Impact of male trait exaggeration on sex-biased gene expression and genome architecture in a water strider.

BACKGROUND: Exaggerated secondary sexual traits are widespread in nature and often evolve under strong directional sexual selection. Although heavily studied from both theoretical and empirical viewpoints, we have little understanding of how sexual selection influences sex-biased gene regulation during the development of exaggerated secondary sexual phenotypes, and how these changes are reflected in genomic architecture. This is primarily due to the limited availability of representative genomes and associated tissue and sex transcriptomes to study the development of these traits. Here we present the genome and developmental transcriptomes, focused on the legs, of the water strider Microvelia longipes, a species where males exhibit strikingly long third legs compared to females, which they use as weapons. RESULTS: We generated a high-quality genome assembly with 90% of the sequence captured in 13 scaffolds. The most exaggerated legs in males were particularly enriched in both sex-biased and leg-biased genes, indicating a specific signature of gene expression in association with trait exaggeration. We also found that male-biased genes showed patterns of fast evolution compared to non-biased and female-biased genes, indicative of directional or relaxed purifying selection. By contrast to male-biased genes, female-biased genes that are expressed in the third legs, but not the other legs, are over-represented in the X chromosome compared to the autosomes. An enrichment analysis for sex-biased genes along the chromosomes revealed also that they arrange in large genomic regions or in small clusters of two to four consecutive genes. The number and expression of these enriched regions were often associated with the exaggerated legs of males, suggesting a pattern of common regulation through genomic proximity in association with trait exaggeration. CONCLUSION: Our findings indicate how directional sexual selection may drive sex-biased gene expression and genome architecture along the path to trait exaggeration and sexual dimorphism.

Sex allocation plasticity on a transcriptome scale: Socially sensitive gene expression in a simultaneous hermaphrodite.

Phenotypic plasticity can enable organisms to produce optimal phenotypes in multiple environments. A crucial life history trait that is often highly plastic is sex allocation, which in simultaneous hermaphrodites describes the relative investment into the male versus female sex functions. Theory predicts-and morphological evidence supports-that greater investment into the male function is favoured with increasing group size, due to the increasing importance of sperm competition for male reproductive success. Here, we performed a genome-wide gene expression assay to test for such sex allocation plasticity in a model simultaneous hermaphrodite, the free-living flatworm Macrostomum lignano. Based on RNA-Seq data from 16 biological replicates spanning four different group size treatments, we demonstrate that at least 10% of the >75,000 investigated transcripts in M. lignano are differentially expressed according to the social environment, rising to >30% of putative gonad-specific transcripts (spermatogenesis and oogenesis candidates) and tail-specific transcripts (seminal fluid candidates). This transcriptional response closely corresponds to the expected shift away from female and towards male reproductive investment with increasing sperm competition level. Using whole-mount in situ hybridization, we then confirm that many plastic transcripts exhibit the expected organ-specific expression, and RNA interference of selected testis- and ovary-specific candidates establishes that these indeed function in gametogenesis pathways. We conclude that a large proportion of sex-specific transcripts in M. lignano are differentially expressed according to the prevailing ecological conditions and that these are functionally relevant to key reproductive phenotypes. Our study thus begins to bridge organismal and molecular perspectives on sex allocation plasticity.

Environmental Sources of Bacteria and Genetic Variation in Behavior Influence Host-Associated Microbiota.

In many organisms, host-associated microbial communities are acquired horizontally after birth. This process is believed to be shaped by a combination of environmental and host genetic factors. We examined whether genetic variation in animal behavior could affect the composition of the animal's microbiota in different environments. The freshwater crustacean Daphnia magna is primarily planktonic but exhibits variation in the degree to which it browses in benthic sediments. We performed an experiment with clonal lines of D. magna showing different levels of sediment-browsing intensity exposed to either bacteria-rich or bacteria-poor sediment or whose access to sediments was prevented. We found that the bacterial composition of the environment and genotype-specific browsing intensity together influence the composition of the Daphnia-associated bacterial community. Exposure to more diverse bacteria did not lead to a more diverse microbiome, but greater abundances of environment-specific bacteria were found associated with host genotypes that exhibited greater browsing behavior. Our results indicate that, although there is a great deal of variation between individuals, behavior can mediate genotype-by-environment interaction effects on microbiome composition.IMPORTANCE An animal's behavior can affect its risk of infection, but it is not well understood how behavior affects microbiome composition. The aquatic crustacean Daphnia exhibits genetic variation in the extent to which it browses in the sediment at the bottoms of ponds. We show that this behavior affects the Daphnia microbiome, indicating that genetic variation among individuals may affect microbiome composition and the movement of bacteria in different environments.

Positional RNA-Seq identifies candidate genes for phenotypic engineering of sexual traits.

INTRODUCTION: RNA interference (RNAi) of trait-specific genes permits the manipulation of specific phenotypic traits ("phenotypic engineering") and thus represents a powerful tool to test trait function in evolutionary studies. The identification of suitable candidate genes, however, often relies on existing functional gene annotation, which is usually limited in emerging model organisms, especially when they are only distantly related to traditional genetic model organisms. A case in point is the free-living flatworm Macrostomum lignano (Lophotrochozoa: Platyhelminthes: Rhabditophora), an increasingly powerful model organism for evolutionary studies of sex in simultaneous hermaphrodites. To overcome the limitation of sparse functional annotation, we have performed a positional RNA-Seq analysis on different body fragments in order to identify organ-specific candidate transcripts. We then performed gene expression (in situ hybridization) and gene function (RNAi) analyses on 23 candidate transcripts, both to evaluate the predictive potential of this approach and to obtain preliminary functional characterizations of these candidate genes. RESULTS: We identified over 4000 transcripts that could be expected to show specific expression in different reproductive organs (including testis, ovary and the male and female genital systems). The predictive potential of the method could then be verified by confirming organ-specific expression for several candidate transcripts, some of which yielded interesting trait-specific knock-down phenotypes that can now be followed up in future phenotypic engineering studies. CONCLUSIONS: Our positional RNA-Seq analysis represents a highly useful resource for the identification of candidate transcripts for functional and phenotypic engineering studies in M. lignano, and it has already been used successfully in several studies. Moreover, this approach can overcome some inherent limitations of homology-based candidate selection and thus should be applicable to a broad range of emerging model organisms.

De novo transcriptome hybrid assembly and validation in the European earwig (Dermaptera, Forficula auricularia).

BACKGROUND: The European earwig (Forficula auricularia) is an established system for studies of sexual selection, social interactions and the evolution of parental care. Despite its scientific interest, little knowledge exists about the species at the genomic level, limiting the scope of molecular studies and expression analyses of genes of interest. To overcome these limitations, we sequenced and validated the transcriptome of the European earwig. METHODOLOGY AND PRINCIPAL FINDINGS: To obtain a comprehensive transcriptome, we sequenced mRNA from various tissues and developmental stages of female and male earwigs using Roche 454 pyrosequencing and Illumina HiSeq. The reads were de novo assembled independently and screened for possible microbial contamination and repeated elements. The remaining contigs were combined into a hybrid assembly and clustered to reduce redundancy. A comparison with the eukaryotic core gene dataset indicates that we sequenced a substantial part of the earwig transcriptome with a low level of fragmentation. In addition, a comparative analysis revealed that more than 8,800 contigs of the hybrid assembly show significant similarity to insect-specific proteins and those were assigned for Gene Ontology terms. Finally, we established a quantitative PCR test for expression stability using commonly used housekeeping genes and applied the method to five homologs of known sex-biased genes of the honeybee. The qPCR pilot study confirmed sex specific expression and also revealed significant expression differences between the brain and antenna tissue samples. CONCLUSIONS: By employing two different sequencing approaches and including samples obtained from different tissues, developmental stages, and sexes, we were able to assemble a comprehensive transcriptome of F. auricularia. The transcriptome presented here offers new opportunities to study the molecular bases and evolution of parental care and sociality in arthropods.

Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein.

BACKGROUND: Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. RESULTS: In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. CONCLUSION: Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.