Phylogenomic analysis of Fundulidae (Teleostei: Cyprinodotiformes) using RNA-sequencing data.

Fishes of the New World cyprinodontiform family Fundulidae display a wide variety of tolerance to environmental conditions, making them a valuable model system for comparative, evolutionary, and environmental studies. Despite numerous attempts to resolve the phylogenetic relationships of family Fundulidae, the basal structure of the phylogeny remains unresolved. The lack of a robust and fully resolved phylogeny for family Fundulidae and its most speciose genus Fundulus is an impediment to future research. This study utilized novel RNA-sequencing data for phylogenetic inference among16 members of Fundulidae to better refine the basal nodes of the family and confront long-standing questions regarding (1) the monophyletic status of genus Fundulus, and validity of the Lucania and recently synonymized Adinia genera; (2) the relationship of the west coast endemic Fundulus parvipinnis and F. lima to other Fundulus species; and (3) the validity of subgeneric classifications. In addition, previously published nuclear gene sequences for 32 Fundulidae species were re-analyzed in combination with novel RNA-sequencing data. Maximum likelihood and Bayesian analyses generated identical phylogenies with strong statistical support at nearly all nodes, demonstrating the utility of RNA-sequencing data in constructing robust phylogenies not achievable by previous methods. While many past hypothesized evolutionary relationships for Fundulidae were reinforced, several alternative relationships are hypothesized at basal nodes resulting in a re-analysis of the deeper structure of family Fundulidae. These results reveal family Fundulidae as a paraphyletic grouping of members of genus Fundulus and Lucania and supports the previous synonymy of genus Adinia with genus Fundulus.

Genomic and physiological footprint of the Deepwater Horizon oil spill on resident marsh fishes.

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Genomic mechanisms of evolved physiological plasticity in killifish distributed along an environmental salinity gradient.

Adaptive variation tends to emerge clinally along environmental gradients or discretely among habitats with limited connectivity. However, in Atlantic killifish (Fundulus heteroclitus), a population genetic discontinuity appears in the absence of obvious barriers to gene flow along parallel salinity clines and coincides with a physiologically stressful salinity. We show that populations resident on either side of this discontinuity differ in their abilities to compensate for osmotic shock and illustrate the physiological and functional genomic basis of population variation in hypoosmotic tolerance. A population native to a freshwater habitat, upstream of the genetic discontinuity, exhibits tolerance to extreme hypoosmotic challenge, whereas populations native to brackish or marine habitats downstream of the discontinuity lose osmotic homeostasis more severely and take longer to recover. Comparative transcriptomics reveals a core transcriptional response associated with acute and acclimatory responses to hypoosmotic shock and posits unique mechanisms that enable extreme osmotic tolerance. Of the genes that vary in expression among populations, those that are putatively involved in physiological acclimation are more likely to exhibit nonneutral patterns of divergence between freshwater and brackish populations. It is not the well-known effectors of osmotic acclimation, but rather the lesser-known immediate-early responses, that appear important in contributing to population differences.

Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus.

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.

Independently evolved pollution resistance in four killifish populations is largely explained by few variants of large effect.

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human-altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts that adaptation is due to a few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype-phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and using RAD-seq genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB-126. We found that one to two large-effect QTL loci accounted for resistance to PCB-mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling. One QTL locus was shared across all populations and another was shared across three populations. One QTL locus showed strong signatures of recent natural selection in the corresponding wild population but another QTL locus did not. Some candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB-126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB-126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex.

Common functional targets of adaptive micro- and macro-evolutionary divergence in killifish.

Environmental salinity presents a key barrier to dispersal for most aquatic organisms, and adaptation to alternate osmotic environments likely enables species diversification. Little is known of the functional basis for derived tolerance to environmental salinity. We integrate comparative physiology and functional genomics to explore the mechanistic underpinnings of evolved variation in osmotic plasticity within and among two species of killifish; Fundulus majalis harbours the ancestral mainly salt-tolerant phenotype, whereas Fundulus heteroclitus harbours a derived physiology that retains extreme salt tolerance but with expanded osmotic plasticity towards the freshwater end of the osmotic continuum. Common-garden comparative hypo-osmotic challenge experiments show that F. heteroclitus is capable of remodelling gill epithelia more quickly and at more extreme osmotic challenge than F. majalis. We detect an unusual pattern of baseline transcriptome divergence, where neutral evolutionary processes appear to govern expression divergence within species, but patterns of divergence for these genes between species do not follow neutral expectations. During acclimation, genome expression profiling identifies mechanisms of acclimation-associated response that are conserved within the genus including regulation of paracellular permeability. In contrast, several responses vary among species including those putatively associated with cell volume regulation, and these same mechanisms are targets for adaptive physiological divergence along osmotic gradients within F. heteroclitus. As such, the genomic and physiological mechanisms that are associated with adaptive fine-tuning within species also contribute to macro-evolutionary divergence as species diversify across osmotic niches.

Independently evolved pollution resistance in four killifish populations is largely explained by few variants of large effect.

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human-altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts the influence of few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype-phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB-126. We found that a few large-effect QTL loci accounted for resistance to PCB-mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling, where some (but not all) of these QTL loci were shared across all populations, and some (but not all) of these loci showed signatures of recent natural selection in the corresponding wild population. Some strong candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB-126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB-126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex.

Salinity- and population-dependent genome regulatory response during osmotic acclimation in the killifish (Fundulus heteroclitus) gill.

The killifish Fundulus heteroclitus is abundant in osmotically dynamic estuaries and it can quickly adjust to extremes in environmental salinity. We performed a comparative osmotic challenge experiment to track the transcriptomic and physiological responses to two salinities throughout a time course of acclimation, and to explore the genome regulatory mechanisms that enable extreme osmotic acclimation. One southern and one northern coastal population, known to differ in their tolerance to hypo-osmotic exposure, were used as our comparative model. Both populations could maintain osmotic homeostasis when transferred from 32 to 0.4 p.p.t., but diverged in their compensatory abilities when challenged down to 0.1 p.p.t., in parallel with divergent transformation of gill morphology. Genes involved in cell volume regulation, nucleosome maintenance, ion transport, energetics, mitochondrion function, transcriptional regulation and apoptosis showed population- and salinity-dependent patterns of expression during acclimation. Network analysis confirmed the role of cytokine and kinase signaling pathways in coordinating the genome regulatory response to osmotic challenge, and also posited the importance of signaling coordinated through the transcription factor HNF-4α. These genome responses support hypotheses of which regulatory mechanisms are particularly relevant for enabling extreme physiological flexibility.

Evaluation of pituitary function after traumatic brain injury in childhood.

OBJECTIVES: Post-traumatic hypopituitarism is well described amongst adult traumatic brain injury (TBI) survivors. We aimed to determine the prevalence and clinical significance of pituitary dysfunction after head injury in childhood. DESIGN: Retrospective exploratory study. PATIENTS: 33 survivors of accidental head injury (27 boys). Mean (range) age at study was 13·4 years (5·4-21·7 years) and median (range) interval since injury 4·3 years (1·4-7·8 years). Functional outcome at study: 15 good recovery, 16 moderate disability, two severe disability. MEASUREMENTS: Early morning urine osmolality and basal hormone evaluation were followed by the gonadotrophin releasing hormone (GnRH) and insulin tolerance (n = 25) or glucagon tests (if previous seizures, n = 8). Subjects were not primed. Head injury details were extracted from patient records. RESULTS: No subject had short stature (mean height SD score +0·50, range -1·57 to +3·00). Suboptimal GH responses (<5 µg/l) occurred in six peri-pubertal boys (one with slow growth on follow-up) and one postpubertal adolescent (peak GH 3·2 µg/l). Median peak cortisol responses to insulin tolerance or glucagon tests were 538 and 562 nm. Nine of twenty-five and two of eight subjects had suboptimal responses, respectively, two with high basal cortisol levels. None required routine glucocorticoid replacement. In three, steroid cover was recommended for moderate/severe illness or injury. One boy was prolactin deficient. Other basal endocrine results and GnRH-stimulated LH and FSH were appropriate for age, sex and pubertal stage. Abnormal endocrine findings were unrelated to the severity or other characteristics of TBI or functional outcome. CONCLUSIONS: No clinically significant endocrinopathy was identified amongst survivors of accidental childhood TBI, although minor pituitary hormone abnormalities were observed.

Draft genome assemblies using sequencing reads from Oxford Nanopore Technology and Illumina platforms for four species of North American Fundulus killifish.

BACKGROUND: Whole-genome sequencing data from wild-caught individuals of closely related North American killifish species (Fundulus xenicus, Fundulus catenatus, Fundulus nottii, and Fundulus olivaceus) were obtained using long-read Oxford Nanopore Technology (ONT) PromethION and short-read Illumina platforms. FINDINGS: Draft de novo reference genome assemblies were generated using a combination of long and short sequencing reads. For each species, the PromethION platform was used to generate 30-45× sequence coverage, and the Illumina platform was used to generate 50-160× sequence coverage. Illumina-only assemblies were fragmented with high numbers of contigs, while ONT-only assemblies were error prone with low BUSCO scores. The highest N50 values, ranging from 0.4 to 2.7 Mb, were from assemblies generated using a combination of short- and long-read data. BUSCO scores were consistently >90% complete using the Eukaryota database. CONCLUSIONS: High-quality genomes can be obtained from a combination of using short-read Illumina data to polish assemblies generated with long-read ONT data. Draft assemblies and raw sequencing data are available for public use. We encourage use and reuse of these data for assembly benchmarking and other analyses.

Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin.

PURPOSE: Angiogenesis is a target for the treatment of cancer and other diseases, and its complex biology suggests that establishing the appropriate dose and schedule for antiangiogenic treatment will require extensive study. We present the initial results of a dose-finding clinical trial of recombinant human endostatin (rh-Endo) that examined potential surrogates for response to antiangiogenic therapy. PATIENTS AND METHODS: Twenty-five patients were treated with escalating doses of rh-Endo. Positron emission tomography (PET) was used to assess tumor blood flow (with [15O]H2O) and metabolism (with [18F]fluorodeoxyglucose) before the start of therapy and then every 4 weeks. To directly assess the effects of rh-Endo on endothelial cells within the tumors, biopsy specimens of tumor tissue were obtained before therapy and again at 8 weeks and evaluated for endothelial cell and tumor cell apoptosis. RESULTS: Tumor blood flow and metabolism as measured by PET scans generally decreased with increasing doses of rh-Endo; however, the effects were complex and in some analyses nonlinear. Tumor biopsy analysis revealed a significant increase in tumor cell apoptosis (P =.027) and endothelial cell apoptosis (P =.027) after 8 weeks of therapy. However, there was no statistically significant relationship between rh-Endo dose and induction of tumor cell or endothelial cell apoptosis. CONCLUSION: These initial data suggest that rh-Endo has measurable effects on tumor blood flow and metabolism and induces endothelial and tumor cell apoptosis even in the absence of demonstrable anticancer effects. Further study and validation of these biomarkers in the context of antiangiogenic therapy will be required.

Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin.

Several tumors, including mesothelioma and ovarian cancer, can overexpress mesothelin, a glycosylphosphatidylinositol-linked differentiation glycoprotein. The membrane-bound type of mesothelin is found in the blood of cancer patients at a very low level, which makes mesothelin a good candidate for targeted therapy of certain cancers. An antimesothelin disulfide-linked Fv (SS1 Fv) was fused to a truncated mutant of Pseudomonas exotoxin A to produce the recombinant immunotoxin SS1(dsFv)-PE38, which has a high binding affinity to mesothelin (Kd = 0.7 nM). Our studies in vitro showed that SS1(dsFv)-PE38 is significantly more cytotoxic to the high-mesothelin-producing NCI-H226 human non-small cell lung cancer cells than to human lung adenocarcinoma PC14PE6 cells, which do not express mesothelin. When administered at a nontoxic dose of 500 microg/kg on days 7, 9, and 11 to nude mice injected i.v. with the two human lung cancer cell lines, SS1(dsFv)-PE38 selectively inhibited experimental lung metastases produced by the mesothelin-producing NCI-H226 cells. Our data indicate that mesothelin-producing squamous cell carcinoma of the lung may be a good target for this immunotoxin.

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression.

BACKGROUND: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. RESULTS: We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism. CONCLUSION: The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at http://genomics.rsmas.miami.edu/funnybase/super_craw4/. Data and source code are available by request at jpaschall@bioinfobase.umkc.edu.

Assessment of antiangiogenic effect using 99mTc-EC-endostatin.

PURPOSE: Tumor vascular density may provide a prognostic indicator of metastatic potential or survival. The purpose of this study was to develop 99mTc-ethylenedicysteine-endostatin (99mTc-EC-endostatin) for the evaluation of anti-angiogenesis therapy. METHOD: 99mTc-EC-endostatin was prepared by conjugating ethylenedicysteine (EC) to endostatin, followed by adding pertechnetate and tin chloride. Radiochemical purity was > 95%. In vitro cell viability, affinity and TUNEL assays were performed. Tissue distribution and planar imaging of radiolabeled endostatin were determined in tumor-bearing rats. To assess anti-angiogenic treatment response, rats were treated with endostatin, paclitaxel and saline, followed by imaging with 99mTc-EC-endostatin. Tumor response to endostatin therapy in tumor-bearing animal models was assessed by correlating tumor uptake dose with microvessel density, VEGF, bFGF and IL-8 expression during endostatin therapy. RESULTS: In vitro cell viability and TUNEL assays indicated no marked difference between EC-endostatin and endostatin. Cellular uptake assay suggests that endostatin binds to endostatin receptor. Biodistribution of 99mTc-EC-endostatin in tumor-bearing rats showed increased tumor-to-tissue count density ratios as a function of time. Tumor uptake (%ID/g) of 99mTc-EC-endostatin was 0.2-0.5. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-endostatin. The optimal time for imaging using radiolabeled endostatin was 2 hrs. 99mTc-EC-endostatin could assess treatment response. There was a correlation between tumor uptake and cellular targets expression. CONCLUSION: The results indicate that it is feasible to use 99mTc-EC-endostatin to assess efficiency of anti-angiogenesis therapy.

Landscape influences on climate-related lake shrinkage at high latitudes.

Climate-related declines in lake area have been identified across circumpolar regions and have been characterized by substantial spatial heterogeneity. An improved understanding of the mechanisms underlying lake area trends is necessary to predict where change is most likely to occur and to identify implications for high latitude reservoirs of carbon. Here, using a population of ca. 2300 lakes with statistically significant increasing and decreasing lake area trends spanning longitudinal and latitudinal gradients of ca. 1000 km in Alaska, we present evidence for a mechanism of lake area decline that involves the loss of surface water to groundwater systems. We show that lakes with significant declines in lake area were more likely to be located: (1) in burned areas; (2) on coarser, well-drained soils; and (3) farther from rivers compared to lakes that were increasing. These results indicate that postfire processes such as permafrost degradation, which also results from a warming climate, may promote lake drainage, particularly in coarse-textured soils and farther from rivers where overland flooding is less likely and downslope flow paths and negative hydraulic gradients between surface water and groundwater systems are more common. Movement of surface water to groundwater systems may lead to a deepening of subsurface flow paths and longer hydraulic residence time which has been linked to increased soil respiration and CO2 release to the atmosphere. By quantifying relationships between statewide coarse resolution maps of landscape characteristics and spatially heterogeneous responses of lakes to environmental change, we provide a means to identify at-risk lakes and landscapes and plan for a changing climate.

Genetic variation among interconnected populations of Catostomus occidentalis: implications for distinguishing impacts of contaminants from biogeographical structuring.

Exposure to contaminants can affect survivorship, recruitment, reproductive success, mutation rates and migration, and may play a significant role in the partitioning of genetic variation among exposed and nonexposed populations. However, the application of molecular population genetic data to evaluate such influences has been uncommon and often flawed. We tested whether patterns of genetic variation among native fish populations (Sacramento sucker, Catostomus occidentalis) in the Central Valley of California were consistent with long-term pesticide exposure history, or primarily with expectations based on biogeography. Field sampling was designed to rigorously test for both geographical and contamination influences. Fine-scale structure of these interconnected populations was detected with both amplified fragment length polymorphisms (AFLP) and microsatellite markers, and patterns of variation elucidated by the two marker systems were highly concordant. Analyses indicated that biogeographical hypotheses described the data set better than hypotheses relating to common historical pesticide exposure. Downstream populations had higher genetic diversity than upstream populations, regardless of exposure history, and genetic distances showed that populations from the same river system tended to cluster together. Relatedness among populations reflected primarily directions of gene flow, rather than convergence among contaminant-exposed populations. Watershed geography accounted for significant partitioning of genetic variation among populations, whereas contaminant exposure history did not. Genetic patterns indicating contaminant-induced selection, increased mutation rates or recent bottlenecks were weak or absent. We stress the importance of testing contaminant-induced genetic change hypotheses within a biogeographical context. Strategic application of molecular markers for analysis of fine-scale structure, and for evaluating contaminant impacts on gene pools, is discussed.