Comparative analysis of the molecular starvation response of Southern Ocean copepods.

Large lipid-storing copepods dominate mesozooplankton biomass in the polar oceans and form a critical link between primary production and higher trophic levels. The ecological success of these species depends on their ability to survive periods of food deprivation in a highly seasonal environment, but the molecular changes that mediate starvation tolerance in these taxa are unknown. We conducted starvation experiments for two dominant Southern Ocean copepods, Calanoides acutus and Calanus propinquus, allowing us to compare the molecular starvation response between species. These species differ in life history, diet and metabolic traits, and expressed overlapping but distinct transcriptomic responses to starvation. Most starvation-response genes were species-specific, but we identified a conserved core set of starvation-response genes related to RNA and protein metabolism. We used phylotranscriptomics to place these results in the context of copepod evolution and found that starvation-response genes are under strong purifying selection at the sequence level and stabilizing selection at the expression level, consistent with their role in mediating essential biological functions. Selection on starvation-response genes was especially strong in our focal lipid-storing lineage relative to other copepod taxa, underscoring the significance of starvation tolerance for these species. We also found that certain key lipid enzymes (elongases and desaturases) have experienced diversification and positive selection in lipid-storing lineages, reflecting the unique lipid storage needs of these animals. Our results shed light on the molecular adaptations of high-latitude zooplankton to variable food conditions and suggest that starvation-response genes are under particularly strong sequence and expression constraints.

Diel metabolic patterns revealed by in situ transcriptome and proteome in a vertically migratory copepod.

Zooplankton undergo a diel vertical migration (DVM) which exposes them to gradients of light, temperature, oxygen, and food availability on a predictable daily schedule. Disentangling the co-varying and potentially synergistic interactions on metabolic rates has proven difficult, despite the importance of this migration for the delivery of metabolic waste products to the distinctly different daytime (deep) and nighttime (surface) habitats. This study examines the transcriptomic and proteomic profiles of the circumglobal migratory copepod, Pleuromamma xiphias, over the diel cycle. The transcriptome showed that 96% of differentially expressed genes were upregulated during the middle of the day - the period often considered to be of lowest zooplankton activity. The changes in protein abundance were more spread out over time, peaking (42% of comparisons) in the early evening. Between 9:00 and 15:00, both the transcriptome and proteome datasets showed increased expression related to chitin synthesis and degradation. Additionally, at 09:00 and 22:00, there were increases in myosin and vitellogenin proteins, potentially linked to the stress of migration and/or reproductive investment. Based on protein abundances detected, there is an inferred switch in broad metabolic processes, shifting from electron transport system in the day to glycolysis and glycogen mobilization in the afternoon/evening. These observations provide evidence of the diel impact of DVM on transcriptomic and proteomic pathways that likely influence metabolic processes and subsequent excretion products, and clarify how this behaviour results in the direct rapid transport of waste metabolites from the surface to the deep ocean.

Chromatin dynamics enable transcriptional rhythms in the cnidarian Nematostella vectensis.

In animals, circadian rhythms are driven by oscillations in transcription, translation, and proteasomal degradation of highly conserved genes, resulting in diel cycles in the expression of numerous clock-regulated genes. Transcription is largely regulated through the binding of transcription factors to cis-regulatory elements within accessible regions of the chromatin. Chromatin remodeling is linked to circadian regulation in mammals, but it is unknown whether cycles in chromatin accessibility are a general feature of clock-regulated genes throughout evolution. To assess this, we applied an ATAC-seq approach using Nematostella vectensis, grown under two separate light regimes (light:dark (LD) and constant darkness (DD)). Based on previously identified N. vectensis circadian genes, our results show the coupling of chromatin accessibility and circadian transcription rhythmicity under LD conditions. Out of 180 known circadian genes, we were able to list 139 gene promoters that were highly accessible compared to common promoters. Furthermore, under LD conditions, we identified 259 active enhancers as opposed to 333 active enhancers under DD conditions, with 171 enhancers shared between the two treatments. The development of a highly reproducible ATAC-seq protocol integrated with published RNA-seq and ChIP-seq databases revealed the enrichment of transcription factor binding sites (such as C/EBP, homeobox, and MYB), which have not been previously associated with circadian signaling in cnidarians. These results provide new insight into the regulation of cnidarian circadian machinery. Broadly speaking, this supports the notion that the association between chromatin remodeling and circadian regulation arose early in animal evolution as reflected in this non-bilaterian lineage.

Plasticity in parental effects confers rapid larval thermal tolerance in the estuarine anemone Nematostella vectensis.

Parental effects can prepare offspring for different environments and facilitate survival across generations. We exposed parental populations of the estuarine anemone, Nematostella vectensis, from Massachusetts to elevated temperatures and quantified larval mortality across a temperature gradient. We found that parental exposure to elevated temperatures resulted in a consistent increase in larval thermal tolerance, as measured by the temperature at which 50% of larvae die (LT50), with a mean increase in LT50 of 0.3°C. Larvae from subsequent spawns returned to baseline thermal thresholds when parents were returned to normal temperatures, indicating plasticity in these parental effects. Histological analyses of gametogenesis in females suggested that these dynamic shifts in larval thermal tolerance may be facilitated by maternal effects in non-overlapping gametic cohorts. We also compared larvae from North Carolina (a genetically distinct population with higher baseline thermal tolerance) and Massachusetts parents, and observed that larvae from heat-exposed Massachusetts parents had thermal thresholds comparable to those of larvae from unexposed North Carolina parents. North Carolina parents also increased larval thermal tolerance under the same high-temperature regime, suggesting that plasticity in parental effects is an inherent trait for N. vectensis Overall, we find that larval thermal tolerance in N. vectensis shows a strong genetic basis and can be modulated by parental effects. Further understanding of the mechanisms behind these shifts can elucidate the fate of thermally sensitive ectotherms in a rapidly changing thermal environment.

Chromatin Dynamics and Gene Expression Response to Heat Exposure in Field-Conditioned versus Laboratory-Cultured Nematostella vectensis.

Organisms' survival is associated with the ability to respond to natural or anthropogenic environmental stressors. Frequently, these responses involve changes in gene regulation and expression, consequently altering physiology, development, or behavior. Here, we present modifications in response to heat exposure that mimics extreme summertime field conditions of lab-cultured and field-conditioned Nematostella vectensis. Using ATAC-seq and RNA-seq data, we found that field-conditioned animals had a more concentrated reaction to short-term thermal stress, expressed as enrichment of the DNA repair mechanism pathway. By contrast, lab animals had a more diffuse reaction that involved a larger number of differentially expressed genes and enriched pathways, including amino acid metabolism. Our results demonstrate that pre-conditioning affects the ability to respond efficiently to heat exposure in terms of both chromatin accessibility and gene expression and reinforces the importance of experimentally addressing ecological questions in the field.

Environmental entrainment demonstrates natural circadian rhythmicity in the cnidarian Nematostella vectensis.

Considerable advances in chronobiology have been made through controlled laboratory studies, but distinct temporal rhythms can emerge under natural environmental conditions. Lab-reared Nematostella vectensis sea anemones exhibit circadian behavioral and physiological rhythms. Given that these anemones inhabit shallow estuarine environments subject to tidal inputs, it was unclear whether circadian rhythmicity would persist following entrainment in natural conditions, or whether circatidal periodicity would predominate. Nematostella were conditioned within a marsh environment, where they experienced strong daily temperature cycles as well as brief tidal flooding around the full and new moons. Upon retrieval, anemones exhibited strong circadian (∼24 h) activity rhythms under a light-dark cycle or continuous darkness, but reduced circadian rhythmicity under continuous light. However, some individuals in each light condition showed circadian rhythmicity, and a few individuals showed circatidal rhythmicity. Consistent with the behavioral studies, a large number of transcripts (1640) exhibited diurnal rhythmicity compared with very few (64) with semidiurnal rhythmicity. Diurnal transcripts included core circadian regulators, and 101 of 434 (23%) genes that were previously found to be upregulated by exposure to ultraviolet radiation. Together, these behavioral and transcriptional studies show that circadian rhythmicity predominates and suggest that solar radiation drives physiological cycles in this sediment-dwelling subtidal animal.

Exposure to CO2 influences metabolism, calcification and gene expression of the thecosome pteropod Limacina retroversa.

Thecosomatous pteropods, a group of aragonite shell-bearing zooplankton, are becoming an important sentinel organism for understanding the influence of ocean acidification on pelagic organisms. These animals show vulnerability to changing carbonate chemistry conditions, are geographically widespread, and are both biogeochemically and trophically important. The objective of this study was to determine how increasing duration and severity of CO2 treatment influence the physiology of the thecosome Limacina retroversa, integrating both gene expression and organism-level (respiration and calcification) metrics. We exposed pteropods to over-saturated, near-saturated or under-saturated conditions and sampled individuals at 1, 3, 7, 14 and 21 days of exposure to test for the effect of duration. We found that calcification was affected by borderline and under-saturated conditions by week two, while respiration appeared to be more strongly influenced by an interaction between severity and duration of exposure, showing complex changes by one week of exposure. The organismal metrics were corroborated by specific gene expression responses, with increased expression of biomineralization-associated genes in the medium and high treatments throughout and complex changes in metabolic genes corresponding to both captivity and CO2 treatment. Genes associated with other physiological processes such as lipid metabolism, neural function and ion pumping had complex responses, influenced by both duration and severity. Beyond these responses, our findings detail the captivity effects for these pelagic organisms, providing information to contextualize the conclusions of previous studies, and emphasizing a need for better culturing protocols.

Phylogenetic analysis of cnidarian peroxiredoxins and stress-responsive expression in the estuarine sea anemone Nematostella vectensis.

Peroxiredoxins (PRXs) are a family of antioxidant enzymes present in all domains of life. To date, the diversity and function of peroxiredoxins within animals have only been studied in a few model species. Thus, we sought to characterize peroxiredoxin diversity in cnidarians and to gain insight into their function in one cnidarian-the sea anemone Nematostella vectensis. Phylogenetic analysis using all six known PRX subfamilies (PRX1-4, PRX5, PRX6, PRXQ/AHPE1, TPX, BCP-PRXQ) revealed that like bilaterians, cnidarians contain representatives from three subfamilies (PRX1-4, PRX5, PRX6). Within the PRX1-4 subfamily, cnidarian sequences fall into two clades: PRX4, and a cnidarian-specific clade, which we term CNID-PRX. This phylogenetic analysis demonstrates that the three PRX subfamilies present in Bilateria were also present in the last common ancestor of the Cnidaria and Bilateria, and further that diversification of the PRX1-4 subfamily has occurred within the cnidarian lineage. We next examined the impact of decreased salinity, increased temperature, and peroxide exposure on the expression of four prx genes in N. vectensis (cnid-prx, prx4, prx5, and prx6). These genes exhibited unique expression patterns in response to these environmental stressors. Expression of prx4 decreased with initial exposure to elevated temperature, cnid-prx increased with exposure to elevated temperatures as well as with hydrogen peroxide exposure, and expression of all prxs transiently decreased with reduced salinity. Predicted subcellular localization patterns also varied among PRX proteins. Together these results provide evidence that peroxiredoxins in N. vectensis serve distinct physiological roles and lay a groundwork for understanding how peroxiredoxins mediate cnidarian developmental processes and environmental responses.

Maternal polycyclic aromatic hydrocarbon (PAH) transfer and effects on offspring of copepods exposed to dispersed oil with and without oil droplets.

Copepods of the genus Calanus have the potential for accumulating lipophilic oil components due to their high lipid content and found to filter and ingest oil droplets during exposure. As female copepods produce eggs at the expense of lipid storage, there is a concern for transfer of lipophilic contaminants to offspring. To assess the potential for maternal transfer of oil components, ovigerous female copepods (Calanus finmarchicus) were exposed to filtered and unfiltered oil dispersions for 4 days, collected and eggs maintained in clean seawater and hatching and gene expression examined in hatched nauplii. Oil droplet exposure contributed to polycyclic aromatic hydrocarbon (PAH) uptake in dispersion-treated adult copepods, as displayed through PAH body residue analyses and fluorescence microscopy. Applying the latter methodology, transfer of heavy PAH from copepod mothers to offspring were detected Subtle effects were observed in offspring as evidenced by a temporal reduction in hatching success appear to be occurring only when mothers were exposed to the unfiltered oil dispersions. Offspring reared in clean water through to late naupliar stages were collected for RNA extraction and preparation of libraries for high-throughput transcriptome sequencing. Differentially expressed genes were identified through pairwise comparisons between treatments. Among these, several expressed genes have known roles in responses to chemical stress including xenobiotic metabolism enzymes, antioxidants, chaperones, and components of the inflammatory response. While gene expression results suggest a transgenerational activation of stress responses, the increase in relatively small number of differentially expressed genes suggests a minor long-term effect on offspring following maternal exposure.

Aryl hydrocarbon receptor (AHR) in the cnidarian Nematostella vectensis: comparative expression, protein interactions, and ligand binding.

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachealess). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, ß-naphthoflavone). Together, our data suggest that AHR's original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian-bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates.

Transcriptional profiling of reproductive development, lipid storage and molting throughout the last juvenile stage of the marine copepod Calanus finmarchicus.

INTRODUCTION: Calanus finmarchicus, a highly abundant copepod that is an important primary consumer in North Atlantic ecosystems, has a flexible life history in which copepods in the last juvenile developmental stage (fifth copepodid, C5) may either delay maturation and enter diapause or molt directly into adults. The factors that regulate this developmental plasticity are poorly understood, and few tools have been developed to assess the physiological condition of individual copepods. RESULTS: We sampled a cultured population of C. finmarchicus copepods daily throughout the C5 stage and assessed molt stage progression, gonad development and lipid storage. We used high-throughput sequencing to identify genes that were differentially expressed during progression through the molt stage and then used qPCR to profile daily expression of individual genes. Based on expression profiles of twelve genes, samples were statistically clustered into three groups: (1) an early period occurring prior to separation of the cuticle from the epidermis (apolysis) when expression of genes associated with lipid synthesis and transport (FABP and ELOV) and two nuclear receptors (ERR and HR78) was highest, (2) a middle period of rapid change in both gene expression and physiological condition, including local minima and maxima in several nuclear receptors (FTZ-F1, HR38b, and EcR), and (3) a late period when gonads were differentiated and expression of genes associated with molting (Torso-like, HR38a) peaked. The ratio of Torso-like to HR38b strongly differentiated the early and late groups. CONCLUSIONS: This study provides the first dynamic profiles of gene expression anchored with morphological markers of lipid accumulation, development and gonad maturation throughout a copepod molt cycle. Transcriptomic profiling revealed significant changes over the molt cycle in genes with presumed roles in lipid synthesis, molt regulation and gonad development, suggestive of a coupling of these processes in Calanus finmarchicus. Finally, we identified gene expression profiles that strongly differentiate between early and late development within the C5 copepodid stage. We anticipate that these findings and continued development of robust gene expression biomarkers that distinguish between diapause preparation and continuous development will ultimately enable novel studies of the intrinsic and extrinsic factors that govern diapause initiation in Calanus finmarchicus.

Establishing a model organism: a report from the first annual Nematostella meeting.

The sea anemone Nematostella vectensis has developed into a model organism for studying genome evolution and animal development.

Sea butterflies in a pickle: reliable biomarkers and seasonal sensitivity of Limacina retroversa to ocean acidification in the Gulf of Maine.

The passive dissolution of anthropogenically produced CO2 into the ocean system is reducing ocean pH and changing a suite of chemical equilibria, with negative consequences for some marine organisms, in particular those that bear calcium carbonate shells. Although our monitoring of these chemical changes has improved, we have not developed effective tools to translate observations, which are typically of the pH and carbonate saturation state, into ecologically relevant predictions of biological risks. One potential solution is to develop bioindicators: biological variables with a clear relationship to environmental risk factors that can be used for assessment and management. Thecosomatous pteropods are a group of pelagic shelled marine gastropods, whose biological responses to CO2 have been suggested as potential bioindicators of ocean acidification owing to their sensitivity to acidification in both the laboratory and the natural environment. Using five CO2 exposure experiments, occurring across four seasons and running for up to 15 days, we describe a consistent relationship between saturation state, shell transparency and duration of exposure, as well as identify a suite of genes that could be used for biological monitoring with further study. We clarify variations in thecosome responses due to seasonality, resolving prior uncertainties and demonstrating the range of their phenotypic plasticity. These biomarkers of acidification stress can be implemented into ecosystem models and monitoring programmes in regions where pteropods are found, whilst the approach will serve as an example for other regions on how to bridge the gap between point-based chemical monitoring and biologically relevant assessments of ecosystem health.

Effects of petrogenic pollutants on North Atlantic and Arctic Calanus copepods: From molecular mechanisms to population impacts.

Oil and gas industries in the Northern Atlantic Ocean have gradually moved closer to the Arctic areas, a process expected to be further facilitated by sea ice withdrawal caused by global warming. Copepods of the genus Calanus hold a key position in these cold-water food webs, providing an important energetic link between primary production and higher trophic levels. Due to their ecological importance, there is a concern about how accidental oil spills and produced water discharges may impact cold-water copepods. In this review, we summarize the current knowledge of the toxicity of petroleum on North Atlantic and Arctic Calanus copepods. We also review how recent development of high-quality transcriptomes from RNA-sequencing of copepods have identified genes regulating key biological processes, like molting, diapause and reproduction in Calanus copepods, to suggest linkages between exposure, molecular mechanisms and effects on higher levels of biological organization. We found that the available ecotoxicity threshold data for these copepods provide valuable information about their sensitivity to acute petrogenic exposures; however, there is still insufficient knowledge regarding underlying mechanisms of toxicity and the potential for long-term implications of relevance for copepod ecology and phenology. Copepod transcriptomics has expanded our understanding of how key biological processes are regulated in cold-water copepods. These advances can improve our understanding of how pollutants affect biological processes, and thus provide the basis for new knowledge frameworks spanning the effect continuum from molecular initiating events to adverse effects of regulatory relevance. Such efforts, guided by concepts such as adverse outcome pathways (AOPs), enable standardized and transparent characterization and evaluation of knowledge and identifies research gaps and priorities. This review suggests enhancing mechanistic understanding of exposure-effect relationships to better understand and link biomarker responses to adverse effects to improve risk assessments assessing ecological effects of pollutant mixtures, like crude oil, in Arctic areas.

A report from the second Nematostella vectensis research conference.

This report summarizes information discussed at the second Nematostella vectensis research conference, which took place on August 27, 2012 in Boston, MA, USA. The startlet sea anemone Nematostella is emerging as one of leading model organisms among cnidarians, in part because of the extensive genome and transcriptome resources that are becoming available for Nematostella, which were the focus of several presentations. In addition, research was presented on the use of Nematostella in developmental, regeneration, signal transduction, host-symbiont, and gene-environment interaction studies.

Sensory conflict disrupts circadian rhythms in the sea anemone Nematostella vectensis.

Circadian clocks infer time of day by integrating information from cyclic environmental factors called zeitgebers, including light and temperature. Single zeitgebers entrain circadian rhythms, but few studies have addressed how multiple, simultaneous zeitgeber cycles interact to affect clock behavior. Misalignment between zeitgebers ('sensory conflict') can disrupt circadian rhythms, or alternatively clocks may privilege information from one zeitgeber over another. Here, we show that temperature cycles modulate circadian locomotor rhythms in Nematostella vectensis, a model system for cnidarian circadian biology. We conduct behavioral experiments across a comprehensive range of light and temperature cycles and find that Nematostella's circadian behavior is disrupted by chronic misalignment between light and temperature, which involves disruption of the endogenous clock itself rather than a simple masking effect. Sensory conflict also disrupts the rhythmic transcriptome, with numerous genes losing rhythmic expression. However, many metabolic genes remained rhythmic and in-phase with temperature, and other genes even gained rhythmicity, implying that some rhythmic metabolic processes persist even when behavior is disrupted. Our results show that a cnidarian clock relies on information from light and temperature, rather than prioritizing one signal over the other. Although we identify limits to the clock's ability to integrate conflicting sensory information, there is also a surprising robustness of behavioral and transcriptional rhythmicity.

Almost all living things exhibit circadian rhythms ­ internally driven biological processes ­ which regulate important bodily functions, including sleep and wake cycles, over a roughly 24-hour period. Circadian clocks govern these rhythms by receiving information from the environment that allows them to tell what time of day it is. Two of the most important environmental signals, known as 'zeitgebers' ­ meaning 'time giver' ­ are light and temperature. In nature, circadian clocks must integrate information from multiple zeitgebers simultaneously. Typically, over a 24-hour period, temperature increases and decreases with the light cycle, getting warmer during the day and colder at night. However, artificial light pollution and circadian disruption ­ such as shift work ­ can impact the natural relationship between light and temperature. This 'sensory conflict', where two zeitgebers provide conflicting information about the time of day, can impact ecosystems such as coral reefs; and is also linked to poor health in humans. How circadian clocks behave in complex multi-zeitgeber environments and specifically, whether they prioritize one zeitgeber over another is not fully understood. To investigate how cnidarians ­ a group of marine animals including corals and jellyfish ­ respond to sensory conflict, Berger and Tarrant varied the relationship between light and temperature cycles using the sea anemone Nematostella vectensis as a model system. Nematostella is a nocturnal cnidarian, meaning it moves most at night. First, Berger and Tarrant kept Nematostella in dark conditions with 24-hour temperature cycles ­ starting cold, increasing to a peak in the middle of the day before decreasing towards the end of the day. Monitoring Nematostella movement revealed that they moved most during the cold phase, showing that temperature cycles alone can maintain rhythmic behavior. Similarly, when temperature and light cycles were aligned such that both rose and fell together, nocturnal behavior was preserved. However, when large misalignments between light and temperature cycles were introduced ­ such that temperature decreased during light periods and increased in the dark ­ nocturnal behavior was almost completely lost. This suggests that both light and temperature interact to produce complex patterns of circadian behavior, with neither signal being prioritized over the other. Additionally, Berger and Tarrant investigated how sensory conflict impacts the activity of Nematostella genes. While many genes remained rhythmic, suggesting some gene expression persists when behavior is disturbed, others that were rhythmic became arrhythmic. In contrast, a selection of genes that do not normally display rhythmic behavior gained rhythmic expression. Genes related to protein metabolism and other energy-intensive processes were particularly disrupted. In an increasingly 24/7 society, it is important to understand how complex multi-sensory environments impact circadian rhythms and as a result, health and fitness. The findings show that certain light and temperature regimes severely disrupt Nematostella behavior and could be useful in predicting how other organisms might respond to disruptions such as light pollution. In the future, such information could be used to design optimal light regimes for ecosystems in which the relationship between light cycles and other environmental signals is disrupted by human behavior.

Nematostella vectensis exhibits an enhanced molecular stress response upon co-exposure to highly weathered oil and surface UV radiation.

Crude oil released into the environment undergoes weathering processes that gradually change its composition and toxicity. Co-exposure to petroleum mixtures and other stressors, including ultraviolet (UV) radiation, may lead to synergistic effects and increased toxicity. Laboratory studies should consider these factors when testing the effects of oil exposure on aquatic organisms. Here, we study transcriptomic responses of the estuarine sea anemone Nematostella vectensis to naturally weathered oil, with or without co-exposure to environmental levels of UV radiation. We find that co-exposure greatly enhances the response. We use bioinformatic analyses to identify molecular pathways implicated in this response, which suggest phototoxicity and oxidative damage as mechanisms for the enhanced stress response. Nematostella's stress response shares similarities with the vertebrate oxidative stress response, implying deep conservation of certain stress pathways in animals. We show that exposure to weathered oil along with surface-level UV exposure has substantial physiological consequences in a model cnidarian.

Correlated evolution of androgen receptor and aromatase revisited.

Conserved interactions among proteins or other molecules can provide strong evidence for coevolution across their evolutionary history. Diverse phylogenetic methods have been applied to identify potential coevolutionary relationships. In most cases, these methods minimally require comparisons of orthologous sequences and appropriate controls to separate effects of selection from the overall evolutionary relationships. In vertebrates, androgen receptor (AR) and cytochrome p450 aromatase (CYP19) share an affinity for androgenic steroids, which serve as receptor ligands and enzyme substrates. In a recent study, Tiwary and Li (Tiwary BK, Li W-H. 2009. Parallel evolution between aromatase and androgen receptor in the animal kingdom. Mol Biol Evol. 26:123-129) reported that AR and CYP19 displayed a signature of ancient and conserved interactions throughout all the Eumetazoa (i.e., cnidarians, protostomes, and deuterostomes). Because these findings conflicted with a number of previous studies, we reanalyzed the data set used by Tiwary and Li. First, our analyses demonstrate that the invertebrate genes used in the previous analysis are not orthologous sequences but instead represent a diverse set of nuclear receptors and CYP enzymes with no confirmed or hypothesized relationships with androgens. Second, we show that 1) their analytical approach, which measures correlations in evolutionary distances between proteins, potentially led to spurious significant relationships due simply to conserved domains and 2) control comparisons provide positive evidence for a strong influence of evolutionary history. We discuss how corrections to this method and analysis of key taxa (e.g., duplications in the teleost fish and suiform lineages) can inform investigations of the coevolutionary relationships between AR and aromatase.

Ecdysteroid receptor from the American lobster Homarus americanus: EcR/RXR isoform cloning and ligand-binding properties.

In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.