A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Gene Expression Modularity Reveals Footprints of Polygenic Adaptation in Theobroma cacao.

Separating footprints of adaptation from demography is challenging. When selection has acted on a single locus with major effect, this issue can be alleviated through signatures left by selective sweeps. However, as adaptation is often driven by small allele frequency shifts at many loci, studies focusing on single genes are able to identify only a small portion of genomic variants responsible for adaptation. In face of this challenge, we utilize coexpression information to search for signals of polygenetic adaptation in Theobroma cacao, a tropical tree species that is the source of chocolate. Using transcriptomics and a weighted correlation network analysis, we group genes with similar expression patterns into functional modules. We then ask whether modules enriched for specific biological processes exhibit cumulative effects of differential selection in the form of high FST and dXY between populations. Indeed, modules putatively involved in protein modification, flowering, and water transport show signs of polygenic adaptation even though individual genes that are members of those groups do not bear strong signatures of selection. Modeling of demography, background selection, and the effects of genomic features reveal that these patterns are unlikely to arise by chance. We also find that specific modules are enriched for signals of strong or relaxed purifying selection, with one module bearing signs of adaptive differentiation and an excess of deleterious mutations. Our results provide insight into polygenic adaptation and contribute to understanding of population structure, demographic history, and genome evolution in T. cacao.

Resistance Genes Affect How Pathogens Maintain Plant Abundance and Diversity.

AbstractSpecialized pathogens are thought to maintain plant community diversity; however, most ecological studies treat pathogens as a black box. Here we develop a theoretical model to test how the impact of specialized pathogens changes when plant resistance genes (R-genes) mediate susceptibility. This work synthesizes two major hypotheses: the gene-for-gene model of pathogen resistance and the Janzen-Connell hypothesis of pathogen-mediated coexistence. We examine three scenarios. First, R-genes do not affect seedling survival; in this case, pathogens promote diversity. Second, seedlings are protected from pathogens when their R-gene alleles and susceptibility differ from those of nearby conspecific adults, thereby reducing transmission. If resistance is not costly, pathogens are less able to promote diversity because populations with low R-gene diversity suffer higher mortality, putting those populations at a disadvantage and potentially causing their exclusion. R-gene diversity may also be reduced during population bottlenecks, creating a priority effect. Third, when R-genes affect survival but resistance is costly, populations can avoid extinction by losing resistance alleles, as they cease paying a cost that is unneeded. Thus, the impact pathogens can have on tree diversity depends on the mechanism of plant-pathogen interactions. Future empirical studies should examine which of these scenarios most closely reflects the real world.

Host plant defense produces species-specific alterations to flight muscle protein structure and flight-related fitness traits of two armyworms.

Insects manifest phenotypic plasticity in their development and behavior in response to plant defenses, via molecular mechanisms that produce tissue-specific changes. Phenotypic changes might vary between species that differ in their preferred hosts and these effects could extend beyond larval stages. To test this, we manipulated the diet of southern armyworm (SAW; Spodoptera eridania) and fall armyworm (FAW; Spodoptera frugiperda) using a tomato mutant for jasmonic acid plant defense pathway (def1), and wild-type plants, and then quantified gene expression of Troponin t (Tnt) and flight muscle metabolism of the adult insects. Differences in Tnt spliceform ratios in insect flight muscles correlate with changes to flight muscle metabolism and flight muscle output. We found that SAW adults reared on induced def1 plants had a higher relative abundance (RA) of the A isoform of Troponin t (Tnt A) in their flight muscles; in contrast, FAW adults reared on induced def1 plants had a lower RA of Tnt A in their flight muscles compared with adults reared on def1 and controls. Although mass-adjusted flight metabolic rate showed no independent host plant effects in either species, higher flight metabolic rates in SAW correlated with increased RA of Tnt A Flight muscle metabolism also showed an interaction of host plants with Tnt A in both species, suggesting that host plants might be influencing flight muscle metabolic output by altering Tnt This study illustrates how insects respond to variation in host plant chemical defense by phenotypic modifications to their flight muscle proteins, with possible implications for dispersal.

Resistant and susceptible cacao genotypes exhibit defense gene polymorphism and unique early responses to Phytophthora megakarya inoculation.

KEY MESSAGE: Key genes potentially involved in cacao disease resistance were identified by transcriptomic analysis of important cacao cultivars. Defense gene polymorphisms were identified which could contribute to pathogen recognition capacity. Cacao suffers significant annual losses to the water mold Phytophthora spp. (Oomycetes). In West Africa, P. megakarya poses a major threat to farmer livelihood and the stability of cocoa production. As part of a long-term goal to define key disease resistance genes in cacao, here we use a transcriptomic analysis of the disease-resistant cacao clone SCA6 and the susceptible clone NA32 to characterize basal differences in gene expression, early responses to infection, and polymorphisms in defense genes. Gene expression measurements by RNA-seq along a time course revealed the strongest transcriptomic response 24 h after inoculation in the resistant genotype. We observed strong regulation of several pathogenesis-related genes, pattern recognition receptors, and resistance genes, which could be critical for the ability of SCA6 to combat infection. These classes of genes also showed differences in basal expression between the two genotypes prior to infection, suggesting that prophylactic expression of defense-associated genes could contribute to SCA6's broad-spectrum disease resistance. Finally, we analyzed polymorphism in a set of defense-associated receptors, identifying coding variants between SCA6 and NA32 which could contribute to unique capacities for pathogen recognition. This work is an important step toward characterizing genetic differences underlying a successful defense response in cacao.

Enzyme polymorphism, oxygen and injury: a lipidomic analysis of flight-induced oxidative damage in a succinate dehydrogenase d (Sdhd)-polymorphic insect.

When active tissues receive insufficient oxygen to meet metabolic demand, succinate accumulates and has two fundamental effects: it causes ischemia-reperfusion injury while also activating the hypoxia-inducible factor pathway (HIF). The Glanville fritillary butterfly (Melitaea cinxia) possesses a balanced polymorphism in Sdhd, shown previously to affect HIF pathway activation and tracheal morphology and used here to experimentally test the hypothesis that variation in succinate dehydrogenase affects oxidative injury. We stimulated butterflies to fly continuously in a respirometer (3 min duration), which typically caused episodes of exhaustion and recovery, suggesting a potential for cellular injury from hypoxia and reoxygenation in flight muscles. Indeed, flight muscle from butterflies flown on consecutive days had lipidome profiles similar to those of rested paraquat-injected butterflies, but distinct from those of rested untreated butterflies. Many butterflies showed a decline in flight metabolic rate (FMR) on day 2, and there was a strong inverse relationship between the ratio of day 2 to day 1 FMR and the abundance of sodiated adducts of phosphatidylcholines and co-enzyme Q (CoQ). This result is consistent with elevation of sodiated lipids caused by disrupted intracellular ion homeostasis in mammalian tissues after hypoxia-reperfusion. Butterflies carrying the Sdhd M allele had a higher abundance of lipid markers of cellular damage, but the association was reversed in field-collected butterflies, where focal individuals typically flew for seconds at a time rather than continuously. These results indicate that Glanville fritillary flight muscles can be injured by episodes of high exertion, but injury severity appears to be determined by an interaction between SDH genotype and behavior (prolonged versus intermittent flight).

Origin and diversification of wings: Insights from a neopteran insect.

Winged insects underwent an unparalleled evolutionary radiation, but mechanisms underlying the origin and diversification of wings in basal insects are sparsely known compared with more derived holometabolous insects. In the neopteran species Oncopeltus fasciatus, we manipulated wing specification genes and used RNA-seq to obtain both functional and genomic perspectives. Combined with previous studies, our results suggest the following key steps in wing origin and diversification. First, a set of dorsally derived outgrowths evolved along a number of body segments including the first thoracic segment (T1). Homeotic genes were subsequently co-opted to suppress growth of some dorsal flaps in the thorax and abdomen. In T1 this suppression was accomplished by Sex combs reduced, that when experimentally removed, results in an ectopic T1 flap similar to prothoracic winglets present in fossil hemipteroids and other early insects. Global gene-expression differences in ectopic T1 vs. T2/T3 wings suggest that the transition from flaps to wings required ventrally originating cells, homologous with those in ancestral arthropod gill flaps/epipods, to migrate dorsally and fuse with the dorsal flap tissue thereby bringing new functional gene networks; these presumably enabled the T2/T3 wing's increased size and functionality. Third, "fused" wings became both the wing blade and surrounding regions of the dorsal thorax cuticle, providing tissue for subsequent modifications including wing folding and the fit of folded wings. Finally, Ultrabithorax was co-opted to uncouple the morphology of T2 and T3 wings and to act as a general modifier of hindwings, which in turn governed the subsequent diversification of lineage-specific wing forms.

Locomotion in response to shifting climate zones: not so fast.

Although a species' locomotor capacity is suggestive of its ability to escape global climate change, such a suggestion is not necessarily straightforward. Species vary substantially in locomotor capacity, both ontogenetically and within/among populations, and much of this variation has a genetic basis. Accordingly, locomotor capacity can and does evolve rapidly, as selection experiments demonstrate. Importantly, even though this evolution of locomotor capacity may be rapid enough to escape changing climate, genetic correlations among traits (often due to pleiotropy) are such that successful or rapid dispersers are often limited in colonization or reproductive ability, which may be viewed as a trade-off. The nuanced assessment of this variation and evolution is reviewed for well-studied models: salmon, flying versus flightless insects, rodents undergoing experimental evolution, and metapopulations of butterflies. This work reveals how integration of physiology with population biology and functional genomics can be especially informative.

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells.

Cigarette smoke-induced protein aggregation damages the lung cells in emphysema and COPD; however, lung cancer cells continue to thrive, evolving to persist in the toxic environment. Here, we showed that upon the cigarette smoke condensate exposure, A549 lung cancer cells exhibit better survival and reduced level of protein aggregation when compared to non-cancerous Beas-2B and H-6053 cells. Our data suggests that upregulation of efflux pumps in cancer cells assists in reducing smoke toxicity. Specifically, we demonstrated that inhibition of the ABCG2 transporter in A549 by febuxostat or its downregulation by shRNA-mediated RNA interference resulted in a significant increase in protein aggregation due to smoke exposure.

Insect Flight: State of the Field and Future Directions.

The evolution of flight in an early winged insect ancestral lineage is recognized as a key adaptation explaining the unparalleled success and diversification of insects. Subsequent transitions and modifications to flight machinery, including secondary reductions and losses, also play a central role in shaping the impacts of insects on broadscale geographic and ecological processes and patterns in the present and future. Given the importance of insect flight, there has been a centuries-long history of research and debate on the evolutionary origins and biological mechanisms of flight. Here, we revisit this history from an interdisciplinary perspective, discussing recent discoveries regarding the developmental origins, physiology, biomechanics, and neurobiology and sensory control of flight in a diverse set of insect models. We also identify major outstanding questions yet to be addressed and provide recommendations for overcoming current methodological challenges faced when studying insect flight, which will allow the field to continue to move forward in new and exciting directions. By integrating mechanistic work into ecological and evolutionary contexts, we hope that this synthesis promotes and stimulates new interdisciplinary research efforts necessary to close the many existing gaps about the causes and consequences of insect flight evolution.

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes.

Nucleotide polymorphism at a gene (Pgi) under balancing selection in a butterfly metapopulation.

The Glanville fritillary butterfly (Melitaea cinxia, Nymphalidae) has a large, well-studied metapopulation in the Aland Islands in Finland. Previous studies have found that the common allozyme genotypes at the phosphoglucose isomerase (PGI) locus are associated with individual variation in performance and fitness, with phenotypic data suggesting ongoing balancing selection via heterozygote advantage. Here, we analyze nucleotide polymorphism in the coding region of the Pgi gene. Pgi is exceptionally polymorphic, in contrast to three other metabolic genes (Mdh, Idh, and Gapdh) with low levels of polymorphism. Most of the variation is due to two common haplotype clades, which are highly divergent and exhibit extensive linkage disequilibrium. These two clades correspond to the two most common allozyme alleles previously studied. Molecular tests of selection and coalescence simulations indicate that patterns of nucleotide polymorphism depart from neutrality and are consistent with long-term balancing selection. The split between the two main haplotype clades is estimated to predate the last common ancestor of a clade of five extant Melitaea species. Comparative structural analysis of Pgi polymorphism in M. cinxia and the unrelated Colias eurytheme butterfly suggests a similar but not identical target of balancing selection. Our results indicate convergent evolution between these two species at both the phenotypic and molecular levels.

Functional genomics of life history variation in a butterfly metapopulation.

In fragmented landscapes, small populations frequently go extinct and new ones are established with poorly understood consequences for genetic diversity and evolution of life history traits. Here, we apply functional genomic tools to an ecological model system, the well-studied metapopulation of the Glanville fritillary butterfly. We investigate how dispersal and colonization select upon existing genetic variation affecting life history traits by comparing common-garden reared 2-day adult females from new populations with those from established older populations. New-population females had higher expression of abdomen genes involved in egg provisioning and thorax genes involved in the maintenance of flight muscle proteins. Physiological studies confirmed that new-population butterflies have accelerated egg maturation, apparently regulated by higher juvenile hormone titer and angiotensin converting enzyme mRNA, as well as enhanced flight metabolism. Gene expression varied between allelic forms of two metabolic genes (Pgi and Sdhd), which themselves were associated with differences in flight metabolic rate, population age and population growth rate. These results identify likely molecular mechanisms underpinning life history variation that is maintained by extinction-colonization dynamics in metapopulations.

Body weight-dependent troponin T alternative splicing is evolutionarily conserved from insects to mammals and is partially impaired in skeletal muscle of obese rats.

Do animals know at a physiological level how much they weigh, and, if so, do they make homeostatic adjustments in response to changes in body weight? Skeletal muscle is a likely tissue for such plasticity, as weight-bearing muscles receive mechanical feedback regarding body weight and consume ATP in order to generate forces sufficient to counteract gravity. Using rats, we examined how variation in body weight affected alternative splicing of fast skeletal muscle troponin T (Tnnt3), a component of the thin filament that regulates the actin-myosin interaction during contraction and modulates force output. In response to normal growth and experimental body weight increases, alternative splicing of Tnnt3 in rat gastrocnemius muscle was adjusted in a quantitative fashion. The response depended on weight per se, as externally attached loads had the same effect as an equal change in actual body weight. Examining the association between Tnnt3 alternative splicing and ATP consumption rate, we found that the Tnnt3 splice form profile had a significant association with nocturnal energy expenditure, independently of effects of weight. For a subset of the Tnnt3 splice forms, obese Zucker rats failed to make the same adjustments; that is, they did not show the same relationship between body weight and the relative abundance of five Tnnt3 ß splice forms (i.e. Tnnt3 ß2-ß5 and ß8), four of which showed significant effects on nocturnal energy expenditure in Sprague-Dawley rats. Heavier obese Zucker rats displayed certain splice form relative abundances (e.g. Tnnt3 ß3) characteristic of much lighter, lean animals, resulting in a mismatch between body weight and muscle molecular composition. Consequently, we suggest that body weight-inappropriate skeletal muscle Tnnt3 expression in obesity is a candidate mechanism for muscle weakness and reduced mobility. Weight-dependent quantitative variation in Tnnt3 alternative splicing appears to be an evolutionarily conserved feature of skeletal muscle and provides a quantitative molecular marker to track how an animal perceives and responds to body weight.

Alleles in metabolic and oxygen-sensing genes are associated with antagonistic pleiotropic effects on life history traits and population fitness in an ecological model insect.

Genes with opposing effects on fitness at different life stages are the mechanistic basis for evolutionary theories of aging and life history. Examples come from studies of mutations in model organisms, but there is little knowledge of genetic bases of life history tradeoffs in natural populations. Here, we test the hypothesis that alleles affecting oxygen sensing in Glanville fritillary butterflies have opposing effects on larval versus adult fitness-related traits. Intermediate-frequency alleles in Succinate dehydrogenase d, and to a lesser extent Hypoxia inducible factor 1α, are associated in larvae with variation in metabolic rate and activation of the hypoxia inducible factor (HIF) pathway, which affects tracheal development and delivery of oxygen to adult flight muscles. A dominant Sdhd allele is likely to cause antagonistic pleiotropy for fitness through its opposing effects on larval metabolic and growth rate versus adult flight and dispersal, and may have additional effects arising from sensitivity to low-iron host plants. Prior results in Glanville fritillaries indicate that fitness of alleles in Sdhd and another antagonistically pleiotropic metabolic gene, Phosphoglucose isomerase, depend strongly on the size and distribution of host plant patches. Hence, these intermediate-frequency alleles are involved in ecoevolutionary dynamics involving life history tradeoffs.

Discovery of antitumor lectins from rainforest tree root transcriptomes.

Glycans are multi-branched sugars that are displayed from lipids and proteins. Through their diverse polysaccharide structures they can potentiate a myriad of cellular signaling pathways involved in development, growth, immuno-communication and survival. Not surprisingly, disruption of glycan synthesis is fundamental to various human diseases; including cancer, where aberrant glycosylation drives malignancy. Here, we report the discovery of a novel mannose-binding lectin, ML6, which selectively recognizes and binds to these irregular tumor-specific glycans to elicit potent and rapid cancer cell death. This lectin was engineered from gene models identified in a tropical rainforest tree root transcriptome and is unusual in its six canonical mannose binding domains (QxDxNxVxY), each with a unique amino acid sequence. Remarkably, ML6 displays antitumor activity that is >105 times more potent than standard chemotherapeutics, while being almost completely inactive towards non-transformed, healthy cells. This activity, in combination with results from glycan binding studies, suggests ML6 differentiates healthy and malignant cells by exploiting divergent glycosylation pathways that yield naïve and incomplete cell surface glycans in tumors. Thus, ML6 and other high-valence lectins may serve as novel biochemical tools to elucidate the glycomic signature of different human tumors and aid in the rational design of carbohydrate-directed therapies. Further, understanding how nature evolves proteins, like ML6, to combat the changing defenses of competing microorganisms may allow for fundamental advances in the way we approach combinatorial therapies to fight therapeutic resistance in cancer.

Enhanced heat tolerance of viral-infected aphids leads to niche expansion and reduced interspecific competition.

Vector-borne pathogens are known to alter the phenotypes of their primary hosts and vectors, with implications for disease transmission as well as ecology. Here we show that a plant virus, barley yellow dwarf virus, increases the surface temperature of infected host plants (by an average of 2 °C), while also significantly enhancing the thermal tolerance of its aphid vector Rhopalosiphum padi (by 8 °C). This enhanced thermal tolerance, which was associated with differential upregulation of three heat-shock protein genes, allowed aphids to occupy higher and warmer regions of infected host plants when displaced from cooler regions by competition with a larger aphid species, R. maidis. Infection thereby led to an expansion of the fundamental niche of the vector. These findings show that virus effects on the thermal biology of hosts and vectors can influence their interactions with one another and with other, non-vector organisms.

Flight metabolic rate and Pgi genotype influence butterfly dispersal rate in the field.

Dispersal is a key life-history trait, especially in species inhabiting fragmented landscapes. The process of dispersal is affected by a suite of morphological, physiological, and behavioral traits, all of which have a more or less complex genetic basis and are affected by the prevailing environmental conditions. To be able to identify genetic and phenotypic effects on dispersal, movements have to be recorded over relevant spatial and temporal scales. We used harmonic radar to track free-flying Glanville fritillary butterflies (Melitaea cinxia) released in the field and reconstructed their flight tracks for several hours. Flight track lengths for individual butterflies ranged from tens of meters to several kilometers. Butterflies were most mobile at midday and in intermediate temperatures. Flight metabolic rate (MR), measured prior to the tracking, explained variation in mobility at all scales studied. One-third of the variation in the distance moved in one hour could be attributed to variation in flight MR. Heterozygous individuals at a single nucleotide polymorphism in the phosphoglucose isomerase (Pgi) gene moved longer distances in the morning and at lower ambient temperatures than homozygous individuals. A similar genotype x temperature interaction was found to affect the metabolic rate. Our results establish connections from molecular variation in a single gene to flight physiology and movement behavior at the landscape level. These results indicate a fitness advantage to the heterozygous genotype in low temperatures and suggest a mechanism by which varying environmental conditions maintain genetic polymorphism in populations.

Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.

Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.

Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing.

We present a de novo assembly of a eukaryote transcriptome using 454 pyrosequencing data. The Glanville fritillary butterfly (Melitaea cinxia; Lepidoptera: Nymphalidae) is a prominent species in population biology but had no previous genomic data. Sequencing runs using two normalized complementary DNA collections from a genetically diverse pool of larvae, pupae, and adults yielded 608,053 expressed sequence tags (mean length = 110 nucleotides), which assembled into 48,354 contigs (sets of overlapping DNA segments) and 59,943 singletons. BLAST comparisons confirmed the accuracy of the sequencing and assembly, and indicated the presence of c. 9000 unique genes, along with > 6000 additional microarray-confirmed unannotated contigs. Average depth of coverage was 6.5-fold for the longest 4800 contigs (348-2849 bp in length), sufficient for detecting large numbers of single nucleotide polymorphisms. Oligonucleotide microarray probes designed from the assembled sequences showed highly repeatable hybridization intensity and revealed biological differences among individuals. We conclude that 454 sequencing, when performed to provide sufficient coverage depth, allows de novo transcriptome assembly and a fast, cost-effective, and reliable method for development of functional genomic tools for nonmodel species. This development narrows the gap between approaches based on model organisms with rich genetic resources vs. species that are most tractable for ecological and evolutionary studies.