Ianina Altshuler

An Integrated Multi-Disciplinary Approach for Studying Multiple Stressors in Freshwater Ecosystems: Daphnia as a Model Organism

The increased overexploitation of freshwater ecosystems and their extended watersheds often generates a cascade of anthropogenic stressors (e.g., acidification, eutrophication, metal contamination, Ca decline, changes in the physical environment, introduction of invasive species, over-harvesting of resources). The combined effect of these stressors is particularly difficult to study, requiring a coordinated multi-disciplinary effort and insights from various sub-disciplines of biology, including ecology, evolution, toxicology, and genetics. It also would benefit from a well-developed and broadly accepted model systems. The freshwater crustacean Daphnia is an excellent model organism for studying multiple stressors because it has been a chosen focus of study in all four of these fields. Daphnia is a widespread keystone species in most freshwater ecosystems, where it is routinely exposed to a multitude of anthropogenic and natural stressors. It has a fully sequenced genome, a well-understood life history and ecology, and a huge library of responses to toxicity. To make the case for its value as a model species, we consider the joint and separate effects of natural and three anthropogenic stressors-climatic change, calcium decline, and metal contaminants on daphniids. We propose that integrative approaches marrying various subfields of biology can advance our understanding of the combined effects of stressors. Such approaches can involve the measuring of multiple responses at several levels of biological organization from molecules to natural populations. For example, novel interdisciplinary approaches such as transcriptome profiling and mutation accumulation experiments can offer insights into how multiple stressors influence gene transcription and mutation rates across genomes, and, thus, help determine the causal mechanism between environmental stressors and population/community effects as well as long-term evolutionary patterns.

Changes in water chemistry can disable plankton prey defenses

The effectiveness of antipredator defenses is greatly influenced by the environment in which an organism lives. In aquatic ecosystems, the chemical composition of the water itself may play an important role in the outcome of predator–prey interactions by altering the ability of prey to detect predators or to implement defensive responses once the predator’s presence is perceived. Here, we demonstrate that low calcium concentrations (<1.5 mg/L) that are found in many softwater lakes and ponds disable the ability of the water flea, Daphnia pulex to respond effectively to its predator, larvae of the phantom midge, Chaoborus americanus. This low-calcium environment prevents development of the prey’s normal array of induced defenses, which include an increase in body size, formation of neck spines, and strengthening of the carapace. We estimate that this inability to access these otherwise effective defenses results in a 50–186% increase in the vulnerability of the smaller juvenile instars of Daphnia, the stages most susceptible to Chaoborus predation. Such a change likely contributes to the observed lack of success of daphniids in most low-calcium freshwater environments, and will speed the loss of these important zooplankton in lakes where calcium levels are in decline.

The Evolutionary History of Sarco(endo)plasmic Calcium ATPase (SERCA)

Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na+/K+ transporters, H+/K+ transporters, and plasma membrane Ca2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca2+ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

Synergistic interactions of biotic and abiotic environmental stressors on gene expression

Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis - cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) - using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors.

Mechanisms of subzero growth in the cryophile Planococcus halocryophilus determined through proteomic analysis

The eurypsychrophilic bacterium Planococcus halocryophilus is capable of growth down to -15°C, making it ideal for studying adaptations to subzero growth. To increase our understanding of the mechanisms and pathways important for subzero growth, we performed proteomics on P. halocryophilus grown at 23°C, 23°C with 12% w/v NaCl and -10°C with 12% w/v NaCl. Many proteins with increased abundances at -10°C versus 23°C also increased at 23C-salt versus 23°C, indicating a closely tied relationship between salt and cold stress adaptation. Processes which displayed the largest changes in protein abundance were peptidoglycan and fatty acid (FA) synthesis, translation processes, methylglyoxal metabolism, DNA repair and recombination, and protein and nucleotide turnover. We identified intriguing targets for further research at -10°C, including PlsX and KASII (FA metabolism), DD-transpeptidase and MurB (peptidoglycan synthesis), glyoxalase family proteins (reactive electrophile response) and ribosome modifying enzymes (translation turnover). PemK/MazF may have a crucial role in translational reprogramming under cold conditions. At -10°C P. halocryophilus induces stress responses, uses resources efficiently, and carefully controls its growth and metabolism to maximize subzero survival. The present study identifies several mechanisms involved in subzero growth and enhances our understanding of cold adaptation.

Macroinvertebrate and soil prokaryote communities in the forest–tundra ecotone of the Subarctic Yukon

The forest–tundra interface is the world’s largest ecotone, and is globally important due to its biodiversity, climatic sensitivity, and natural resources. The ecological communities which characterize this ecotone, and which provide local and global ecosystem services, are affected by environmental variation at multiple scales. We explored correlations between environmental variables and macroinvertebrate and soil prokaryote communities in the forest–tundra ecotone of the Yukon, Canada. We found that each tussock tundra site possessed a distinct community of macroinvertebrates and prokaryotes, and therefore represented a unique contribution to regional biodiversity. Prokaryote diversity increased with active layer depth, which could be an effect of temperature, or could be evidence of a species-area effect. Prokaryote diversity decreased with lichen cover, which could be due to antimicrobial properties of lichen. The macroinvertebrate community composition was affected by proximity to a human disturbance, the Dempster Highway. Both macroinvertebrate and prokaryote community compositions changed along the latitudinal transect, as the biome transitioned from taiga to tundra. We also found that the abundance of carnivores relative to herbivores decreased with latitude, which adds to recent evidence that predation decreases with latitude. Our survey yielded new insights about how macro- and microorganisms vary together and independently in relation to environmental variables at multiple scales in a forest–tundra ecotone.

In Situ Field Sequencing and Life Detection in Remote (79°26′N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities

Significant progress is being made in the development of the next generation of low cost life detection instrumentation with much smaller size, mass and energy requirements. Here, we describe in situ life detection and sequencing in the field in soils over laying ice wedges in polygonal permafrost terrain on Axel Heiberg Island, located in the Canadian high Arctic (79°26′N), an analog to the polygonal permafrost terrain observed on Mars. The life detection methods used here include (1) the cryo-iPlate for culturing microorganisms using diffusion of in situ nutrients into semi-solid media (2) a Microbial Activity Microassay (MAM) plate (BIOLOG Ecoplate) for detecting viable extant microorganisms through a colourimetric assay, and (3) the Oxford Nanopore MinION for nucleic acid detection and sequencing of environmental samples and the products of MAM plate and cryo-iPlate. We obtained 39 microbial isolates using the cryo-iPlate, which included several putatively novel strains based on the 16S rRNA gene, including a Pedobacter sp. (96% closest similarity in GenBank) which we partially genome sequenced using the MinION. The MAM plate successfully identified an active community capable of L-serine metabolism, which was used for metagenomic sequencing with the MinION to identify the active and enriched community. A metagenome on environmental ice wedge soil samples was completed, with base calling and uplink/downlink carried out via satellite internet. Validation of MinION sequencing using the Illumina MiSeq platform was consistent with the results obtained with the MinION. The instrumentation and technology utilized here is pre-existing, low cost, low mass, low volume, and offers the prospect of equipping micro-rovers and micro-penetrators with aggressive astrobiological capabilities. Since potentially habitable astrobiology targets have been identified (RSLs on Mars, near subsurface water ice on Mars, the plumes and oceans of Europa and Enceladus), future astrobiology missions will certainly target these areas and there is a need for direct life detection instrumentation.

Gene expression variation in duplicate lactate dehydrogenase genes: do ecological species show distinct responses?

Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5–7 mg/l or 2–3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments.

Comparative Transcriptomics of Cold Growth and Adaptive Features of a Eury- and Steno-Psychrophile

Permafrost subzero environments harbor diverse, active communities of microorganisms. However, our understanding of the subzero growth, metabolisms, and adaptive properties of these microbes remains very limited. We performed transcriptomic analyses on two subzero-growing permafrost isolates with different growth profiles in order to characterize and compare their cold temperature growth and cold-adaptive strategies. The two organisms, Rhodococcus sp. JG3 (-5 to 30°C) and Polaromonas sp. Eur3 1.2.1 (-5 to 22°C), shared several common responses during low temperature growth, including induction of translation and ribosomal processes, upregulation of nutrient transport, increased oxidative and osmotic stress responses, and stimulation of polysaccharide capsule synthesis. Recombination appeared to be an important adaptive strategy for both isolates at low temperatures, likely as a mechanism to increase genetic diversity and the potential for survival in cold systems. While Rhodococcus sp. JG3 favored upregulating iron and amino acid transport, sustaining redox potential, and modulating fatty acid synthesis and composition during growth at -5°C compared to 25°C, Polaromonas sp. Eur3 1.2.1 increased the relative abundance of transcripts involved in primary energy metabolism and the electron transport chain, in addition to signal transduction and peptidoglycan synthesis at 0°C compared to 20°C. The increase in energy metabolism may explain why Polaromonas sp. Eur3 1.2.1 is able to sustain growth rates at 0°C comparable to those at higher temperatures. For Rhodococcus sp. JG3, flexibility in use of carbon sources, iron acquisition, control of membrane fatty acid composition, and modulating redox and co-factor potential may be ways in which this organism is able to sustain growth over a wider range of temperatures. Increasing our understanding of the microbes in these habitats helps us better understand active pathways and metabolisms in extreme environments. Identifying novel, thermolabile, and cold-active enzymes from studies such as this is also of great interest to the biotechnology and food industries.

Microbial Life in Permafrost

Permafrost is a hostile environment that harbors a diverse and active microbial community. Next generation sequencing studies have demonstrated a wide diversity of microorganisms present in Arctic, Antarctic and high altitude permafrost soils. In situ activity of these microorganisms has been demonstrated through multiple lines of evidence. Radiolabeled studies and stable isotope probing have established that active respiration and DNA replication occur in permafrost soils under frozen conditions. Furthermore, microorganisms capable of subzero growth have been isolated from permafrost samples. These isolates have adapted to the permafrost environment through a multitude of molecular changes, such as increased expression of cold shock and metabolite transport proteins, reduced fatty acid saturation in the membrane, and presence of temperature specific isozymes. Recent studies have focused on permafrost thaw due to anthropogenic climate change. The subsequent thaw of frozen organic carbon stores in permafrost is thought to increase microbial activity and emissions of greenhouse gases to the atmosphere. As the permafrost thaws, the microbial community changes in terms of diversity and functional potential in response to warmer temperatures, and increased carbon and water availability.