RESUMO
Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.
Assuntos
Chironomidae/citologia , Chironomidae/genética , Puffs Cromossômicos/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Puffs Cromossômicos/química , Puffs Cromossômicos/genética , Genes de Insetos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/química , Ribonucleoproteínas/genéticaRESUMO
In large-scale mutagenesis screens performed in 1979-1980 at the EMBL in Heidelberg, we isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants we characterized could be assigned to 120 genes and represent the majority of such genes in the genome. These mutants subsequently provided a rich resource for understanding many fundamental developmental processes, such as the transcriptional hierarchies controlling segmentation, the establishment of cell states by signaling pathways, and the differentiation of epithelial cells. Most of the Heidelberg genes are now molecularly known, and many of them are conserved in other animals, including humans. Although the screens were initially driven entirely by curiosity, the mutants now serve as models for many human diseases. In this review, we describe the rationale of the screening procedures and provide a classification of the genes on the basis of their initial phenotypes and the subsequent molecular analyses.
Assuntos
Drosophila/genética , Testes Genéticos , Mutação/genética , Animais , Genes de Insetos , Mutagênese/genética , Transdução de Sinais/genéticaRESUMO
Almost 20 years have passed since the first reference genome assemblies were published for Plasmodium falciparum, the deadliest malaria parasite, and Anopheles gambiae, the most important mosquito vector of malaria in sub-Saharan Africa. Reference genomes now exist for all human malaria parasites and nearly half of the ~40 important vectors around the world. As a foundation for genetic diversity studies, these reference genomes have helped advance our understanding of basic disease biology and drug and insecticide resistance, and have informed vaccine development efforts. Population genomic data are increasingly being used to guide our understanding of malaria epidemiology, for example by assessing connectivity between populations and the efficacy of parasite and vector interventions. The potential value of these applications to malaria control strategies, together with the increasing diversity of genomic data types and contexts in which data are being generated, raise both opportunities and challenges in the field. This Review discusses advances in malaria genomics and explores how population genomic data could be harnessed to further support global disease control efforts.
Assuntos
Malária/parasitologia , Metagenômica/tendências , Mosquitos Vetores/genética , Plasmodium falciparum/genética , Animais , Anopheles/genética , Antimaláricos/farmacologia , Resistência a Medicamentos , Genes de Insetos , Genes de Protozoários , Humanos , Malária/prevenção & controle , Vacinas Antimaláricas , Plasmodium falciparum/efeitos dos fármacosRESUMO
With detailed data on gene expression accessible from an increasingly broad array of species, we can test the extent to which our developmental genetic knowledge from model organisms predicts expression patterns and variation across species. But to know when differences in gene expression across species are significant, we first need to know how much evolutionary variation in gene expression we expect to observe. Here we provide an answer by analyzing RNAseq data across twelve species of Hawaiian Drosophilidae flies, focusing on gene expression differences between the ovary and other tissues. We show that over evolutionary time, there exists a cohort of ovary specific genes that is stable and that largely corresponds to described expression patterns from laboratory model Drosophila species. Our results also provide a demonstration of the prediction that, as phylogenetic distance increases, variation between species overwhelms variation between tissue types. Using ancestral state reconstruction of expression, we describe the distribution of evolutionary changes in tissue-biased expression, and use this to identify gains and losses of ovary-biased expression across these twelve species. We then use this distribution to calculate the evolutionary correlation in expression changes between genes, and demonstrate that genes with known interactions in D. melanogaster are significantly more correlated in their evolution than genes with no or unknown interactions. Finally, we use this correlation matrix to infer new networks of genes that share evolutionary trajectories, and we present these results as a dataset of new testable hypotheses about genetic roles and interactions in the function and evolution of the Drosophila ovary.
Assuntos
Drosophila melanogaster , Ovário , Animais , Feminino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Filogenia , Havaí , Genes de Insetos , Evolução Molecular , Drosophila/genética , Expressão GênicaRESUMO
The study of insect social behavior has offered tremendous insight into the molecular mechanisms mediating behavioral and phenotypic plasticity. Genomic applications to the study of eusocial insect species, in particular, have led to several hypotheses for the processes underlying the molecular evolution of behavior. Advances in understanding the genetic control of social organization have also been made, suggesting an important role for supergenes in the evolution of divergent behavioral phenotypes. Intensive study of social phenotypes across species has revealed that behavior and caste are controlled by an interaction between genetic and environmentally mediated effects and, further, that gene expression and regulation mediate plastic responses to environmental signals. However, several key methodological flaws that are hindering progress in the study of insect social behavior remain. After reviewing the current state of knowledge, we outline ongoing challenges in experimental design that remain to be overcome in order to advance the field.
Assuntos
Comportamento Animal , Genes de Insetos , Genoma de Inseto , Insetos/genética , Comportamento Social , Animais , Evolução Biológica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interação Gene-Ambiente , FenótipoRESUMO
The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.
Assuntos
Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Macrófagos/fisiologia , Animais , Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Genes de Insetos , Genes fos , Análise de Sequência de RNA , Tetraspaninas , Fatores de Transcrição/metabolismoRESUMO
Access to hitherto unexploited ecological opportunities is associated with phenotypic evolution and often results in significant lineage diversification. Yet our understanding of the mechanisms underlying such adaptive traits remains limited. Water striders have been able to exploit the water-air interface, primarily facilitated by changes in the density of hydrophobic bristles and a significant increase in leg length. These two traits are functionally correlated and are both necessary for generating efficient locomotion on the water surface. Whether bristle density and leg length have any cellular or developmental genetic mechanisms in common is unknown. Here, we combine comparative genomics and transcriptomics with functional RNA interference assays to examine the developmental genetic and cellular mechanisms underlying the patterning of the bristles and the legs in Gerris buenoi and Mesovelia mulsanti, two species of water striders. We found that two duplication events in the genes beadex and taxi led to a functional expansion of the paralogs, which affected bristle density and leg length. We also identified genes for which no function in bristle development has been previously described in other insects. Interestingly, most of these genes play a dual role in regulating bristle development and leg length. In addition, these genes play a role in regulating cell division. This result suggests that cell division may be a common mechanism through which these genes can simultaneously regulate leg length and bristle density. We propose that pleiotropy, through which gene function affects the development of multiple traits, may play a prominent role in facilitating access to unexploited ecological opportunities and species diversification.
Assuntos
Heterópteros/fisiologia , Locomoção , Água , Animais , Genes de Insetos , Heterópteros/anatomia & histologia , Heterópteros/embriologia , Proteínas de Insetos/genética , TranscriptomaRESUMO
Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 (Kr-h1), broad, and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonataE93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant (Met), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1, broad, and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1, broad, and E93 in the evolution of insect metamorphosis.
Assuntos
Evolução Biológica , Metamorfose Biológica/genética , Odonatos/crescimento & desenvolvimento , Asas de Animais , Animais , Feminino , Perfilação da Expressão Gênica , Genes de Insetos , Masculino , Odonatos/genética , Interferência de RNARESUMO
Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.
Assuntos
Borboletas/genética , Borboletas/efeitos da radiação , Genes de Troca , Iridescência/genética , Enxofre/química , Raios Ultravioleta , Animais , Sistemas CRISPR-Cas/genética , Cromossomos/genética , Genes de Insetos , Loci Gênicos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Iridescência/efeitos da radiação , Masculino , Comportamento Sexual Animal/fisiologia , Especificidade da Espécie , Simpatria/genética , Asas de Animais/metabolismoRESUMO
The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.
Assuntos
Abelhas , Sistema Enzimático do Citocromo P-450 , Evolução Molecular , Genes de Insetos , Inativação Metabólica , Proteínas de Insetos , Inseticidas , Animais , Abelhas/enzimologia , Abelhas/genética , Sequência Conservada , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Inseticidas/metabolismo , Inseticidas/toxicidade , Neonicotinoides/metabolismo , Neonicotinoides/toxicidade , FilogeniaRESUMO
Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.
Assuntos
Drosophila melanogaster/genética , Fertilidade/genética , Genes Essenciais/fisiologia , Genes de Insetos/fisiologia , Reprodução/genética , Animais , Sistemas CRISPR-Cas/genética , Evolução Molecular , Mutação da Fase de Leitura , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Letais/fisiologia , Infertilidade Masculina/genética , Masculino , Filogenia , Interferência de RNA , Espermatogênese/genética , Testículo/anatomia & histologia , Testículo/metabolismoRESUMO
Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.
Assuntos
Blattellidae/metabolismo , Ácidos Graxos/metabolismo , Hidrocarbonetos/metabolismo , Atrativos Sexuais/metabolismo , Caracteres Sexuais , Comportamento Sexual Animal , Animais , Blattellidae/genética , Blattellidae/fisiologia , Feminino , Genes de Insetos , Diferenciação Sexual/genéticaRESUMO
Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-ß-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-ß-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-ß-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-ß-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.
Assuntos
Alquil e Aril Transferases/metabolismo , Afrodisíacos/antagonistas & inibidores , Borboletas , Feromônios/metabolismo , Alquil e Aril Transferases/genética , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Borboletas/genética , Borboletas/metabolismo , Evolução Molecular , Feminino , Genes de Insetos , Masculino , Feromônios/farmacologia , Filogenia , Comportamento Sexual Animal/efeitos dos fármacos , Especificidade da EspécieRESUMO
Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper, Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals' ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance.
Assuntos
Hemípteros/efeitos dos fármacos , Resistência a Inseticidas/efeitos dos fármacos , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Oryza/parasitologia , Peroxirredoxinas/fisiologia , Adaptação Biológica/efeitos dos fármacos , Adaptação Biológica/genética , Alelos , Animais , Mapeamento Cromossômico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Genes Modificadores/efeitos dos fármacos , Genes Modificadores/fisiologia , Estudos de Associação Genética , Aptidão Genética/efeitos dos fármacos , Hemípteros/fisiologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Oryza/efeitos dos fármacos , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/metabolismo , Testes de ToxicidadeRESUMO
The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.
Assuntos
Mecanismo Genético de Compensação de Dose/genética , Drosophila melanogaster/genética , Genes de Insetos/genética , Animais , Animais Geneticamente Modificados , Austrália , Calliphoridae/genética , Dípteros/genética , Proteínas de Drosophila/genética , Feminino , Genes Ligados ao Cromossomo X/genética , Masculino , Pupa/genética , Interferência de RNA/fisiologia , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Ovinos , Fatores de Transcrição/genética , Cromossomo X/genéticaRESUMO
High-throughput genetic screens are powerful methods to interrogate gene function on a genome-wide scale and identify genes responsible to certain stresses. Here, we developed a piggyBac strategy to deliver pooled sgRNA libraries stably into cell lines. We used this strategy to conduct a screen based on genome-wide clustered regularly interspaced short palindromic repeat technology (CRISPR)-Cas9 in Bombyx mori cells. We first constructed a single guide RNA (sgRNA) library containing 94,000 sgRNAs, which targeted 16,571 protein-coding genes. We then generated knockout collections in BmE cells using the piggyBac transposon. We identified 1006 genes that are essential for cell viability under normal growth conditions. Of the identified genes, 82.4% (829 genes) were homologous to essential genes in seven animal species. We also identified 838 genes whose loss facilitated cell growth. Next, we performed context-specific positive screens for resistance to biotic or nonbiotic stresses using temperature and baculovirus separately, which identified several key genes and pathways from each screen. Collectively, our results provide a novel and versatile platform for functional annotations of B. mori genomes and deciphering key genes responsible for various conditions. This study also shows the effectiveness, practicality, and convenience of genome-wide CRISPR screens in nonmodel organisms.
Assuntos
Bombyx/genética , Sistemas CRISPR-Cas , Genes Essenciais , Genes de Insetos , Animais , Bombyx/virologia , Linhagem Celular , Sobrevivência Celular/genética , Genoma de Inseto , Interações Hospedeiro-Patógeno , RNA , Estresse Fisiológico/genética , TemperaturaRESUMO
Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3'-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3'-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3'- and 5'-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors.
Assuntos
Genes de Insetos/genética , Genes Virais/genética , Insetos Vetores/genética , Insetos Vetores/virologia , Tenuivirus/genética , Animais , Humanos , MicroRNAs/genética , Replicação Viral/genéticaRESUMO
Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.
Assuntos
Homeostase/fisiologia , Intestinos/fisiologia , MicroRNAs/genética , Tripanossomíase Africana/parasitologia , Moscas Tsé-Tsé/genética , Moscas Tsé-Tsé/parasitologia , Animais , Animais Geneticamente Modificados , Microbioma Gastrointestinal/fisiologia , Genes de Insetos , Insetos Vetores/genética , Insetos Vetores/parasitologia , TrypanosomaRESUMO
Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These 'biotypes' have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system's complex dual role in interacting with both beneficial and harmful microbes.
Assuntos
Afídeos/microbiologia , Carga Bacteriana/genética , Enterobacteriaceae/imunologia , Imunidade Inata/genética , Simbiose , Animais , Afídeos/classificação , Afídeos/genética , Afídeos/imunologia , Carga Bacteriana/fisiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/citologia , Enterobacteriaceae/genética , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes de Insetos/genética , Variação Genética/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Especificidade da Espécie , Simbiose/genética , Simbiose/imunologiaRESUMO
The development of dimorphic adult sexes is a critical process for most animals, one that is subject to intense selection. Work in vertebrate and insect model species has revealed that sex determination mechanisms vary widely among animal groups. However, this variation is not uniform, with a limited number of conserved factors. Therefore, sex determination offers an excellent context to consider themes and variations in gene network evolution. Here we review the literature describing sex determination in diverse insects. We have screened public genomic sequence databases for orthologs and duplicates of 25 genes involved in insect sex determination, identifying patterns of presence and absence. These genes and a 3.5 reference set of 43 others were used to infer phylogenies and compared to accepted organismal relationships to examine patterns of congruence and divergence. The function of candidate genes for roles in sex determination (virilizer, female-lethal-2-d, transformer-2) and sex chromosome dosage compensation (male specific lethal-1, msl-2, msl-3) were tested using RNA interference in the milkweed bug, Oncopeltus fasciatus. None of these candidate genes exhibited conserved roles in these processes. Amidst this variation we wish to highlight the following themes for the evolution of sex determination: (1) Unique features within taxa influence network evolution. (2) Their position in the network influences a component's evolution. Our analyses also suggest an inverse association of protein sequence conservation with functional conservation.