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An amendment to this paper has been published and can be accessed via the original article.
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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.
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Genoma de Inseto , Características de História de Vida , Tisanópteros/fisiologia , Transcriptoma , Animais , Produtos Agrícolas , Comportamento Alimentar , Cadeia Alimentar , Imunidade Inata/genética , Percepção , Filogenia , Reprodução/genética , Tisanópteros/genética , Tisanópteros/imunologiaRESUMO
BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.
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Genoma , Heterópteros/genética , Heterópteros/fisiologia , Proteínas de Insetos/genética , Adaptação Fisiológica , Animais , Evolução Molecular , Genômica , Heterópteros/classificação , Fenótipo , FilogeniaRESUMO
In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.
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Blastoderma/embriologia , Conexinas/metabolismo , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Tribolium/embriologia , Animais , Membrana Celular/fisiologia , Conexinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Microtúbulos/metabolismo , Morfogênese/genética , Interferência de RNA , RNA Interferente PequenoRESUMO
BACKGROUND: Nicrophorus vespilloides eggs are deposited into the soil in close proximity to the decomposing vertebrate carcasses that these insects use as an obligate resource to rear their offspring. Eggs in this environment potentially face significant risks from the bacteria that proliferate in the grave-soil environment following nutrient influx from the decomposing carcass. Our aims in this paper are twofold: first, to examine the fitness effects of grave-soil bacteria to eggs, and second, to quantify egg immunocompetence as a defence against these bacteria. RESULTS: Our results provide strong evidence that grave-soil microbes significantly reduce the survival of Nicrophorus eggs. Females provided with microbe rich carcasses to rear broods laid fewer eggs that were less likely to hatch than females given uncontaminated carcasses. Furthermore, we show that egg hatch success is significantly reduced by bacterial exposure. Using a split-brood design, which controlled for intrinsic differences in eggs produced by different females, we found that eggs washed free of surface-associated bacteria show increased survival compared to unwashed eggs. By contrast, eggs exposed to the entomopathogen Serratia marcescens show decreased survival compared to unexposed eggs. We next tested the immune competence of eggs under challenge from bacterial infection, and found that eggs lacked endogenous production of antimicrobial peptides, despite well-developed responses in larvae. Finally, we found that despite lacking immunity, N. vespilloides eggs produce an extraembryonic serosa, indicating that the serosa has lost its immune inducing capacity in this species. CONCLUSIONS: The dependency on ephemeral resources might strongly select for fast developing animals. Our results suggest that Nicrophorus carrion beetles, and other species developing on ephemeral resources, face a fundamental trade-off between egg immunity and development time.
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Fenômenos Fisiológicos Bacterianos , Besouros/crescimento & desenvolvimento , Besouros/microbiologia , Microbiologia do Solo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Cadáver , Besouros/química , Besouros/imunologia , Feminino , Larva/crescimento & desenvolvimento , Óvulo/fisiologiaRESUMO
Cytonuclear discordance in contact zones between related lineages is common, with mitochondrial clines often being displaced from clines in nuclear allele frequency. Proposed explanations for such a pattern include adaptive introgression of mtDNA or a neutral wake of mtDNA being left behind following hybrid zone movement. However, studies investigating these hypotheses are rare. Our previous survey of genetic variation in the long-toed salamander (Ambystoma macrodactylum) highlighted a potential case of cytonuclear discordance between two lineages in western Canada. Here, we use additional markers and samples to clarify the extent of this discordance. We simultaneously assess the feeding performance of individuals in a common environment to test for an association between mitotype and individual performance. The genetic results confirm a general pattern of cytonuclear discordance in the focal region. However, we also observed more limited introgression of a diagnostic nuclear marker. Intriguingly, although there were differences in individual performance associated with the transition between mitotypes, these differences were not fully explained by mitotype. Instead, the lowest performance was observed in individuals demonstrating the greatest mismatch between mtDNA and all nuclear markers, suggesting the potential for cytonuclear incompatibilities to be acting. These results highlight the complexity of understanding the causes and consequences of mtDNA introgression and cytonuclear discordance in contact zones.
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Ambystoma/genética , Núcleo Celular/genética , DNA Mitocondrial/genética , Genética Populacional , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Canadá , Comportamento Alimentar , Feminino , Marcadores Genéticos , Variação Genética , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Developmental time is a key life-history trait with large effects on Darwinian fitness. In many insects, developmental time is currently under strong selection to minimize ecological mismatches in seasonal timing induced by climate change. The genetic basis of responses to such selection, however, is poorly understood. To address this problem, we set up a long-term evolve-and-resequence experiment in the beetle Tribolium castaneum and selected replicate, outbred populations for fast or slow embryonic development. The response to this selection was substantial and embryonic developmental timing of the selection lines started to diverge during dorsal closure. Pooled whole-genome resequencing, gene expression analysis and an RNAi screen pinpoint a 222 bp deletion containing binding sites for Broad and Tramtrack upstream of the ecdysone degrading enzyme Cyp18a1 as a main target of selection. Using CRISPR/Cas9 to reconstruct this allele in the homogenous genetic background of a laboratory strain, we unravel how this single deletion advances the embryonic ecdysone peak inducing dorsal closure and show that this allele accelerates larval development but causes a trade-off with fecundity. Our study uncovers a life-history allele of large effect and reveals the evolvability of developmental time in a natural insect population.
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Besouros , Tribolium , Animais , Ecdisona , Alelos , Insetos , Tribolium/genéticaRESUMO
Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.
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Embrião não Mamífero/anatomia & histologia , Membrana Serosa/embriologia , Tribolium/embriologia , Animais , Evolução Biológica , Quitina Sintase/genética , Desidratação , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Insetos/embriologia , Óvulo/citologia , Interferência de RNA , Tribolium/genéticaRESUMO
Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria. For Oncopeltus, RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays and describe two novel AMP families called Serosins and Ovicins. For both insects, quantitative polymerase chain reaction shows immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
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Anti-Infecciosos , Heterópteros , Tribolium , Animais , Anti-Infecciosos/metabolismo , Imunidade , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos/metabolismo , Membrana Serosa/metabolismo , Tribolium/genéticaRESUMO
BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.
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Evolução Molecular , Genoma de Inseto , Hemípteros/genética , Sequência de Aminoácidos , Animais , Dedos de Zinco CYS2-HIS2 , Comportamento Alimentar , Dosagem de Genes , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Genes Homeobox , Hemípteros/crescimento & desenvolvimento , Hemípteros/metabolismo , Pigmentação/genética , Olfato , Fatores de Transcrição/genéticaRESUMO
In order to survive microbe encounters, insects rely on both physical barriers as well as local and systemic immune responses. Most research focusses on adult or larval defenses however, whereas insect eggs are also in need of protection. Lately, the defense of eggs against microbes has received an increasing amount of attention, be it through endogenous egg defenses, trans-generational immune priming (TGIP) or parental investment. Here we studied the endogenous immune response in eggs and adults of Tenebrio molitor. We show that many immune genes are induced in both adults and eggs. Furthermore, we show that eggs reach comparable levels of immune gene expression as adults. These findings show that the eggs of Tenebrio are capable of an impressive endogenous immune response, and indicate that such inducible egg defenses are likely common in insects.
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Peptídeos Catiônicos Antimicrobianos/biossíntese , Tenebrio/imunologia , Zigoto/imunologia , Animais , Proteínas do Ovo/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Imunidade , Imunização , Proteínas de Insetos/imunologia , LarvaRESUMO
The burying beetle Nicrophorus vespilloides has emerged as a model system for the investigation of adaptations that allow the utilization of carrion as a diet and as a resource for reproduction. The survival of beetles and their offspring given their exposure to soil-dwelling and cadaver-borne microbes requires mechanisms that reduce bacterial contamination in the diet and that achieve sanitation of the microhabitat. To explore the role of antimicrobial peptides (AMPs) in this context, we analyzed burying beetle males and females at different stages of their breeding cycle using the RNA-Seq and proteomics approaches. To address variation in immune functions, we investigated the impact of adult sex, the presence or absence of offspring (social context), and the presence of carrion (environmental context) on the expression of the identified immune effector genes. We found that particular AMPs are sex-specific and tightly regulated by the presence of a carcass or offspring and identified the two most context-dependent antimicrobial proteins in anal secretions. The context-specific expression dynamics of particular AMPs and lysozymes reveals a complex regulatory system, reflecting adaptations to specific ecological niches. This study highlights how burying beetles cope with microorganisms found on carrion and identifies candidates for both internal and external immunity.
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Peptídeos Catiônicos Antimicrobianos/genética , Besouros/genética , Proteômica , Reprodução/genética , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Cadáver , Besouros/microbiologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Larva/genética , Larva/microbiologia , Masculino , Caracteres Sexuais , Microbiologia do SoloRESUMO
Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs. We therefore set out to investigate serosal cuticle formation and function in a beetle (Tribolium castaneum) using RNAi-mediated knockdown of three candidate genes known to structure chitin in the adult cuticle, and we aimed to identify other serosal cuticle genes using RNA sequencing. Knockdown of Knickkopf (TcKnk-1) or Retroactive (TcRtv) affects the laminar structure of the serosal cuticle, as revealed by Transmission Electron Microscopy in knockdown eggs. In the absence of this laminar structure, significantly fewer eggs survive at low humidity compared to wild-type eggs. Survival in dry conditions is also adversely affected when cross-linking among proteins and chitin is prevented by Laccase2 (TcLac-2) RNAi. Finally, we compare the transcriptomes of wild-type eggs to serosa-less eggs and find serosa-biased expression of 21 cuticle-related genes including structural components, chitin deacetylases and chitinases. Our data indicate that the serosal cuticle utilizes the same machinery for structuring the cuticle as adults. We demonstrate that the structure of the cuticle is crucial for desiccation resistance, and we put forward the serosal cuticle of Tribolium as an excellent model to study the ecological properties of the insect cuticle.
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Proteínas de Insetos/fisiologia , Tribolium/fisiologia , Animais , Dessecação , Feminino , Expressão Gênica , Umidade , Masculino , Óvulo/fisiologia , Óvulo/ultraestrutura , Interferência de RNA , Análise de Sequência de RNA , Membrana Serosa/metabolismo , Tribolium/ultraestruturaRESUMO
Drosophila larvae and adults possess a potent innate immune response, but the response of Drosophila eggs is poor. In contrast to Drosophila, eggs of the beetle Tribolium are protected by a serosa, an extraembryonic epithelium that is present in all insects except higher flies. In this study, we test a possible immune function of this frontier epithelium using Tc-zen1 RNAi-mediated deletion. First, we show that bacteria propagate twice as fast in serosa-less eggs. Then, we compare the complete transcriptomes of wild-type, control RNAi, and Tc-zen1 RNAi eggs before and after sterile or septic injury. Infection induces genes involved in Toll and IMD-signaling, melanisation, production of reactive oxygen species and antimicrobial peptides in wild-type eggs but not in serosa-less eggs. Finally, we demonstrate constitutive and induced immune gene expression in the serosal epithelium using in situ hybridization. We conclude that the serosa provides insect eggs with a full-range innate immune response.
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Epitélio/embriologia , Membranas Extraembrionárias/imunologia , Imunidade Inata , Óvulo/imunologia , Membrana Serosa/imunologia , Tribolium/embriologia , Tribolium/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Contagem de Colônia Microbiana , Epitélio/efeitos dos fármacos , Epitélio/imunologia , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/microbiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes de Insetos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Hibridização In Situ , Óvulo/efeitos dos fármacos , Óvulo/microbiologia , Interferência de RNA , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/imunologia , Sepse/microbiologia , Sepse/patologia , Análise de Sequência de RNA , Membrana Serosa/efeitos dos fármacos , Membrana Serosa/embriologia , Membrana Serosa/microbiologia , Tribolium/efeitos dos fármacos , Tribolium/genéticaRESUMO
Innate immunity is common to all metazoans and serves as a first line of defense against pathogens. Although the immune response of adult and larval insects has been well characterized, it remains unknown whether the insect egg is able to mount an immune response. Contrary to Drosophila, Tribolium eggs develop an extraembryonic epithelium, the serosa. Epithelia are well known for their ability to fight infection, so the serosa has the potential to protect the embryo against pathogens. To test this hypothesis we created serosa-less eggs by Tc-zen1 parental RNAi. We found that the Tribolium egg upregulates several immune genes to comparable levels as adults in response to infection. Drosophila eggs and serosa-less Tribolium eggs, however, show little to no upregulation of any of the tested immune genes. We conclude that the extraembryonic serosa is crucial for the early immune competence of the Tribolium egg.