Cave beetle lineages gained genes before going down under: An example of repeated genomic exaptation?

The adaptation of animals to subterranean habitats like caves and aquifers stereotypically leads to dramatic trait-loss consequences like the lack of eyes and body pigmentation. These body plan regression trends are expected to be tied to gene loss as well. Indeed, previous studies documented the degeneration of vision genes in obligate cave dwellers. Contradicting this picture, the first broad-scale comparative transcriptome-wide study of gene content evolution in separate subterranean Australian and Mediterranean beetle clades unearthed evidence of global gene gain and retention. This suggests that the transition to cave life may be more contingent on gene repertoire expansion than contraction. Future studies, however, will need to examine how much the observed patterns of gene content evolution reflect subfunctionalization and fitness-securing genetic redundancy outcomes following gene duplication as opposed to adaptive trajectories.

Close to complete conservation of the brachyceran opsin repertoire in the stalk-eyed fly Teleopsis dalmanni.

Due to the unique morphology of their adult visual system, stalk-eyed flies represent an important model of exaggerated trait evolution through sexual selection. Early physiological measurements indicated wavelength sensitivity peaks in the ultraviolet (360 nm), blue (450), blue-green (490 nm), and red (>550 nm) ranges in the compound eye retina of the stalk-eyed fly Teleopsis dalmanni, consistent with the trichromatic color and broad range motion detection vision system of brachyceran Diptera. A previous study of dipteran opsin gene diversification, however, detected only homologs of members of the long wavelength range sensitive opsin subfamilies Rh2 and Rh6 in T. dalmanni. Here, I report findings from analyzing the most recent T. dalmanni genome assembly, which revealed the conservation of most brachyceran opsin homologs except for the UV wavelength range-sensitive homolog Rh4. These results and other examples highlight the caution that needs to be applied to gene loss conclusions.

Parallel Losses of Blue Opsin Correlate with Compensatory Neofunctionalization of UV-Opsin Gene Duplicates in Aphids and Planthoppers.

Expanding on previous efforts to survey the visual opsin repertoires of the Hemiptera, this study confirms that homologs of the UV- and LW-opsin subfamilies are conserved in all Hemiptera, while the B-opsin subfamily is missing from the Heteroptera and subgroups of the Sternorrhyncha and Auchenorrhyncha, i.e., aphids (Aphidoidea) and planthoppers (Fulgoroidea), respectively. Unlike in the Heteroptera, which are characterized by multiple independent expansions of the LW-opsin subfamily, the lack of B-opsin correlates with the presence of tandem-duplicated UV-opsins in aphids and planthoppers. Available data on organismal wavelength sensitivities and retinal gene expression patterns lead to the conclusion that, in both groups, one UV-opsin paralog shifted from ancestral UV peak sensitivity to derived blue sensitivity, likely compensating for the lost B-opsin. Two parallel bona fide tuning site substitutions compare to 18 non-corresponding amino acid replacements in the blue-shifted UV-opsin paralogs of aphids and planthoppers. Most notably, while the aphid blue-shifted UV-opsin clade is characterized by a replacement substitution at one of the best-documented UV/blue tuning sites (Rhodopsin site 90), the planthopper blue-shifted UV-opsin paralogs retained the ancestral lysine at this position. Combined, the new findings identify aphid and planthopper UV-opsins as a new valuable data sample for studying adaptive opsin evolution.

Global Gene Expression Analysis Reveals Complex Cuticle Organization of the Tribolium Compound Eye.

The red flour beetle Tribolium castaneum is a resource-rich model for genomic and developmental studies. To extend previous studies on Tribolium eye development, we produced transcriptomes for normal-eyed and eye-depleted heads of pupae and adults to identify differentially transcript-enriched (DE) genes in the visual system. Unexpectedly, cuticle-related genes were the largest functional class in the pupal compound eye DE gene population, indicating differential enrichment in three distinct cuticle components: clear lens facet cuticle, highly melanized cuticle of the ocular diaphragm, which surrounds the Tribolium compound eye for internal fortification, and newly identified facet margins of the tanned cuticle, possibly enhancing external fortification. Phylogenetic, linkage, and high-throughput gene knockdown data suggest that most cuticle proteins (CPs) expressed in the Tribolium compound eye stem from the deployment of ancient CP genes. Consistent with this, TcasCPR15, which we identified as the major lens CP gene in Tribolium, is a beetle-specific but pleiotropic paralog of the ancient CPR RR-2 CP gene family. The less abundant yet most likely even more lens-specific TcasCP63 is a member of a sprawling family of noncanonical CP genes, documenting a role of local gene family expansions in the emergence of the Tribolium compound eye CP repertoire. Comparisons with Drosophila and the mosquito Anopheles gambiae reveal a steady turnover of lens-enriched CP genes during insect evolution.

Coming into clear sight at last: Ancestral and derived events during chelicerate visual system development.

Pioneering molecular work on chelicerate visual system development in the horseshoe crab Limulus polyphemus surprised with the possibility that this process may not depend on the deeply conserved retinal determination function of Pax6 transcription factors. Genomic, transcriptomic, and developmental studies in spiders now reveal that the arthropod Pax6 homologs eyeless and twin of eyeless act as ancestral determinants of the ocular head segment in chelicerates, which clarifies deep gene regulatory and structural homologies and recommends more unified terminologies in the comparison of arthropod visual systems. Following this phylotypic stage, chelicerate visual system development differs fundamentally from other arthropods during the compartmentalization of the ocular segment in that eye and optic neuropil primordia originate independently from each other. Comparative analyses of this phase identified further gene regulatory homologies but also major differences, most notably the possibly compensatory replacement of Pax6 by Pax2 in lateral eye specification. Also see the video abstract here: https://youtu.be/Hdfr3z5kEXU.

Deep Conservation of Hid-Like RHG Gene Family Homologs in Winged Insects Revealed by "Taxon Hopping" BLAST.

Together with sickle (skl), the Drosophila paralogs reaper (rpr), head involution defective (hid), and grim (RHG) control a critical switch in the induction of programmed cell death. RHG homologs have been identified in other dipteran and lepidopteran species but not beyond. Revisiting this issue with a "taxon hopping" BLAST search strategy in current genome and transcriptome resources, I detected high confidence RHG homologs in Coleoptera, Hymenoptera, Hemiptera, and Dictyoptera. Analyses of gene structure and protein sequence conservation revealed aconserved splicing pattern and highly conserved amino acid residues at both the N- and C-terminal ends that identify hid as the most ancestrally organized RHG gene family member in Drosophila. hid-like RHG homologs were also detected in mosquitoes, redefining their michelob_x (mx) genes as an expansion of derived RHG homologs. Only singleton homologs were detected in the large majority of other insect clades. Lepidopteran RHG homologs, however, stand out by producing an evolutionarily-derived splice isoform, identified in previous work, in addition to the newly detected hid-like isoform. Exceptional sequence diversification of select RHG homologs at the family- and genus-level explain their previous elusiveness in important insect genome model species like the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Combined, these findings expand the minimal age of the RHG gene family by about 100 million years and open new avenues for molecular cell death studies in insects.

Semper's cells in the insect compound eye: Insights into ocular form and function.

The arthropod compound eye represents one of two major eye types in the animal kingdom and has served as an essential experimental paradigm for defining fundamental mechanisms underlying sensory organ formation, function, and maintenance. One of the most distinguishing features of the compound eye is the highly regular array of lens facets that define individual eye (ommatidial) units. These lens facets are produced by a deeply conserved quartet of cuticle-secreting cells, called Semper cells (SCs). Also widely known as cone cells, SCs were originally identified for their secretion of the dioptric system, i.e. the corneal lens and underlying crystalline cones. Additionally, SCs are now known to execute a diversity of patterning and glial functions in compound eye development and maintenance. Here, we present an integrated account of our current knowledge of SC multifunctionality in the Drosophila compound eye, highlighting emerging gene regulatory modules that may drive the diverse roles for these cells. Drawing comparisons with other deeply conserved retinal glia in the vertebrate single lens eye, this discussion speaks to glial cell origins and opens new avenues for understanding sensory system support programs.

The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Evolutionary conservation of opsin gene expression patterns in the compound eyes of darkling beetles.

Recent large-scale studies of opsin gene contents in representatives of the largest order of insects, the Coleoptera (beetles), revealed that the blue wavelength-sensitive (B) opsin subfamily is absent in this clade, while the ultraviolet- (UV) and long wavelength-sensitive (LW) opsin subfamilies are broadly conserved with gene duplications possibly reintroducing blue sensitivity in select subclades. Little is known yet, however, how opsin genes are expressed in the compound eyes of beetles. In a previous study, we analyzed opsin gene expression in the red flour beetle Tribolium castaneum, a member of the family of darkling beetles (Tenebrionidae), and found that a singleton LW opsin homolog is homogeneously expressed in all photoreceptors of the compound eye retina with a singleton UV opsin homolog being co-expressed in the R7 subtype photoreceptors. To probe for the evolutionary conservation of these expression patterns, we isolated complete opsin transcript sequences from three additional species in the subfamily Tenebrionidae (Tribolium confusum, Tenebrio molitor, Zophobas morio) and studied their expression via whole mount in situ hybridization in the pupal retina. These experiments revealed very similar, if not identical, photoreceptor subtype-specific expression patterns in all three species compared with T. castaneum. Documenting a deep conservation of photoreceptor subtype-specific opsin gene expression in this range of darkling beetles, our study provides a first point of reference for broader comparative studies of retinal organization in the Coleoptera.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Genomic signatures of globally enhanced gene duplicate accumulation in the megadiverse higher Diptera fueling intralocus sexual conflict resolution.

Gene duplication is an important source of evolutionary innovation. To explore the relative impact of gene duplication during the diversification of major insect model system lineages, we performed a comparative analysis of lineage-specific gene duplications in the fruit fly Drosophila melanogaster (Diptera: Brachycera), the mosquito Anopheles gambiae (Diptera: Culicomorpha), the red flour beetle Tribolium castaneum (Coleoptera), and the honeybee Apis mellifera (Hymenoptera). Focusing on close to 6,000 insect core gene families containing maximally six paralogs, we detected a conspicuously higher number of lineage-specific duplications in Drosophila (689) compared to Anopheles (315), Tribolium (386), and Apis (223). Based on analyses of sequence divergence, phylogenetic distribution, and gene ontology information, we present evidence that an increased background rate of gene duplicate accumulation played an exceptional role during the diversification of the higher Diptera (Brachycera), in part by providing enriched opportunities for intralocus sexual conflict resolution, which may have boosted speciation rates during the early radiation of the megadiverse brachyceran subclade Schizophora.

Towards reconstructing the dipteran demise of an ancient essential gene: E3 ubiquitin ligase Murine double minute.

Genome studies have uncovered many examples of essential gene loss, raising the question of how ancient genes transition from essentiality to dispensability. We explored this process for the deeply conserved E3 ubiquitin ligase Murine double minute (Mdm), which is lacking in Drosophila despite the conservation of its main regulatory target, the cellular stress response gene p53. Conducting gene expression and knockdown experiments in the red flour beetle Tribolium castaneum, we found evidence that Mdm has remained essential in insects where it is present. Using bioinformatics approaches, we confirm the absence of the Mdm gene family in Drosophila, mapping its loss to the stem lineage of schizophoran Diptera and Pipunculidae (big-headed flies), about 95-85 million years ago. Intriguingly, this gene loss event was preceded by the de novo origin of the gene Companion of reaper (Corp), a novel p53 regulatory factor that is characterized by functional similarities to vertebrate Mdm2 despite lacking E3 ubiquitin ligase protein domains. Speaking against a 1:1 compensatory gene gain/loss scenario, however, we found that hoverflies (Syrphidae) and pointed-wing flies (Lonchopteridae) possess both Mdm and Corp. This implies that the two p53 regulators have been coexisting for ~ 150 million years in select dipteran clades and for at least 50 million years in the lineage to Schizophora and Pipunculidae. Given these extensive time spans of Mdm/Corp coexistence, we speculate that the loss of Mdm in the lineage to Drosophila involved further acquisitions of compensatory gene activities besides the emergence of Corp. Combined with the previously noted reduction of an ancestral P53 contact domain in the Mdm homologs of crustaceans and insects, we conclude that the loss of the ancient Mdm gene family in flies was the outcome of incremental functional regression over long macroevolutionary time scales.

The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila.

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.

Sawfly Genomes Reveal Evolutionary Acquisitions That Fostered the Mega-Radiation of Parasitoid and Eusocial Hymenoptera.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.