Major changes in domain arrangements are associated with the evolution of termites.

Domains as functional protein units and their rearrangements along the phylogeny can shed light on the functional changes of proteomes associated with the evolution of complex traits like eusociality. This complex trait is associated with sterile soldiers and workers, and long-lived, highly fecund reproductives. Unlike in Hymenoptera (ants, bees, and wasps), the evolution of eusociality within Blattodea, where termites evolved from within cockroaches, was accompanied by a reduction in proteome size, raising the question of whether functional novelty was achieved with existing rather than novel proteins. To address this, we investigated the role of domain rearrangements during the evolution of termite eusociality. Analysing domain rearrangements in the proteomes of three solitary cockroaches and five eusocial termites, we inferred more than 5,000 rearrangements over the phylogeny of Blattodea. The 90 novel domain arrangements that emerged at the origin of termites were enriched for several functions related to longevity, such as protein homeostasis, DNA repair, mitochondrial activity, and nutrient sensing. Many domain rearrangements were related to changes in developmental pathways, important for the emergence of novel castes. Along with the elaboration of social complexity, including permanently sterile workers and larger, foraging colonies, we found 110 further domain arrangements with functions related to protein glycosylation and ion transport. We found an enrichment of caste-biased expression and splicing within rearranged genes, highlighting their importance for the evolution of castes. Furthermore, we found increased levels of DNA methylation among rearranged compared to non-rearranged genes suggesting fundamental differences in their regulation. Our findings indicate the importance of domain rearrangements in the generation of functional novelty necessary for termite eusociality to evolve.

More effective transposon regulation in fertile, long-lived termite queens than in sterile workers.

Transposable elements (TEs) are mobile genetic sequences, which can cause the accumulation of genomic damage in the lifetime of an organism. The regulation of TEs, for instance via the piRNA-pathway, is an important mechanism to protect the integrity of genomes, especially in the germ-line where mutations can be transmitted to offspring. In eusocial insects, soma and germ-line are divided among worker and reproductive castes, so one may expect caste-specific differences in TE regulation to exist. To test this, we compared whole-genome levels of repeat element transcription in the fat body of female workers, kings and five different queen stages of the higher termite, Macrotermes natalensis. In this species, queens can live over 20 years, maintaining near maximum reproductive output, while sterile workers only live weeks. We found a strong, positive correlation between TE expression and the expression of neighbouring genes in all castes. However, we found substantially higher TE activity in workers than in reproductives. Furthermore, TE expression did not increase with age in queens, despite a sevenfold increase in overall gene expression, due to a significant upregulation of the piRNA-pathway in 20-year-old queens. Our results suggest a caste- and age-specific regulation of the piRNA-pathway has evolved in higher termites that is analogous to germ-line-specific activity in solitary organisms. In the fat body of these termite queens, an important metabolic tissue for maintaining their extreme longevity and reproductive output, an efficient regulation of TEs likely protects genome integrity, thus further promoting reproductive fitness even at high age.

Evidence for reduced immune gene diversity and activity during the evolution of termites.

The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well-studied immune systems of cells and individuals, little is known about the origins of immunity during the transition to eusociality, a major evolutionary transition comparable to the evolution of multicellular organisms from single-celled ancestors. We aimed to tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We find that key transitions in termite sociality are correlated with immune gene family contractions. In cross-species comparisons of immune gene expression, we find evidence for a caste-specific social defence system in termites, which appears to operate at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity may have entailed a fundamental reshaping of the immune system optimized for group over individual defence.

No obvious transcriptome-wide signature of indirect selection in termites.

The evolution of sterile helper castes in social insects implies selection on genes that underlie variation in this nonreproductive phenotype. These focal genes confer no direct fitness and are presumed to evolve through indirect fitness effects on the helper's reproducing relatives. This separation of a gene's phenotypic effect on one caste and its fitness effect on another suggests that genes for this and other forms of reproductive altruism are buffered from selection and will thus evolve closer to the neutral rate than genes directly selected for selfish reproduction. We test this hypothesis by comparing the strength of selection at loci associated in their expression with reproductive versus sterile castes in termites. Specifically, we gather caste-biased gene expression data from four termite transcriptomes and measure the global dN/dS ratio across gene sets and phylogenetic lineages. We find that the majority of examined orthologous gene groups show patterns of nucleotide substitution that are consistent with strong purifying selection and display little evidence for distinct signatures of direct versus indirect selection in reproductive and sterile castes. For one particular species (Reticulitermes flavipes), the strength of purifying selection is relaxed in a reproductive nymph-biased gene set, which opposes the nearly neutral idea. In other species, the synonymous rate (dS) alone was often found to be the highest in the sterile worker caste, suggesting a more subtle signature of indirect selection or an altogether different relationship between caste-biased expression and rates of molecular evolution.

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.

Comparative analysis of lincRNA in insect species.

BACKGROUND: The ever increasing availability of genomes makes it possible to investigate and compare not only the genomic complements of genes and proteins, but also of RNAs. One class of RNAs, the long noncoding RNAs (lncRNAs) and, in particular, their subclass of long intergenic noncoding RNAs (lincRNAs) have recently gained much attention because of their roles in regulation of important biological processes such as immune response or cell differentiation and as possible evolutionary precursors for protein coding genes. lincRNAs seem to be poorly conserved at the sequence level but at least some lincRNAs have conserved structural elements and syntenic genomic positions. Previous studies showed that transposable elements are a main contribution to the evolution of lincRNAs in mammals. In contrast, plant lincRNA emergence and evolution has been linked with local duplication events. However, little is known about their evolutionary dynamics in general and in insect genomes in particular. RESULTS: Here we compared lincRNAs between seven insect genomes and investigated possible evolutionary changes and functional roles. We find very low sequence conservation between different species and that similarities within a species are mostly due to their association with transposable elements (TE) and simple repeats. Furthermore, we find that TEs are less frequent in lincRNA exons than in their introns, indicating that TEs may have been removed by selection. When we analysed the predicted thermodynamic stabilities of lincRNAs we found that they are more stable than their randomized controls which might indicate some selection pressure to maintain certain structural elements. We list several of the most stable lincRNAs which could serve as prime candidates for future functional studies. We also discuss the possibility of de novo protein coding genes emerging from lincRNAs. This is because lincRNAs with high GC content and potentially with longer open reading frames (ORF) are candidate loci where de novo gene emergence might occur. CONCLUSION: The processes responsible for the emergence and diversification of lincRNAs in insects remain unclear. Both duplication and transposable elements may be important for the creation of new lincRNAs in insects.

Reproductive workers show queenlike gene expression in an intermediately eusocial insect, the buff-tailed bumble bee Bombus terrestris.

Bumble bees represent a taxon with an intermediate level of eusociality within Hymenoptera. The clear division of reproduction between a single founding queen and the largely sterile workers is characteristic for highly eusocial species, whereas the morphological similarity between the bumble bee queen and the workers is typical for more primitively eusocial hymenopterans. Also, unlike other highly eusocial hymenopterans, division of labour among worker subcastes is plastic and not predetermined by morphology or age. We conducted a differential expression analysis based on RNA-seq data from 11 combinations of developmental stage and caste to investigate how a single genome can produce the distinct castes of queens, workers and males in the buff-tailed bumble bee Bombus terrestris. Based on expression patterns, we found males to be the most distinct of all adult castes (2411 transcripts differentially expressed compared to nonreproductive workers). However, only relatively few transcripts were differentially expressed between males and workers during development (larvae: 71 and pupae: 162). This indicates the need for more distinct expression patterns to control behaviour and physiology in adults compared to those required to create different morphologies. Among female castes, reproductive workers and their nonreproductive sisters displayed differential expression in over ten times more transcripts compared to the differential expression found between reproductive workers and their mother queen. This suggests a strong shift towards a more queenlike behaviour and physiology when a worker becomes fertile. This contrasts with eusocial species where reproductive workers are more similar to nonreproductive workers than the queen.

Genomic signatures of eusocial evolution in insects.

The genomes of eusocial insects allow the production and regulation of highly distinct phenotypes, largely independent of genotype. Although rare, eusociality has evolved convergently in at least three insect orders (Hymenoptera, Blattodea and Coleoptera). Despite such disparate origins, eusocial phenotypes show remarkable similarity, exhibiting long-lived reproductives and short-lived sterile workers and soldiers. In this article, we review current knowledge on genomic signatures of eusocial evolution. We confirm that especially an increased regulatory complexity and the adaptive evolution of chemical communication are common to several origins of eusociality. Furthermore, colony life itself can shape genomes of divergent taxa in a similar manner. Future research should be geared towards generating more high-quality genomic resources, especially in hitherto understudied clades, such as ambrosia beetles and termites. The application of more sophisticated tools such as machine learning techniques may allow the detection of more subtle convergent genomic footprints of eusociality.

Disentangling specific and unspecific components of innate immune memory in a copepod-tapeworm system.

Evidence that the innate immune system can respond with forms of memory upon reinfection has been accumulating over the past few years. These phenomena of "immune priming" in invertebrates, and "trained immunity" in vertebrates, are contrary to previous belief that immune memory and specificity are restricted to the adaptive immune system. However, while trained immunity is usually a response with rather low specificity, immune priming has shown highly specific responses in certain species. To date, it is largely unknown how specificity in innate immune memory can be achieved in response to different parasite types. Here, we revisited a system where an exceptionally high degree of innate immune specificity had been demonstrated for the first time, consisting of the copepod Macrocyclops albidus and its natural parasite, the tapeworm Schistocephalus solidus. Using homologous (same family) vs. heterologous (different family) priming-challenge experiments, we first confirm that copepods exposed to the same parasite family benefit from reduced secondary infections. We further focused on exposed-but-not-infected copepods in primary exposure to employ a transcriptomic approach, distinguishing between immunity that was either specific or unspecific regarding the discrimination between tapeworm types. A weighted gene co-expression network (WGCN) revealed differences between specific and unspecific immunity; while both involved histone modification regulation, specific immunity involved gene-splicing factors, whereas unspecific immunity was primarily involved in metabolic shift. We found a functional enrichment in spliceosome in specific immunity, whereas oxidative phosphorylation and carbon metabolism were enriched in unspecific immunity. Our findings allow discrimination of specific and unspecific components of an innate immune memory, based on gene expression networks, and deepen our understanding of basic aspects of immune systems.

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.

Live-bearing cockroach genome reveals convergent evolutionary mechanisms linked to viviparity in insects and beyond.

Live birth (viviparity) has arisen repeatedly and independently among animals. We sequenced the genome and transcriptome of the viviparous Pacific beetle-mimic cockroach and performed comparative analyses with two other viviparous insect lineages, tsetse flies and aphids, to unravel the basis underlying the transition to viviparity in insects. We identified pathways undergoing adaptive evolution for insects, involved in urogenital remodeling, tracheal system, heart development, and nutrient metabolism. Transcriptomic analysis of cockroach and tsetse flies revealed that uterine remodeling and nutrient production are increased and the immune response is altered during pregnancy, facilitating structural and physiological changes to accommodate and nourish the progeny. These patterns of convergent evolution of viviparity among insects, together with similar adaptive mechanisms identified among vertebrates, highlight that the transition to viviparity requires changes in urogenital remodeling, enhanced tracheal and heart development (corresponding to angiogenesis in vertebrates), altered nutrient metabolism, and shifted immunity in animal systems.

Complex regulatory role of DNA methylation in caste- and age-specific expression of a termite.

The reproductive castes of eusocial insects are often characterized by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite Macrotermes natalensis. We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens.

Eusocial Transition in Blattodea: Transposable Elements and Shifts of Gene Expression.

(1) Unravelling the molecular basis underlying major evolutionary transitions can shed light on how complex phenotypes arise. The evolution of eusociality, a major evolutionary transition, has been demonstrated to be accompanied by enhanced gene regulation. Numerous pieces of evidence suggest the major impact of transposon insertion on gene regulation and its role in adaptive evolution. Transposons have been shown to be play a role in gene duplication involved in the eusocial transition in termites. However, evidence of the molecular basis underlying the eusocial transition in Blattodea remains scarce. Could transposons have facilitated the eusocial transition in termites through shifts of gene expression? (2) Using available cockroach and termite genomes and transcriptomes, we investigated if transposons insert more frequently in genes with differential expression in queens and workers and if those genes could be linked to specific functions essential for eusocial transition. (3) The insertion rate of transposons differs among differentially expressed genes and displays opposite trends between termites and cockroaches. The functions of termite transposon-rich queen- and worker-biased genes are related to reproduction and ageing and behaviour and gene expression, respectively. (4) Our study provides further evidence on the role of transposons in the evolution of eusociality, potentially through shifts in gene expression.

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

Gene Coexpression Network Reveals Highly Conserved, Well-Regulated Anti-Ageing Mechanisms in Old Ant Queens.

Evolutionary theories of ageing predict a reduction in selection efficiency with age, a so-called "selection shadow," due to extrinsic mortality decreasing effective population size with age. Classic symptoms of ageing include a deterioration in transcriptional regulation and protein homeostasis. Understanding how ant queens defy the trade-off between fecundity and lifespan remains a major challenge for the evolutionary theory of ageing. It has often been discussed that the low extrinsic mortality of ant queens, that are generally well protected within the nest by workers and soldiers, should reduce the selection shadow acting on old queens. We tested this by comparing strength of selection acting on genes upregulated in young and old queens of the ant, Cardiocondyla obscurior. In support of a reduced selection shadow, we find old-biased genes to be under strong purifying selection. We also analyzed a gene coexpression network (GCN) with the aim to detect signs of ageing in the form of deteriorating regulation and proteostasis. We find no evidence for ageing. In fact, we detect higher connectivity in old queens indicating increased transcriptional regulation with age. Within the GCN, we discover five highly correlated modules that are upregulated with age. These old-biased modules regulate several antiageing mechanisms such as maintenance of proteostasis, transcriptional regulation, and stress response. We observe stronger purifying selection on central hub genes of these old-biased modules compared with young-biased modules. These results indicate a lack of transcriptional ageing in old C. obscurior queens, possibly facilitated by strong selection at old age and well-regulated antiageing mechanisms.

Transcriptomic Signatures of Ageing Vary in Solitary and Social Forms of an Orchid Bee.

Eusocial insect queens are remarkable in their ability to maximize both fecundity and longevity, thus escaping the typical trade-off between these two traits. Several mechanisms have been proposed to underlie the remolding of the trade-off, such as reshaping of the juvenile hormone (JH) pathway, or caste-specific susceptibility to oxidative stress. However, it remains a challenge to disentangle the molecular mechanisms underlying the remolding of the trade-off in eusocial insects from caste-specific physiological attributes that have subsequently arisen. The socially polymorphic orchid bee Euglossa viridissima represents an excellent model to address the role of sociality per se in longevity as it allows direct comparisons of solitary and social individuals within a common genetic background. We investigated gene expression and JH levels in young and old bees from both solitary and social nests. We found 902 genes to be differentially expressed with age in solitary females, including genes involved in oxidative stress, versus only 100 genes in social dominant females, and 13 genes in subordinate females. A weighted gene coexpression network analysis further highlights pathways related to ageing in this species, including the target of rapamycin pathway. Eleven genes involved in translation, apoptosis, and DNA repair show concurrent age-related expression changes in solitary but not in social females, representing potential differences based on social status. JH titers did not vary with age or social status. Our results represent an important step in understanding the proximate mechanisms underlying the remodeling of the fecundity/longevity trade-off that accompanies the evolutionary transition from solitary life to eusociality.

Deleterious Mutation Accumulation in Arabidopsis thaliana Pollen Genes: A Role for a Recent Relaxation of Selection.

In many studies, sex-related genes have been found to evolve rapidly. We therefore expect plant pollen genes to evolve faster than sporophytic genes. In addition, pollen genes are expressed as haploids which can itself facilitate rapid evolution because recessive advantageous and deleterious alleles are not masked by dominant alleles. However, this mechanism is less straightforward to apply in the model plant species Arabidopsis thaliana. For 1 Myr, A. thaliana has been self-compatible, a life history switch that has caused: a reduction in pollen competition, increased homozygosity, and a dilution of masking in diploid expressed, sporophytic genes. In this study, we have investigated the relative strength of selection on pollen genes compared with sporophytic genes in A. thaliana. We present two major findings: 1) before becoming self-compatible, positive selection was stronger on pollen genes than sporophytic genes for A. thaliana and 2) current polymorphism data indicate that selection is weaker on pollen genes compared with sporophytic genes. This weaker selection on pollen genes can in part be explained by their higher tissue specificity, which in outbreeding plants can be outweighed by the effects of haploid expression and pollen competition. These results indicate that since A. thaliana has become self-compatible, selection on pollen genes has become more relaxed. This has led to higher polymorphism levels and a higher build-up of deleterious mutations in pollen genes compared with sporophytic genes.

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.

High-Speed "4D" Computational Microscopy of Bacterial Surface Motility.

Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven "twitching" motility and flagellum-driven "spinning" and "swarming" motility. Appendage-driven motility is controlled by molecular motors, and analysis of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, we combine electromagnetic field computation and statistical image analysis to generate 3D movies close to a surface at 5 ms time resolution using conventional inverted microscopes. We treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell's equations and compute the diffracted light intensities associated with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calculations between measured 2D microscopy images and a library of computed light intensities, we demonstrate that near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resolution. Comparison between computational reconstructions and detailed hydrodynamic calculations reveals that P. aeruginosa act like low Reynolds number spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN µm. Interestingly, our analysis reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.