Getting a Full Dose? Reconsidering Sex Chromosome Dosage Compensation in the Silkworm, Bombyx mori

Dosage compensation—equalizing gene expression levels in response to differences in gene dose or copy number—is classically considered to play a critical role in the evolution of heteromorphic sex chromosomes. As the X and Y diverge through degradation and gene loss on the Y (or the W in female-heterogametic ZW taxa), it is expected that dosage compensation will evolve to correct for sex-specific differences in gene dose. Although this is observed in some organisms, recent genome-wide expression studies in other taxa have revealed striking exceptions. In particular, reports that both birds and the silkworm moth (Bombyx mori) lack dosage compensation have spurred speculation that this is the rule for all female-heterogametic taxa. Here, we revisit the issue of dosage compensation in silkworm by replicating and extending the previous analysis. Contrary to previous reports, our efforts reveal a pattern typically associated with dosage compensated taxa: the global male:female expression ratio does not differ between the Z and autosomes. We believe the previous report of unequal male:female ratios on the Z reflects artifacts of microarray normalization in conjunction with not testing a major assumption that the male:female global expression ratio was unbiased for autosomal loci. However, we also find that the global Z chromosome expression is significantly reduced relative to autosomes, a pattern not expected in dosage compensated taxa. This combination of male:female parity with an overall reduction in expression for sex-linked loci is not consistent with the prevailing evolutionary theory of sex chromosome evolution and dosage compensation

EST analysis of male accessory glands from Heliconius butterflies with divergent mating systems

<p>Abstract</p> <p>Background</p> <p><it>Heliconius </it>butterflies possess a remarkable diversity of phenotypes, physiologies, and behaviors that has long distinguished this genus as a focal taxon in ecological and evolutionary research. Recently <it>Heliconius </it>has also emerged as a model system for using genomic methods to investigate the causes and consequences of biological diversity. One notable aspect of <it>Heliconius </it>diversity is a dichotomy in mating systems which provides an unusual opportunity to investigate the relationship between sexual selection and the evolution of reproductive proteins. As a first step in pursuing this research, we report the generation and analysis of expressed sequence tags (ESTs) from the male accessory gland of <it>H. erato </it>and <it>H. melpomene</it>, species representative of the two mating systems present in the genus <it>Heliconius</it>.</p> <p>Results</p> <p>We successfully sequenced 933 ESTs clustering into 371 unigenes from <it>H. erato </it>and 1033 ESTs clustering into 340 unigenes from <it>H. melpomene</it>. Results from the two species were very similar. Approximately a third of the unigenes showed no significant BLAST similarity (E-value <10^-5) to sequences in GenBank's non-redundant databases, indicating that a large proportion of novel genes are expressed in <it>Heliconius </it>male accessory glands. In both species only a third of accessory gland unigenes were also found among genes expressed in wing tissue. About 25% of unigenes from both species encoded secreted proteins. This includes three groups of highly abundant unigenes encoding repetitive proteins considered to be candidate seminal fluid proteins; proteins encoded by one of these groups were detected in <it>H. erato </it>spermatophores.</p> <p>Conclusion</p> <p>This collection of ESTs will serve as the foundation for the future identification and evolutionary analysis of male reproductive proteins in <it>Heliconius </it>butterflies. These data also represent a significant advance in the rapidly growing collection of genomic resources available in <it>Heliconius </it>butterflies. As such, they substantially enhance this taxon as a model system for investigating questions of ecological, phenotypic, and genomic diversity.</p

Conserved Patterns of Sex Chromosome Dosage Compensation in the Lepidoptera (WZ/ZZ): Insights from a Moth Neo-Z Chromosome

Where previously described, patterns of sex chromosome dosage compensation in the Lepidoptera (moths and butterflies) have several unusual characteristics. Other female-heterogametic (ZW/ZZ) species exhibit female Z-linked expression that is reduced compared with autosomal expression and male Z expression. In the Lepidoptera, however, Z expression typically appears balanced between sexes but overall reduced relative to autosomal expression, that is Z ≈ ZZ < AA. This pattern is not easily reconciled with theoretical expectations for the evolution of sex chromosome dosage compensation. Moreover, conflicting results linger due to discrepancies in data analyses and tissues sampled among lepidopterans. To address these issues, we performed RNA-seq to analyze sex chromosome dosage compensation in the codling moth, Cydia pomonella, which is a species from the earliest diverging lepidopteran lineage yet examined for dosage compensation and has a neo-Z chromosome resulting from an ancient Z:autosome fusion. While supported by intraspecific analyses, the Z ≈ ZZ < AA pattern was further evidenced by comparative study using autosomal orthologs of C. pomonella neo-Z genes in outgroup species. In contrast, dosage compensation appears to be absent in reproductive tissues. We thus argue that inclusion of reproductive tissues may explain the incongruence from a prior study on another moth species and that patterns of dosage compensation are likely conserved in the Lepidoptera. Notably, this pattern appears convergent with patterns in eutherian mammals (X ≈ XX < AA). Overall, our results contribute to the notion that the Lepidoptera present challenges both to classical theories regarding the evolution of sex chromosome dosage compensation and the emerging view of the association of dosage compensation with sexual heterogamety62174491398103-00514NSF-DEB 145775

Sex Chromosome Dosage Compensation in Heliconius Butterflies: Global yet Still Incomplete?

The evolution of heterogametic sex chromosomes is often-but not always-accompanied by the evolution of dosage compensating mechanisms that mitigate the impact of sex-specific gene dosage on levels of gene expression. One emerging view of this process is that such mechanisms may only evolve in male-heterogametic (XY) species but not in female-heterogametic (ZW) species, which will consequently exhibit "incomplete" sex chromosome dosage compensation. However, recent results suggest that at least some Lepidoptera (moths and butterflies) may prove to be an exception to this prediction. Studies in bombycoid moths indicate the presence of a chromosome-wide epigenetic mechanism that effectively balances Z chromosome gene expression between the sexes by reducing Z-linked expression in males. In contrast, strong sex chromosome dosage effects without any reduction in male Z-linked expression were previously reported in a pyralid moth, suggesting a lack of any such dosage compensating mechanism. Here we report an analysis of sex chromosome dosage compensation in Heliconius butterflies, sampling multiple individuals for several different adult tissues (head, abdomen, leg, mouth, and antennae). Methodologically, we introduce a novel application of linear mixed-effects models to assess dosage compensation, offering a unified statistical framework that can estimate effects specific to chromosome, to sex, and their interactions (i.e., a dosage effect). Our results show substantially reduced Z-linked expression relative to autosomes in both sexes, as previously observed in bombycoid moths. This observation is consistent with an increasing body of evidence that some lepidopteran species possess an epigenetic dosage compensating mechanism that reduces Z chromosome expression in males to levels comparable with females. However, this mechanism appears to be imperfect in Heliconius, resulting in a modest dosage effect that produces an average 5-20% increase in male expression relative to females on the Z chromosome, depending on the tissue. Thus our results in Heliconius reflect a mixture of previous patterns reported for Lepidoptera. In Heliconius, a moderate pattern of incomplete dosage compensation persists apparently despite the presence of an epigenetic dosage compensating mechanism. The chromosomal distributions of sex-biased genes show an excess of male-biased and a dearth of female-biased genes on the Z chromosome relative to autosomes, consistent with predictions of sexually antagonistic evolution.This research was supported in part by a NSF postdoctoral fellowshiptoJ.R.W. (DBI-0905698). RNA sequencing was funded by the “Capacity and Capability Challenge Program” from The Genome Analysis Centre, Norwich, UK. The computing for this project was performed on the Community Cluster at the Center for Research Computing at the University of Kansas. Luiqi (Aloy) Gu provided valuable comments on the manuscript.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/gbe/evv156

Sex Chromosome Dosage Compensation in Heliconius Butterflies: Global yet Still Incomplete?

The evolution of heterogametic sex chromosomes is often—but not always—accompanied by the evolution of dosage compensating mechanisms that mitigate the impact of sex-specific gene dosage on levels of gene expression. One emerging view of this process is that such mechanisms may only evolve in male-heterogametic (XY) species but not in female-heterogametic (ZW) species, which will consequently exhibit “incomplete” sex chromosome dosage compensation. However, recent results suggest that at least some Lepidoptera (moths and butterflies) may prove to be an exception to this prediction. Studies in bombycoid moths indicate the presence of a chromosome-wide epigenetic mechanism that effectively balances Z chromosome gene expression between the sexes by reducing Z-linked expression in males. In contrast, strong sex chromosome dosage effects without any reduction in male Z-linked expression were previously reported in a pyralid moth, suggesting a lack of any such dosage compensating mechanism. Here we report an analysis of sex chromosome dosage compensation in Heliconius butterflies, sampling multiple individuals for several different adult tissues (head, abdomen, leg, mouth, and antennae). Methodologically, we introduce a novel application of linear mixed-effects models to assess dosage compensation, offering a unified statistical framework that can estimate effects specific to chromosome, to sex, and their interactions (i.e., a dosage effect). Our results show substantially reduced Z-linked expression relative to autosomes in both sexes, as previously observed in bombycoid moths. This observation is consistent with an increasing body of evidence that some lepidopteran species possess an epigenetic dosage compensating mechanism that reduces Z chromosome expression in males to levels comparable with females. However, this mechanism appears to be imperfect in Heliconius, resulting in a modest dosage effect that produces an average 5–20% increase in male expression relative to females on the Z chromosome, depending on the tissue. Thus our results in Heliconius reflect a mixture of previous patterns reported for Lepidoptera. In Heliconius, a moderate pattern of incomplete dosage compensation persists apparently despite the presence of an epigenetic dosage compensating mechanism. The chromosomal distributions of sex-biased genes show an excess of male-biased and a dearth of female-biased genes on the Z chromosome relative to autosomes, consistent with predictions of sexually antagonistic evolution

Support for faster and more adaptive Z chromosome evolution in two divergent lepidopteran lineages^*

The rateof divergence for Z or X chromosomes is usually observed to be greater than autosomes, but the proposed evolutionary causes for this pattern vary, as do empirical results from diverse taxa. Even among moths and butterflies (Lepidoptera), which generally share a single‐origin Z chromosome, the handful of available studies give mixed support for faster or more adaptive evolution of the Z chromosome, depending on the species assayed. Here, we examine the molecular evolution of Z chromosomes in two additional lepidopteran species: the Carolina sphinx moth and the monarch butterfly, the latter of which possesses a recent chromosomal fusion yielding a segment of newly Z‐linked DNA. We find evidence for both faster and more adaptive Z chromosome evolution in both species, although this effect is strongest in the neo‐Z portion of the monarch sex chromosome. The neo‐Z is less male‐biased than expected of a Z chromosome, and unbiased and female‐biased genes drive the signal for adaptive evolution here. Together these results suggest that male‐biased gene accumulation and haploid selection have opposing effects on long‐term rates of adaptation and may help explain the discrepancies in previous findings as well as the repeated evolution of neo‐sex chromosomes in Lepidoptera

Investigation of the stability and 1.0 MeV proton radiation resistance of commercially produced hydrogenated amorphous silicon alloy solar cells

The radiation resistance of commercial solar cells fabricated from hydrogenated amorphous silicon alloys is reported. A number of different device structures were irradiated with 1.0 MeV protons. The cells were annealing at 200 C. The annealing time was dependent on proton fluence. Annealing devices for one hour restores cell parameters or fluences below 1(exp 14) cm(exp -2); fluences above 1(exp 14) cm(exp -2) require longer annealing times. A parametric fitting model was used to characterize current mechanisms observed in dark I-V measurements. The current mechanisms were explored with irradiation fluence, and voltage and light soaking times. The thermal generation current density and quality factor increased with proton fluence. Device simulation shows the degradation in cell characteristics may be explained by the reduction of the electric field in the intrinsic layer

Neo-sex Chromosomes in the Monarch Butterfly, Danaus plexippus

We report the discovery of a neo-sex chromosome in the monarch butterfly, Danaus plexippus, and several of its close relatives. Z-linked scaffolds in the D. plexippus genome assembly were identified via sex-specific differences in Illumina sequencing coverage. Additionally, a majority of the D. plexippus genome assembly was assigned to chromosomes based on counts of one-to-one orthologs relative to the butterfly Melitaea cinxia (with replication using two other lepidopteran species), in which genome scaffolds have been mapped to linkage groups. Sequencing coverage-based assessments of Z linkage combined with homology-based chromosomal assignments provided strong evidence for a Z-autosome fusion in the Danaus lineage, involving the autosome homologous to chromosome 21 in M. cinxia. Coverage analysis also identified three notable assembly errors resulting in chimeric Z-autosome scaffolds. Cytogenetic analysis further revealed a large W chromosome that is partially euchromatic, consistent with being a neo-W chromosome. The discovery of a neo-Z and the provisional assignment of chromosome linkage for >90% of D. plexippus genes lays the foundation for novel insights concerning sex chromosome evolution in this female-heterogametic model species for functional and evolutionary genomics

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty.This work was supported by the Balfour-Browne Fund administered by the Department of Zoology at the University of Cambridge. Additional support came from the University of Kansas.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.ibmb.2015.04.00

Evolutionary Proteomics Reveals Distinct Patterns of Complexity and Divergence between Lepidopteran Sperm Morphs

This article has been accepted for publication in Genome Biology and Evolution Published by Oxford University Press. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.Spermatozoa are one of the most strikingly diverse animal cell types. One poorly understood example of this diversity is sperm heteromorphism, where males produce multiple distinct morphs of sperm in a single ejaculate. Typically, only one morph is capable of fertilization and the function of the nonfertilizing morph, called parasperm, remains to be elucidated. Sperm heteromorphism has multiple independent origins, including Lepidoptera (moths and butterflies), where males produce a fertilizing eupyrene sperm and an apyrene parasperm, which lacks a nucleus and nuclear DNA. Here we report a comparative proteomic analysis of eupyrene and apyrene sperm between two distantly related lepidopteran species, the monarch butterfly (Danaus plexippus) and Carolina sphinx moth (Manduca sexta). In both species, we identified ∼700 sperm proteins, with half present in both morphs and the majority of the remainder observed only in eupyrene sperm. Apyrene sperm thus have a distinctly less complex proteome. Gene ontology (GO) analysis revealed proteins shared between morphs tend to be associated with canonical sperm cell structures (e.g., flagellum) and metabolism (e.g., ATP production). GO terms for morph-specific proteins broadly reflect known structural differences, but also suggest a role for apyrene sperm in modulating female neurobiology. Comparative analysis indicates that proteins shared between morphs are most conserved between species as components of sperm, whereas morph-specific proteins turn over more quickly, especially in apyrene sperm. The rapid divergence of apyrene sperm content is consistent with a relaxation of selective constraints associated with fertilization and karyogamy. On the other hand, parasperm generally exhibit greater evolutionary lability, and our observations may therefore reflect adaptive responses to shifting regimes of sexual selection.National Science Foundation award OAC-1541396/ACI-1541396NSF award OAC-1541396/ACI-1541396Syracuse UniversityUniversity of KansasKU Gould Fellowship and National Science Foundation award DEB-1701931Syracuse University FellowshipMarilyn Kerr Fellowshi