Autorecoding A-to-I RNA editing sites in the Adar gene underwent compensatory gains and losses in major insect clades.

As one of the most prevalent RNA modifications in animals, adenosine-to-inosine (A-to-I) RNA editing facilitates the environmental adaptation of organisms by diversifying the proteome in a temporal-spatial manner. In flies and bees, the editing enzyme Adar has independently gained two different autorecoding sites that form an autofeedback loop, stabilizing the overall editing efficiency. This ensures cellular homeostasis by keeping the normal function of target genes. However, in a broader range of insects, the evolutionary dynamics and significance of this Adar autoregulatory mechanism are unclear. We retrieved the genomes of 377 arthropod species covering the five major insect orders (Hemiptera, Hymenoptera, Coleoptera, Diptera, and Lepidoptera) and aligned the Adar autorecoding sites across all genomes. We found that the two autorecoding sites underwent compensatory gains and losses during the evolution of two orders with the most sequenced species (Diptera and Hymenoptera), and that the two editing sites were mutually exclusive among them: One editable site is significantly linked to another uneditable site. This autorecoding mechanism of Adar could flexibly diversify the proteome and stabilize global editing activity. Many insects independently selected different autorecoding sites to achieve a feedback loop and regulate the global RNA editome, revealing an interesting phenomenon during evolution. Our study reveals the evolutionary force acting on accurate regulation of RNA editing activity in insects and thus deepens our understanding of the functional importance of RNA editing in environmental adaptation and evolution.

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera).

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

Convergent Adaptation of Ootheca Formation as a Reproductive Strategy in Polyneoptera.

Insects have evolved numerous adaptations and colonized diverse terrestrial environments. Several polyneopterans, including dictyopterans (cockroaches and mantids) and locusts, have developed oothecae, but little is known about the molecular mechanism, physiological function, and evolutionary significance of ootheca formation. Here, we demonstrate that the cockroach asymmetric colleterial glands produce vitellogenins, proline-rich protein, and glycine-rich protein as major ootheca structural proteins (OSPs) that undergo sclerotization and melanization for ootheca formation through the cooperative protocatechuic acid pathway and dopachrome and dopaminechrome subpathway. Functionally, OSP sclerotization and melanization prevent eggs from losing water at warm and dry conditions, and thus effectively maintain embryo viability. Dictyopterans and locusts convergently evolved vitellogenins, apolipoprotein D, and laminins as OSPs, whereas within Dictyoptera, cockroaches and mantids independently developed glycine-rich protein and fibroins as OSPs. Highlighting the ecological-evolutionary importance, convergent ootheca formation represents a successful reproductive strategy in Polyneoptera that promoted the radiation and establishment of cockroaches, mantids, and locusts.

Methylation patterns of Tf2 retrotransposons linked to rapid adaptive stress response in the brown planthopper (Nilaparvata lugens).

Transposable elements (TEs) exhibit vast diversity across insect orders and are one of the major factors driving insect evolution and speciation. Presence of TEs can be both beneficial and deleterious to their host. While it is well-established that TEs impact life-history traits, adaptations and survivability of insects under hostile environments, the influence of the ecological niche on TE-landscape remains unclear. Here, we analysed the dynamics of Tf2 retrotransposons in the brown planthopper (BPH), under environmental fluctuations. BPH, a major pest of rice, is found in almost all rice-growing ecosystems. We believe genome plasticity, attributed to TEs, has allowed BPH to adapt and colonise novel ecological niches. Our study revealed bimodal age-distribution for Tf2 elements in BPH, indicating the occurrence of two major transpositional events in its evolutionary history and their contribution in shaping BPH genome. While TEs can provide genome flexibility and facilitate adaptations, they impose massive load on the genome. Hence, we investigated the involvement of methylation in modulating transposition in BPH. We performed comparative analyses of the methylation patterns of Tf2 elements in BPH feeding on resistant- and susceptible-rice varieties, and also under pesticide stress, across different life-stages. Results confirmed that methylation, particularly in non-CG context, is involved in TE regulation and dynamics under stress. Furthermore, we observed differential methylation for BPH adults and nymphs, emphasising the importance of screening juvenile life-stages in understanding adaptive-stress-responses in insects. Collectively, this study enhances our understanding of the role of transposons in influencing the evolutionary trajectory and survival strategies of BPH across generations.

High disparity in repellent gland anatomy across major lineages of stick and leaf insects (Insecta: Phasmatodea).

BACKGROUND: Phasmatodea are well known for their ability to disguise themselves by mimicking twigs, leaves, or bark, and are therefore commonly referred to as stick and leaf insects. In addition to this and other defensive strategies, many phasmatodean species use paired prothoracic repellent glands to release defensive chemicals when disturbed by predators or parasites. These glands are considered as an autapomorphic trait of the Phasmatodea. However, detailed knowledge of the gland anatomy and chemical compounds is scarce and only a few species were studied until now. We investigated the repellent glands for a global sampling of stick and leaf insects that represents all major phasmatodean lineages morphologically via µCT scans and analyzed the anatomical traits in a phylogenetic context. RESULTS: All twelve investigated species possess prothoracic repellent glands that we classify into four distinct gland types. 1: lobe-like glands, 2: sac-like glands without ejaculatory duct, 3: sac-like glands with ejaculatory duct and 4: tube-like glands. Lobe-like glands are exclusively present in Timema, sac-like glands without ejaculatory duct are only found in Orthomeria, whereas the other two types are distributed across all other taxa (= Neophasmatodea). The relative size differences of these glands vary significantly between species, with some glands not exceeding in length the anterior quarter of the prothorax, and other glands extending to the end of the metathorax. CONCLUSIONS: We could not detect any strong correlation between aposematic or cryptic coloration of the examined phasmatodeans and gland type or size. We hypothesize that a comparatively small gland was present in the last common ancestor of Phasmatodea and Euphasmatodea, and that the gland volume increased independently in subordinate lineages of the Occidophasmata and Oriophasmata. Alternatively, the stem species of Neophasmatodea already developed large glands that were reduced in size several times independently. In any case, our results indicate a convergent evolution of the gland types, which was probably closely linked to properties of the chemical components and different predator selection pressures. Our study is the first showing the great anatomical variability of repellent glands in stick and leaf insects.

The i5K Initiative: advancing arthropod genomics for knowledge, human health, agriculture, and the environment.

Insects and their arthropod relatives including mites, spiders, and crustaceans play major roles in the world's terrestrial, aquatic, and marine ecosystems. Arthropods compete with humans for food and transmit devastating diseases. They also comprise the most diverse and successful branch of metazoan evolution, with millions of extant species. Here, we describe an international effort to guide arthropod genomic efforts, from species prioritization to methodology and informatics. The 5000 arthropod genomes initiative (i5K) community met formally in 2012 to discuss a roadmap for sequencing and analyzing 5000 high-priority arthropods and is continuing this effort via pilot projects, the development of standard operating procedures, and training of students and career scientists. With university, governmental, and industry support, the i5K Consortium aspires to deliver sequences and analytical tools for each of the arthropod branches and each of the species having beneficial and negative effects on humankind.

Worker Reproduction and Caste Polymorphism Impact Genome Evolution and Social Genes Across the Ants.

Eusocial insects are characterized by several traits, including reproductive division of labor and caste polymorphisms, which likely modulate genome evolution. Concomitantly, evolution may act on specific genes and pathways underlying these novel, sociality-associated phenotypes. Reproductive division of labor should increase the magnitude of genetic drift and reduce the efficacy of selection by reducing effective population size. Caste polymorphism has been associated with relaxed selection and may facilitate directional selection on caste-specific genes. Here, we use comparative analyses of 22 ant genomes to test how reproductive division of labor and worker polymorphism influence positive selection and selection intensity across the genome. Our results demonstrate that worker reproductive capacity is associated with a reduction in the degree of relaxed selection but is not associated with any significant change to positive selection. We find decreases in positive selection in species with polymorphic workers, but no increase in the degree of relaxed selection. Finally, we explore evolutionary patterns in specific candidate genes associated with our focal traits in eusocial insects. Two oocyte patterning genes previously implicated in worker sterility evolve under intensified selection in species with reproductive workers. Behavioral caste genes generally experience relaxed selection associated with worker polymorphism, whereas vestigial and spalt, both associated with soldier development in Pheidole ants, experience intensified selection in worker polymorphic species. These findings expand our understanding of the genetic mechanisms underlying elaborations of sociality. The impacts of reproductive division of labor and caste polymorphisms on specific genes illuminate those genes' roles in generating complex eusocial phenotypes.

Cell-specific expression and individual function of prohormone convertase PC1/3 in Tribolium larval growth highlights major evolutionary changes between beetle and fly neuroendocrine systems.

BACKGROUND: The insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates, ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome. RESULTS: In order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles' viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient yolk to the developing oocytes. Larval RNAi against PC2 produced moulting defects where the larvae were not able to shed their old cuticle. This ecdysis phenotype was also observed in a small subset of PC1/3 knockdown larvae and was strongest in a double knockdown. Unexpectedly, most PC1/3-RNAi larvae showed strongly reduced growth, but went through larval moults despite minimal to zero weight gain. CONCLUSIONS: The cell type-specific expression of PC1/3 and its essential requirement for larval growth highlight the important role of this gene within the insect neuroendocrine system. Genomic conservation in most insect groups suggests that it has a comparable individual function in other insects as well, which has been replaced by alternative mechanisms in flies.

Evolution of Neuropeptide Precursors in Polyneoptera (Insecta).

Neuropeptides are among the structurally most diverse signaling molecules and participate in intercellular information transfer from neurotransmission to intrinsic or extrinsic neuromodulation. Many of the peptidergic systems have a very ancient origin that can be traced back to the early evolution of the Metazoa. In recent years, new insights into the evolution of these peptidergic systems resulted from the increasing availability of genome and transcriptome data which facilitated the investigation of the complete neuropeptide precursor sequences. Here we used a comprehensive transcriptome dataset of about 200 species from the 1KITE initiative to study the evolution of single-copy neuropeptide precursors in Polyneoptera. This group comprises well-known orders such as cockroaches, termites, locusts, and stick insects. Due to their phylogenetic position within the insects and the large number of old lineages, these insects are ideal candidates for studying the evolution of insect neuropeptides and their precursors. Our analyses include the orthologs of 21 single-copy neuropeptide precursors, namely ACP, allatotropin, AST-CC, AST-CCC, CCAP, CCHamide-1 and 2, CNMamide, corazonin, CRF-DH, CT-DH, elevenin, HanSolin, NPF-1 and 2, MS, proctolin, RFLamide, SIFamide, sNPF, and trissin. Based on the sequences obtained, the degree of sequence conservation between and within the different polyneopteran lineages is discussed. Furthermore, the data are used to postulate the individual neuropeptide sequences that were present at the time of the insect emergence more than 400 million years ago. The data confirm that the extent of sequence conservation across Polyneoptera is remarkably different between the different neuropeptides. Furthermore, the average evolutionary distance for the single-copy neuropeptides differs significantly between the polyneopteran orders. Nonetheless, the single-copy neuropeptide precursors of the Polyneoptera show a relatively high degree of sequence conservation. Basic features of these precursors in this very heterogeneous insect group are explained here in detail for the first time.

Evolution of Termite Symbiosis Informed by Transcriptome-Based Phylogenies.

Termitidae comprises ∼80% of all termite species [1] that play dominant decomposer roles in tropical ecosystems [2, 3]. Two major events during termite evolution were the loss of cellulolytic gut protozoans in the ancestor of Termitidae and the subsequent gain in the termitid subfamily Macrotermitinae of fungal symbionts cultivated externally in "combs" constructed within the nest [4, 5]. How these symbiotic transitions occurred remains unresolved. Phylogenetic analyses of mitochondrial data previously suggested that Macrotermitinae is the earliest branching termitid lineage, followed soon after by Sphaerotermitinae [6], which cultivates bacterial symbionts on combs inside its nests [7]. This has led to the hypothesis that comb building was an important evolutionary step in the loss of gut protozoa in ancestral termitids [8]. We sequenced genomes and transcriptomes of 55 termite species and reconstructed phylogenetic trees from up to 4,065 orthologous genes of 68 species. We found strong support for a novel sister-group relationship between the bacterial comb-building Sphaerotermitinae and fungus comb-building Macrotermitinae. This key finding indicates that comb building is a derived trait within Termitidae and that the creation of a comb-like "external rumen" involving bacteria or fungi may not have driven the loss of protozoa from ancestral termitids, as previously hypothesized. Instead, associations with gut prokaryotic symbionts, combined with dietary shifts from wood to other plant-based substrates, may have played a more important role in this symbiotic transition. Our phylogenetic tree provides a platform for future studies of comparative termite evolution and the evolution of symbiosis in this taxon.

Establishment of the mayfly Cloeon dipterum as a new model system to investigate insect evolution.

The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.

Leg deformation during imaginal ecdysis in the downy emerald, Cordulia aenea (Odonata, Corduliidae).

A dragonfly larva migrates from the water to the shore, perches on a plant stem and grasps it with strongly flexed legs. Adult legs inside the larval exoskeleton fit to the larval legs joint-to-joint. The adult emerges with stretched legs. During the molt, an imaginal leg must follow all the angles in exuvial joints. In turn, larval apodemes are withdrawn from imaginal legs. We visualized transient shapes of the imaginal legs by the instant fixation of insects at different moments of the molt, photographed isolated exuvial legs with the imaginal legs inside and then removed the exuvial sheath. Instant shapes of the imaginal tibia show sharp intrapodomere bends copying the angle in the larval femoro-tibial joint. The site of bending shifts distad during the molt. This is possible if the imaginal leg is pliable. The same problem of leg squeezing is also common in hemimetabolous insects as well as in other arthropods, whereas holometabolous insects overcome problems of a tight confinement either by using leg pliability in other ways but not squeezing (cyclorrhaphan flies, mosquitoes) or by pulling hardened legs out without change of their pupal zigzag configuration (moths, ants, honey bees). The pupal legs are not intended to grasp any external substrate.

The wing base of the palaeodictyopteran genus Dunbaria Tillyard: Where are we now?

The structure of insect wing articulation is considered as reliable source of high level characters for phylogenetic analyses. However, the correct identification of homologous structures among the main groups of Pterygota is a hotly debated issue. Therefore, the reconstruction of the wing bases in Paleozoic extinct relatives is of great interest, but at the same time it should be treated with extreme caution due to distortions caused by taphonomic effects. The present study is focused on the wing base in Dunbaria (Spilapteridae). The articulation in Dunbaria quinquefasciata is mainly formed by a prominent upright axillary plate while the humeral plate is markedly reduced. Due to unique preservation of surface relief of the axillary plate, its composition shows a detailed pattern of three fused axillary sclerites and presumable position of the sclerite 3Ax. The obtained structures were compared among Spilapteridae and to other palaeodictyopterans Ostrava nigra (Homoiopteridae) and Namuroningxia elegans (Namuroningxiidae). The comparative study uncovered two patterns of 3Ax in Dunbaria and Namuroningxia, which correspond to their different suprafamilial classification. In contrast to previous studies these new results reveal the homologous structural elements in the wing base between Paleozoic Palaeodictyoptera and their extant relatives of Ephemeroptera, Odonata and Neoptera.

Hold your breath beetle-Mites!

Respiratory gas exchange in insects occurs via a branching tracheal system. The entrances to the air-filled tracheae are the spiracles, which are gate-like structures in the exoskeleton. The open or closed state of spiracles defines the three possible gas exchange patterns of insects. In resting insects, spiracles may open and close over time in a repeatable fashion that results in a discontinuous gas exchange (DGE) pattern characterized by periods of zero organism-to-environment gas exchange. Several adaptive hypotheses have been proposed to explain why insects engage in DGE, but none have attracted overwhelming support. We provide support for a previously untested hypothesis that posits that DGE minimizes the risk of infestation of the tracheal system by mites and other agents. Here, we analyze the respiratory patterns of 15 species of ground beetle (Carabidae), of which more than 40% of individuals harbored external mites. Compared with mite-free individuals, infested one's engaged significantly more often in DGE. Mite-free individuals predominantly employed a cyclic or continuous gas exchange pattern, which did not include complete spiracle closure. Complete spiracle closure may prevent parasites from invading, clogging, or transferring pathogens to the tracheal system or from foraging on tissue not protected by thick chitinous layers.

The multifaceted activity of insect caspases.

Caspases are frequently considered synonymous with apoptotic cell death. Increasing evidence demonstrates that these proteases may exert their activities in non-apoptotic functions. The non-apoptotic roles of caspases may include developmentally regulated autophagy during insect metamorphosis, as well as neuroblast self-renewal and the immune response. Here, we summarize the established knowledge and the recent advances in the multiple roles of insect caspases to highlight their relevance for physiological processes and survival.

Peripheral olfactory signaling in insects.

Olfactory signaling is a crucial component in the life history of insects. The development of precise and parallel mechanisms to analyze the tremendous amount of chemical information from the environment and other sources has been essential to their evolutionary success. Considerable progress has been made in the study of insect olfaction fueled by bioinformatics- based utilization of genomics along with rapid advances in functional analyses. Here we review recent progress in our rapidly emerging understanding of insect peripheral sensory reception and signal transduction. These studies reveal that the nearly unlimited chemical space insects encounter is covered by distinct chemosensory receptor repertoires that are generally derived by species-specific, rapid gene gain and loss, reflecting the evolutionary consequences of adaptation to meet their specific biological needs. While diverse molecular mechanisms have been put forth, often in the context of controversial models, the characterization of the ubiquitous, highly conserved and insect-specific Orco odorant receptor co-receptor has opened the door to the design and development of novel insect control methods to target agricultural pests, disease vectors and even nuisance insects.

U12-type spliceosomal introns of Insecta.

Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.

Early axonogenesis in the embryo of a primitive insect, the silverfish Ctenolepisma longicaudata.

The pattern of axon growth from the population of neurons that pioneers the major axon pathways in the central nervous system is highly conserved in winged insects. This study sought to determine whether the same pattern of axon growth is shared by an apterygotic insect, the silverfish. We have found that homologues to at least nine early differentiating winged insect neurons are present in the silverfish. The axon trajectories and the sequence of axon outgrowth from these neurons are very similar in silverfish and winged insects, suggesting that the pterygotic and apterygotic insects share a common developmental Bauplan for the construction of the central nervous system. Some of these neurons do show differences in several aspects of axon growth, including the relative timing of axonogenesis, the polarity of axon growth and the pattern of axon fasciculation. In addition, a major, early-appearing fascicle in the posterior commissure of the silverfish is pioneered by a neuron which does not appear to have an equivalent in the winged insects. These differences are similar in character to, albeit more pronounced than, differences previously reported between two winged insects, the fruitfly Drosophila and the grasshopper. Some of the features of early central axon growth, that set the silverfish embryo apart from the winged insects, are shared by crustacean embryos, providing support for the claim that insects and crustaceans share a common developmental Bauplan for the construction of central axonal pathways.