Comparative Evolutionary Genomics in Insects.

Genome sequencing quality, in terms of both read length and accuracy, is constantly improving. By combining long-read sequencing technologies with various scaffolding techniques, chromosome-level genome assemblies are now achievable at an affordable price for non-model organisms. Insects represent an exciting taxon for studying the genomic underpinnings of evolutionary innovations, due to ancient origins, immense species-richness, and broad phenotypic diversity. Here we summarize some of the most important methods for carrying out a comparative genomics study on insects. We describe available tools and offer concrete tips on all stages of such an endeavor from DNA extraction through genome sequencing, annotation, and several evolutionary analyses. Along the way we describe important insect-specific aspects, such as DNA extraction difficulties or gene families that are particularly difficult to annotate, and offer solutions. We describe results from several examples of comparative genomics analyses on insects to illustrate the fascinating questions that can now be addressed in this new age of genomics research.

ZW sex-chromosome evolution and contagious parthenogenesis in Artemia brine shrimp.

Eurasian brine shrimp (genus Artemia) have closely related sexual and asexual lineages of parthenogenetic females, which produce rare males at low frequencies. Although they are known to have ZW chromosomes, these are not well characterized, and it is unclear whether they are shared across the clade. Furthermore, the underlying genetic architecture of the transmission of asexuality, which can occur when rare males mate with closely related sexual females, is not well understood. We produced a chromosome-level assembly for the sexual Eurasian species Artemia sinica and characterized in detail the pair of sex chromosomes of this species. We combined this new assembly with short-read genomic data for the sexual species Artemia sp. Kazakhstan and several asexual lineages of Artemia parthenogenetica, allowing us to perform an in-depth characterization of sex-chromosome evolution across the genus. We identified a small differentiated region of the ZW pair that is shared by all sexual and asexual lineages, supporting the shared ancestry of the sex chromosomes. We also inferred that recombination suppression has spread to larger sections of the chromosome independently in the American and Eurasian lineages. Finally, we took advantage of a rare male, which we backcrossed to sexual females, to explore the genetic basis of asexuality. Our results suggest that parthenogenesis is likely partly controlled by a locus on the Z chromosome, highlighting the interplay between sex determination and asexuality.

Transitions to asexuality and evolution of gene expression in Artemia brine shrimp.

While sexual reproduction is widespread among many taxa, asexual lineages have repeatedly evolved from sexual ancestors. Despite extensive research on the evolution of sex, it is still unclear whether this switch represents a major transition requiring major molecular reorganization, and how convergent the changes involved are. In this study, we investigated the phylogenetic relationship and patterns of gene expression of sexual and asexual lineages of Eurasian Artemia brine shrimp, to assess how gene expression patterns are affected by the transition to asexuality. We find only a few genes that are consistently associated with the evolution of asexuality, suggesting that this shift may not require an extensive overhauling of the meiotic machinery. While genes with sex-biased expression have high rates of expression divergence within Eurasian Artemia, neither female- nor male-biased genes appear to show unusual evolutionary patterns after sexuality is lost, contrary to theoretical expectations.

Sex-Biased Gene Expression and Dosage Compensation on the Artemia franciscana Z-Chromosome.

Males and females of Artemia franciscana, a crustacean commonly used in the aquarium trade, are highly dimorphic. Sex is determined by a pair of ZW chromosomes, but the nature and extent of differentiation of these chromosomes is unknown. Here, we characterize the Z chromosome by detecting genomic regions that show lower genomic coverage in female than in male samples, and regions that harbor an excess of female-specific SNPs. We detect many Z-specific genes, which no longer have homologs on the W, but also Z-linked genes that appear to have diverged very recently from their existing W-linked homolog. We assess patterns of male and female expression in two tissues with extensive morphological dimorphism, gonads, and heads. In agreement with their morphology, sex-biased expression is common in both tissues. Interestingly, the Z chromosome is not enriched for sex-biased genes, and seems to in fact have a mechanism of dosage compensation that leads to equal expression in males and in females. Both of these patterns are contrary to most ZW systems studied so far, making A. franciscana an excellent model for investigating the interplay between the evolution of sexual dimorphism and dosage compensation, as well as Z chromosome evolution in general.

Expansions of key protein families in the German cockroach highlight the molecular basis of its remarkable success as a global indoor pest.

The German cockroach, Blattella germanica, is a worldwide pest that infests buildings, including homes, restaurants, and hospitals, often living in unsanitary conditions. As a disease vector and producer of allergens, this species has major health and economic impacts on humans. Factors contributing to the success of the German cockroach include its resistance to a broad range of insecticides, immunity to many pathogens, and its ability, as an extreme generalist omnivore, to survive on most food sources. The recently published genome shows that B. germanica has an exceptionally high number of protein coding genes. In this study, we investigate the functions of the 93 significantly expanded gene families with the aim to better understand the success of B. germanica as a major pest despite such inhospitable conditions. We find major expansions in gene families with functions related to the detoxification of insecticides and allelochemicals, defense against pathogens, digestion, sensory perception, and gene regulation. These expansions might have allowed B. germanica to develop multiple resistance mechanisms to insecticides and pathogens, and enabled a broad, flexible diet, thus explaining its success in unsanitary conditions and under recurrent chemical control. The findings and resources presented here provide insights for better understanding molecular mechanisms that will facilitate more effective cockroach control.

Hemimetabolous genomes reveal molecular basis of termite eusociality.

Around 150 million years ago, eusocial termites evolved from within the cockroaches, 50 million years before eusocial Hymenoptera, such as bees and ants, appeared. Here, we report the 2-Gb genome of the German cockroach, Blattella germanica, and the 1.3-Gb genome of the drywood termite Cryptotermes secundus. We show evolutionary signatures of termite eusociality by comparing the genomes and transcriptomes of three termites and the cockroach against the background of 16 other eusocial and non-eusocial insects. Dramatic adaptive changes in genes underlying the production and perception of pheromones confirm the importance of chemical communication in the termites. These are accompanied by major changes in gene regulation and the molecular evolution of caste determination. Many of these results parallel molecular mechanisms of eusocial evolution in Hymenoptera. However, the specific solutions are remarkably different, thus revealing a striking case of convergence in one of the major evolutionary transitions in biological complexity.

Global Dosage Compensation Is Ubiquitous in Lepidoptera, but Counteracted by the Masculinization of the Z Chromosome.

While chromosome-wide dosage compensation of the X chromosome has been found in many species, studies in ZW clades have indicated that compensation of the Z is more localized and/or incomplete. In the ZW Lepidoptera, some species show complete compensation of the Z chromosome, while others lack full equalization, but what drives these inconsistencies is unclear. Here, we compare patterns of male and female gene expression on the Z chromosome of two closely related butterfly species, Papilio xuthus and Papilio machaon, and in multiple tissues of two moths species, Plodia interpunctella and Bombyx mori, which were previously found to differ in the extent to which they equalize Z-linked gene expression between the sexes. We find that, while some species and tissues seem to have incomplete dosage compensation, this is in fact due to the accumulation of male-biased genes and the depletion of female-biased genes on the Z chromosome. Once this is accounted for, the Z chromosome is fully compensated in all four species, through the up-regulation of Z expression in females and in some cases additional down-regulation in males. We further find that both sex-biased genes and Z-linked genes have increased rates of expression divergence in this clade, and that this can lead to fast shifts in patterns of gene expression even between closely related species. Taken together, these results show that the uneven distribution of sex-biased genes on sex chromosomes can confound conclusions about dosage compensation and that Z chromosome-wide dosage compensation is not only possible but ubiquitous among Lepidoptera.

De Novo Transcriptome Assembly and Sex-Biased Gene Expression in the Cyclical Parthenogenetic Daphnia galeata.

Daphnia species have become models for ecological genomics and exhibit interesting features, such as high phenotypic plasticity and a densely packed genome with many lineage-specific genes. They are also cyclic parthenogenetic, with alternating asexual and sexual cycles and environmental sex determination. Here, we present a de novo transcriptome assembly of over 32,000 D. galeata genes and use it to investigate gene expression in females and spontaneously produced males of two clonal lines derived from lakes in Germany and the Czech Republic. We find that only a low percentage (18%) of genes shows sex-biased expression and that there are many more female-biased gene (FBG) than male-biased gene (MBG). Furthermore, FBGs tend to be more conserved between species than MBGs in both sequence and expression. These patterns may be a consequence of cyclic parthenogenesis leading to a relaxation of purifying selection on MBGs. The two clonal lines show considerable differences in both number and identity of sex-biased genes, suggesting that they may have reproductive strategies differing in their investment in sexual reproduction. Orthologs of key genes in the sex determination and juvenile hormone pathways, which are thought to be important for the transition from asexual to sexual reproduction, are present in D. galeata and highly conserved among Daphnia species.

Variation in the X:Autosome Distribution of Male-Biased Genes among Drosophila melanogaster Tissues and Its Relationship with Dosage Compensation.

Genes that are expressed differently between males and females (sex-biased genes) often show a nonrandom distribution in their genomic location, particularly with respect to the autosomes and the X chromosome. Previous studies of Drosophila melanogaster found a general paucity of male-biased genes on the X chromosome, although this is mainly limited to comparisons of whole flies or body segments containing the reproductive organs. To better understand the chromosomal distribution of sex-biased genes in various tissues, we used a common analysis framework to analyze microarray and RNA sequence data comparing male and female gene expression in individual tissues (brain, Malpighian tubule, and gonads), composite structures (head and gonadectomized carcass), and whole flies. Although there are relatively few sex-biased genes in the brain, there is a strong and highly significant enrichment of male-biased genes on the X chromosome. A weaker enrichment of X-linked male-biased genes is seen in the head, suggesting that most of this signal comes from the brain. In all other tissues, there is either no departure from the random expectation or a significant paucity of male-biased genes on the X chromosome. The brain and head also differ from other tissues in that their male-biased genes are significantly closer to binding sites of the dosage compensation complex. We propose that the interplay of dosage compensation and sex-specific regulation can explain the observed differences between tissues and reconcile disparate results reported in previous studies.

Population- and sex-biased gene expression in the excretion organs of Drosophila melanogaster.

Within species, levels of gene expression typically vary greatly between tissues, sexes, individuals, and populations. To investigate gene expression variation between sexes and populations in a single somatic tissue, we performed a quantitative analysis of the Malpighian tubule transcriptome in adult males and females of Drosophila melanogaster derived from two distinct populations (one from sub-Saharan Africa and one from northern Europe). We identified 2308 genes that differed in expression between the sexes and 2474 genes that differed in expression between populations at a false discovery rate of 5%. We also identified more than 1000 genes that showed a sex-by-population interaction in their expression. The genes that differed in expression between sexes showed enrichment for a wide variety of functions, although only 55% of them overlapped with sex-biased genes identified in whole-fly studies. The genes expressed differentially between populations included several that were previously implicated in adaptive regulatory evolution, an excess of cytochrome P450 genes, and many genes that were not detected in previous studies of whole flies. Our results demonstrate that there is abundant intraspecific gene expression variation within in a single, somatic tissue and uncover new candidates for adaptive regulatory evolution between populations.

Molecular traces of alternative social organization in a termite genome.

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

Genome-wide transcriptomic responses of the seagrasses Zostera marina and Nanozostera noltii under a simulated heatwave confirm functional types.

Genome-wide transcription analysis between related species occurring in overlapping ranges can provide insights into the molecular basis underlying different ecological niches. The co-occurring seagrass species, Zostera marina and Nanozostera noltii, are found in marine coastal environments throughout the northern hemisphere. Z. marina is often dominant in subtidal environments and subjected to fewer temperature extremes compared to the predominately intertidal and more stress-tolerant N. noltii. We exposed plants of both species to a realistic heat wave scenario in a common-stress-garden experiment. Using RNA-seq (~7million reads/library), four Z. marina and four N. noltii libraries were compared representing northern (Denmark) and southern (Italy) locations within the co-occurring range of the species' European distribution. A total of 8977 expressed genes were identified, of which 78 were directly related to heat stress. As predicted, both species were negatively affected by the heat wave, but showed markedly different molecular responses. In Z. marina the heat response was similar across locations in response to the heatwave at 26°C, with a complex response in functions related to protein folding, synthesis of ribosomal chloroplast proteins, proteins involved in cell wall modification and heat shock proteins (HSPs). In N. noltii the heat response markedly differed between locations, while HSP genes were not induced in either population. Our results suggest that as coastal seawater temperatures increase, Z. marina will disappear along its southern most ranges, whereas N. noltii will continue to move north. As a consequence, sub- and intertidal habitat partitioning may weaken in more northern regions because the higher thermal tolerance of N. noltii provides a competitive advantage in both habitats. Although previous studies have focused on HSPs, the present study clearly demonstrates that a broader examination of stress related genes is necessary.

Identifying core features of adaptive metabolic mechanisms for chronic heat stress attenuation contributing to systems robustness.

The contribution of metabolism to heat stress may play a significant role in defining robustness and recovery of systems; either by providing the energy and metabolites required for cellular homeostasis, or through the generation of protective osmolytes. However, the mechanisms by which heat stress attenuation could be adapted through metabolic processes as a stabilizing strategy against thermal stress are still largely unclear. We address this issue through metabolomic and transcriptomic profiles for populations along a thermal cline where two seagrass species, Zostera marina and Zostera noltii, were found in close proximity. Significant changes captured by these profile comparisons could be detected, with a larger response magnitude observed in northern populations to heat stress. Sucrose, fructose, and myo-inositol were identified to be the most responsive of the 29 analyzed organic metabolites. Many key enzymes in the Calvin cycle, glycolysis and pentose phosphate pathways also showed significant differential expression. The reported comparison suggests that adaptive mechanisms are involved through metabolic pathways to dampen the impacts of heat stress, and interactions between the metabolome and proteome should be further investigated in systems biology to understand robust design features against abiotic stress.

How do new proteins arise?

Proteins are surreptitious cellular agents: while robust against mutations they are very evolvable; most are marginally stable and dynamic but others form a stable cellular matrix. Some genes seem to have emerged de novo from random pieces of genomic DNA, others may have been around for billions of years, virtually unchanged. Genomic and structural data provide new insights on how proteins came such a long way, probably from an initially very small set of domains and domain arrangements: gene duplicates provide the raw material for adaptive transitions (for example from one fold to another) which are very rare, albeit not impossible. 'New' proteins predominantly arise via tinkering, that is by their underlying genes recruiting and adapting smaller fragments of neighbouring DNA or by modular rearrangements of established domain combinations. Such rearrangements arise predominantly via fusion and terminal loss.