, we show that this HCO3 --binding is tightly associated with the progressive blood deoxygenation during diving, therefore, crocodilians differ from the classic vertebrate pattern, where HCO3 - accumulates in the plasma upon excretion from the erythrocytes by the Cl--HCO3 --exchanger.

New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin.

Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in red blood cells upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here, we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on purified Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease O2 saturation of Hb. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas diffusion chamber, we observed similar effects in Hbs of a phylogenetically diverse set of other caiman, alligator and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80-100 million years ago in the common ancestor of crocodilians. Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where the build-up of erytrocytic CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate.

Parental transgenerational epigenetic inheritance related to dietary crude oil exposure in Danio rerio.

Transgenerational inheritance from both parental lines can occur by genetic and epigenetic inheritance. Maternal effects substantially influence offspring survival and fitness. However, investigation of the paternal contribution to offspring success has been somewhat neglected. In the present study, adult zebrafish were separated into female and male groups exposed for 21 days to either a control diet or to a diet containing water accommodated fractions of crude oil. Four F1 offspring groups were obtained: (1) control (non-exposed parents), (2) paternally exposed, (3) maternally exposed and (4) dual-parent-exposed. To determine the maternal and paternal influence on their offspring, we evaluated responses from molecular to whole organismal levels in both generations. Growth rate, hypoxia resistance and heart rate did not differ among parental groups. However, global DNA methylation in heart tissue was decreased in oil-exposed fish compared with control parents. This decrease was accompanied by an upregulation of glycine N-methyltransferase. Unexpectedly, maternal, paternal and dual exposure all enhanced survival of F1 offspring raised in oiled conditions. Regardless of parental exposure, however, F1 offspring exposed to oil exhibited bradycardia. Compared with offspring from control parents, global DNA methylation was decreased in the three offspring groups derived from oil-exposed parents. However, no difference between groups was observed in gene regulation involved in methylation transfer, suggesting that the changes observed in the F1 populations may have been inherited from both parental lines. Phenotypic responses during exposure to persistent environmental stressors in F1 offspring appear to be influenced by maternal and paternal exposure, potentially benefitting offspring populations to survive in challenging environments.

Exposure to Crude Oil Induces Retinal Apoptosis and Impairs Visual Function in Fish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 µg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Parental stressor exposure simultaneously conveys both adaptive and maladaptive larval phenotypes through epigenetic inheritance in the zebrafish (Danio rerio).

Genomic modifications occur slowly across generations, whereas short-term epigenetic inheritance of adaptive phenotypes may be immediately beneficial to large numbers of individuals, acting as a bridge for survival when adverse environments occur. In the present study, crude oil was used as an example of an environmental stressor. Adult zebrafish (P0) were dietarily exposed for 3 weeks to no, low, medium or high concentrations of crude oil. The F1 offspring obtained from the P0 groups were then assessed for transgenerational epigenetic transfer of oil-induced phenotypes. The exposure did not alter body length, body and organ mass or condition factor in the P0 groups. However, the P0 fecundity of both sexes decreased in proportion to the amount of oil fed. The F1 larvae from each P0 were then exposed from 3 hpf to 5 dpf to oil in their ambient water. Remarkably, F1 larvae derived from oil-exposed parents, when reared in oiled water, showed a 30% enhanced survival compared with controls (P<0.001). Unexpectedly, from day 3 to 5 of exposure, F1 larvae from oil-exposed parents showed poorer survival in clean water (up to 55% decreased survival). Additionally, parental oil exposure induced bradycardia (presumably maladaptive) in F1 larvae in both clean and oiled water. We conclude that epigenetic transgenerational inheritance can lead to an immediate and simultaneous inheritance of both beneficial and maladaptive traits in a large proportion of the F1 larvae. The adaptive responses may help fish populations survive when facing transient environmental stressors.

Dietary crude oil exposure during sex differentiation skewed adult sex ratio towards males in the zebrafish.

Dietary crude oil exposure has detrimental morpho-physiological effects in fishes, including endocrine disruption. However, little is known about how it influences sex differentiation and its potential for skewing sex ratios of populations. Appropriate sex ratio is important for maintaining effective population size and structure. Deviations of these ratios can compromise population growth and maintenance and may induce changes in a species' evolutionary trajectory. We assessed the potential of dietary exposure to crude oil (6.5, 11.4, and 17.5 mg/kg food) to alter sex differentiation in the zebrafish (Danio rerio) (20-35 days post fertilization (dpf)) and subsequently skew the adult (90 dpf) sex ratio. Multiple health- and fitness-related phenotypic traits (i.e., body mass and length, condition factor, heart rate, oxygen consumption, and their capacity to cope with hypoxia) were also assessed to better understand the effects of dietary crude oil exposure. We showed that dietary exposure to crude oil during the process of sex differentiation skewed sex ratio towards males (up to 0.34:1 female to male ratio in the highest oil concentration). Remarkably, this effect occurred independently of affecting physiological variables and female gonad characteristics, thus highlighting just how subtle the effects of dietary crude oil exposure can be. Our results suggest that, although fish were in an apparently healthy state during experimentation, sex ratio was still impacted, potentially compromising the resilience of the population. Therefore, considering how complex chemical mixtures affect organisms at several levels (molecular-individual) in experimental designs is warranted to better understand the implications of the exposures and the hazards that populations face in the wild.

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin.

The extraordinary breath-hold diving capacity of crocodilians has been ascribed to a unique mode of allosterically regulating hemoglobin (Hb)-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical phenomenon by performing directed mutagenesis experiments on resurrected ancestral Hbs. Comparisons of Hb function between the common ancestor of archosaurs (the group that includes crocodilians and birds) and the last common ancestor of modern crocodilians revealed that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions represents a croc-specific innovation that evolved in combination with the loss of allosteric regulation by ATP binding. Mutagenesis experiments revealed that evolution of the novel allosteric function in crocodilians and the concomitant loss of ancestral function were not mechanistically coupled and were caused by different sets of substitutions. The gain of bicarbonate sensitivity in crocodilian Hb involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that evolution of the novel protein function was conditional on neutral mutations that produced no adaptive benefit when they first arose but that contributed to a permissive background for subsequent function-altering mutations at other sites. Due to the context dependence of causative substitutions, the unique allosteric properties of crocodilian Hb cannot be easily transplanted into divergent homologs of other species.

Genomic insights into the mystery of mouse mummies on the summits of Atacama volcanoes.

Our understanding of the limits of animal life is continually revised by scientific exploration of extreme environments. Here we report the discovery of mummified cadavers of leaf-eared mice, Phyllotis vaccarum, from the summits of three different Andean volcanoes at elevations 6,029-6,233 m above sea level in the Puna de Atacama in Chile and Argentina. Such extreme elevations were previously assumed to be completely uninhabitable by mammals. In combination with a live-captured specimen of the same species from the nearby summit of Volcán Llullaillaco (6,739 m)1, the summit mummies represent the highest altitude physical records of mammals in the world. We also report a chromosome-level genome assembly for P. vaccarum that, in combination with a whole-genome re-sequencing analysis and radiocarbon dating analysis, provides insights into the provenance and antiquity of the summit mice. Radiocarbon data indicate that the most ancient of the mummies are, at most, a few centuries old. Genomic polymorphism data revealed a high degree of continuity between the summit mice and conspecifics from lower elevations in the surrounding Altiplano. Genomic data also revealed equal numbers of males and females among the summit mice and evidence of close kinship between some individuals from the same summits. These findings bolster evidence for resident populations of Phyllotis at elevations >6,000 m and challenge assumptions about the environmental limits of vertebrate life and the physiological tolerances of small mammals.

Extreme high-elevation mammal surveys reveal unexpectedly high upper range limits of Andean mice.

In the world's highest mountain ranges, uncertainty about the upper elevational range limits of alpine animals represents a critical knowledge gap regarding the environmental limits of life and presents a problem for detecting range shifts in response to climate change. Here we report results of mountaineering mammal surveys in the Central Andes, which led to the discovery of multiple species of mice living at extreme elevations that far surpass previously assumed range limits for mammals. We live-trapped small mammals from ecologically diverse sites spanning >6700 m of vertical relief, from the desert coast of northern Chile to the summits of the highest volcanoes in the Andes. We used molecular sequence data and whole-genome sequence data to confirm the identities of species that represent new elevational records and to test hypotheses regarding species limits. These discoveries contribute to a new appreciation of the environmental limits of vertebrate life.

Altitude acclimatization, hemoglobin-oxygen affinity, and circulatory oxygen transport in hypoxia.

In mammals and other air-breathing vertebrates that live at high altitude, adjustments in convective O2 transport via changes in blood hemoglobin (Hb) content and/or Hb-O2 affinity can potentially mitigate the effects of arterial hypoxemia. However, there are conflicting views about the optimal values of such traits in hypoxia, partly due to the intriguing observation that hypoxia-induced acclimatization responses in humans and other predominantly lowland mammals are frequently not aligned in the same direction as evolved phenotypic changes in high-altitude natives. Here we review relevant theoretical and empirical results and we highlight experimental studies of rodents and humans that provide insights into the combination of hematological changes that help attenuate the decline in aerobic performance in hypoxia. For a given severity of hypoxia, experimental results suggest that optimal values for hematological traits are conditional on the states of other interrelated phenotypes that govern different steps in the O2-transport pathway.

Dietary Exposure to Low Levels of Crude Oil Affects Physiological and Morphological Phenotype in Adults and Their Eggs and Hatchlings of the King Quail (Coturnix chinensis).

Despite the current knowledge of the devastating effects of external exposure to crude oil on animal mortality, the study of developmental, transgenerational effects of such exposure has received little attention. We used the king quail as an animal model to determine if chronic dietary exposure to crude oil in a parental population would affect morpho-physiological phenotypic variables in their immediate offspring generation. Adult quail were separated into three groups: (1) Control, and two experimental groups dietarily exposed for at least 3 weeks to (2) Low (800 PAH ng/g food), or (3) High (2,400 PAH ng/g food) levels of crude oil. To determine the parental influence on their offspring, we measured metabolic and respiratory physiology in exposed parents and in their non-exposed eggs and hatchlings. Body mass and numerous metabolic (e.g., O2 consumption, CO2 production) and respiratory (e.g., ventilation frequency and volume) variables did not vary between control and oil exposed parental groups. In contrast, blood PO2, PCO2, and SO2 varied among parental groups. Notably, water loss though the eggshell was increased in eggs from High oil level exposed parents. Respiratory variables of hatchlings did not vary between populations, but hatchlings obtained from High oil-exposed parents exhibited lower capacities to maintain body temperature while exposed to a cooling protocol in comparison to hatchlings from Low- and Control-derived parents. The present study demonstrates that parental exposure to crude oil via diet impacts some aspects of physiological performance of the subsequent first (F 1 ) generation.

Behavioral consequences of dietary exposure to crude oil extracts in the Siamese fighting fish (Betta splendens).

Uptake by fishes of crude oil and its polycyclic aromatic hydrocarbons (PAHs) components occurs via gills, dietary intake, or diffusion through the skin. Dietary exposure to crude oil and its components is environmentally relevant, and induces physiological and morphological disruptions in fish. However, the impacts of crude oil on fish social and reproductive behaviors and thus the possible influences on reproductive success are poorly understood. As a part of their intraspecific interactions, male Siamese fighting fish (Betta splendens) exhibit highly stereotypic behavioral and territorial displays. This makes this species a tractable model for testing crude oil effects on behavior. After 2 weeks of acclimation at 29 °C, male adult betta fish were divided into three groups and fed for 4 weeks with food spiked with water (control), low oil concentrations or high oil concentrations (∑Total PAH concentrations 340, 3960 or 8820 ng/g dw, respectively) to determine subsequent alterations in behavioral displays. Compared with control fish, the aggressive display of "opercular flaring" was significantly increased (P < 0.03, n = 14-16) in oil-exposed fish. Bubble nest building, as well as testis and brain mass, were significantly reduced in treated fish (P < 0.05). Hematocrit of treated groups was increased significantly (P < 0.02) from 21% in control fish to ∼27% in both oil exposure groups. Dietary exposure over a 4-week period to low, relevant levels of crude oil thus leads to an increase in aggressive behavioral displays, a decrease in reproductive activity and additional morphological changes.