RESUMO
The fall armyworm (FAW) Spodoptera frugiperda is thought to have undergone a rapid 'west-to-east' spread since 2016 when it was first identified in western Africa. Between 2018 and 2020, it was recorded from South Asia (SA), Southeast Asia (SEA), East Asia (EA), and Pacific/Australia (PA). Population genomic analyses enabled the understanding of pathways, population sources, and gene flow in this notorious agricultural pest species. Using neutral single nucleotide polymorphic (SNP) DNA markers, we detected genome introgression that suggested most populations in this study were overwhelmingly C- and R-strain hybrids (n = 252/262). SNP and mitochondrial DNA markers identified multiple introductions that were most parsimoniously explained by anthropogenic-assisted spread, i.e., associated with international trade of live/fresh plants and plant products, and involved 'bridgehead populations' in countries to enable successful pest establishment in neighbouring countries. Distinct population genomic signatures between Myanmar and China do not support the 'African origin spread' nor the 'Myanmar source population to China' hypotheses. Significant genetic differentiation between populations from different Australian states supported multiple pathways involving distinct SEA populations. Our study identified Asia as a biosecurity hotspot and a FAW genetic melting pot, and demonstrated the use of genome analysis to disentangle preventable human-assisted pest introductions from unpreventable natural pest spread.
Assuntos
Comércio , Spodoptera , Animais , Ásia , Austrália , Marcadores Genéticos , Spodoptera/genética , Genética Populacional , Fluxo Gênico , Polimorfismo de Nucleotídeo Único , Espécies IntroduzidasRESUMO
Native to the Americas, the invasive Spodoptera frugiperda (fall armyworm; FAW) was reported in West Africa in 2016, followed by its chronological detection across the Old World and the hypothesis of an eastward Asia expansion. We explored population genomic signatures of American and Old World FAW and identified 12 maternal mitochondrial DNA genome lineages across the invasive range. 870 high-quality nuclear single nucleotide polymorphic DNA markers identified five distinct New World population clusters, broadly reflecting FAW native geographical ranges and the absence of host-plant preferences. We identified unique admixed Old World populations, and admixed and non-admixed Asian FAW individuals, all of which suggested multiple introductions underpinning the pest's global spread. Directional gene flow from the East into eastern Africa was also detected, in contrast to the west-to-east spread hypothesis. Our study demonstrated the potential of population genomic approaches via international partnership to address global emerging pest threats and biosecurity challenges.
Assuntos
Fluxo Gênico , Metagenômica , Spodoptera , África Oriental , Animais , Ásia , Spodoptera/genéticaRESUMO
The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.
Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Spodoptera , Acetilcolinesterase/genética , Animais , Diamida/farmacologia , Genes de Insetos , Genoma de Inseto , Organofosfatos/farmacologia , Mutação Puntual/genética , Mutação Puntual/fisiologia , Piretrinas/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Canais de Sódio Disparados por Voltagem/genética , Sequenciamento Completo do GenomaRESUMO
Stress is a widespread phenomenon that all organisms must endure. Common in nature is oxidative stress, which can interrupt cell homeostasis to cause cell damage and may be derived from respiration or from environmental exposure through diet. As a result of the routine exposure from respiration, many organisms can mitigate the effects of oxidative stress, but less is known about responses to oxidative stress from other sources. Helicoverpa armigera is a major agricultural pest moth that causes significant damage to crops worldwide. Here, we examined the effects of oxidative stress on H. armigera by chronically exposing individuals to paraquat-a free radical producer-and measuring changes in development (weight, developmental rate, lifespan), and gene expression. We found that oxidative stress strongly affected development in H. armigera, with stressed samples spending more time as caterpillars than control samples (>24 vs. ~15 days, respectively) and therefore living longer overall. We found 1,618 up- and 761 down-regulated genes, respectively, in stressed versus control samples. In the up-regulated gene set, was an over-representation of biological processes related to cuticle and chitin development, glycine metabolism, and oxidation-reduction. Oxidative stress clearly impacts physiology and biochemistry in H. armigera and the interesting finding of an extended lifespan in stressed individuals could demonstrate hormesis, the phenomenon whereby toxic compounds can actually be beneficial at low doses. Collectively, our findings provide new insights into physiological and gene expression responses to oxidative stress in invertebrates.
RESUMO
Hybridization between invasive and native species has raised global concern, given the dramatic increase in species range shifts and pest outbreaks due to anthropogenic dispersal. Nevertheless, secondary contact between sister lineages of local and invasive species provides a natural laboratory to understand the factors that determine introgression and the maintenance or loss of species barriers. Here, we characterize the early evolutionary outcomes following secondary contact between invasive Helicoverpa armigera and native H. zea in Brazil. We carried out whole-genome resequencing of Helicoverpa moths from Brazil in two temporal samples: during the outbreak of H. armigera in 2013 and 2017. There is evidence for a burst of hybridization and widespread introgression from local H. zea into invasive H. armigera coinciding with H. armigera expansion in 2013. However, in H. armigera, the admixture proportion and the length of introgressed blocks were significantly reduced between 2013 and 2017, suggesting selection against admixture. In contrast to the genome-wide pattern, there was striking evidence for adaptive introgression of a single region from the invasive H. armigera into local H. zea, including an insecticide resistance allele that increased in frequency over time. In summary, despite extensive gene flow after secondary contact, the species boundaries are largely maintained except for the single introgressed region containing the insecticide-resistant locus. We document the worst-case scenario for an invasive species, in which there are now two pest species instead of one, and the native species has acquired resistance to pyrethroid insecticides through introgression.
Assuntos
Introgressão Genética , Espécies Introduzidas , Mariposas/genética , Adaptação Biológica/genética , Animais , Evolução Biológica , Fluxo Gênico , Genoma de Inseto , Resistência a Inseticidas/genética , Inseticidas , Piretrinas , SimpatriaRESUMO
Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Processamento Alternativo/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Mariposas/genética , Animais , Arizona , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Produtos Agrícolas , Endotoxinas/genética , Endotoxinas/toxicidade , Éxons/genética , Gossypium/genética , Gossypium/parasitologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Índia , Larva/efeitos dos fármacos , Larva/genética , Mariposas/efeitos dos fármacos , Mutação , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/parasitologiaRESUMO
Within the mega-pest lineage of heliothine moths are a number of polyphagous, highly mobile species for which the exchange of adaptive traits through hybridization would affect their properties as pests. The recent invasion of South America by one of the most significant agricultural pests, Helicoverpa armigera, raises concerns for the formation of novel combinations of adaptive genes following hybridization with the closely related Helicoverpa zea To investigate the propensity for hybridization within the genus Helicoverpa, we carried out whole-genome resequencing of samples from six species, focusing in particular upon H. armigera population structure and its relationship with H. zea We show that both H. armigera subspecies have greater genetic diversity and effective population sizes than do the other species. We find no signals for gene flow among the six species, other than between H. armigera and H. zea, with nine Brazilian individuals proving to be hybrids of those two species. Eight had largely H. armigera genomes with some introgressed DNA from H. zea scattered throughout. The ninth resembled an F1 hybrid but with stretches of homozygosity for each parental species that reflect previous hybridization. Regions homozygous for H. armigera-derived DNA in this individual included one containing a gustatory receptor and esterase genes previously associated with host range, while another encoded a cytochrome P450 that confers insecticide resistance. Our data point toward the emergence of novel hybrid ecotypes and highlight the importance of monitoring H. armigera genotypes as they spread through the Americas.
Assuntos
Quimera/genética , Fluxo Gênico , Genoma de Inseto , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Mariposas/genética , Animais , Especificidade da EspécieRESUMO
Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species.
Assuntos
Código de Barras de DNA Taxonômico/normas , Hemípteros/genética , Espécies Introduzidas , Polimorfismo Genético , Pseudogenes , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Evolução Molecular , Genoma Mitocondrial , Hemípteros/classificação , Proteínas de Insetos/genética , SinteniaRESUMO
The Old World bollworm Helicoverpa armigera is now established in Brazil but efforts to identify incursion origin(s) and pathway(s) have met with limited success due to the patchiness of available data. Using international agricultural/horticultural commodity trade data and mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) gene markers, we inferred the origins and incursion pathways into Brazil. We detected 20 mtDNA haplotypes from six Brazilian states, eight of which were new to our 97 global COI-Cyt b haplotype database. Direct sequence matches indicated five Brazilian haplotypes had Asian, African, and European origins. We identified 45 parsimoniously informative sites and multiple substitutions per site within the concatenated (945 bp) nucleotide dataset, implying that probabilistic phylogenetic analysis methods are needed. High diversity and signatures of uniquely shared haplotypes with diverse localities combined with the trade data suggested multiple incursions and introduction origins in Brazil. Increasing agricultural/horticultural trade activities between the Old and New Worlds represents a significant biosecurity risk factor. Identifying pest origins will enable resistance profiling that reflects countries of origin to be included when developing a resistance management strategy, while identifying incursion pathways will improve biosecurity protocols and risk analysis at biosecurity hotspots including national ports.
Assuntos
DNA Mitocondrial/metabolismo , Lepidópteros/genética , Animais , Brasil , Citocromos b/genética , Bases de Dados Factuais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Variação Genética , Haplótipos , Lepidópteros/classificação , FilogeniaRESUMO
Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.
Assuntos
Expressão Gênica , Genoma de Inseto , Manduca/genética , Animais , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Manduca/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Análise de Sequência de DNA , SinteniaRESUMO
We report the complete mitochondrial DNA genome of the soybean stem fly (SSF) Melanagromyza sojae from Brazil Santa Catarina state based on Illumina MiSeq sequence data. The estimated mitogenome is 15 475 base pairs (bp) (KT597923), with 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 tRNAs, and an estimated 579 bp AT-rich control region. Similar to other insects, the SSF mitogenome is A-T bias with 40.9% A, 36.7% T, 13.6% C, and 8.8% G. Molecular characterization of SSF mitogenome will facilitate the development of effective molecular markers, and robust and rapid identification of suspected biosecurity incursions and field infestations of this insect pest.
Assuntos
Dípteros/genética , Genoma Mitocondrial , Animais , Composição de Bases , DNA Mitocondrial/química , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/metabolismo , Dípteros/classificação , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Fases de Leitura Aberta/genética , Filogenia , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , Análise de Sequência de DNARESUMO
The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas/genética , Lepidópteros/genética , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Frequência do Gene , Ligação Genética , Mutação INDEL , Inseticidas/farmacologia , Lepidópteros/efeitos dos fármacos , Lepidópteros/patogenicidade , Plantas Geneticamente Modificadas/genéticaRESUMO
Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Drosophila/genética , Inativação Gênica , RNA Polimerase Dependente de RNA/genética , Transgenes , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs/genética , RNA Interferente Pequeno/genética , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Drosophila melanogaster, along with all insects and the vertebrates, lacks an RdRp gene. We created transgenic strains of Drosophila melanogaster in which the rrf-1 or ego-1 RdRp genes from C. elegans were placed under the control of the yeast GAL4 upstream activation sequence. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. RT-PCR confirmed the successful expression of each RdRp gene. The resulting phenotypes indicated that introduction of the RdRp genes had no effect on D. melanogaster morphological development.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , RNA Polimerase Dependente de RNA/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Caenorhabditis elegans/metabolismo , DNA de Helmintos/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Expressão Gênica , Genes de Helmintos , Masculino , Morfogênese/genética , Morfogênese/fisiologia , Filogenia , Interferência de RNA , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.
Assuntos
Carboxilesterase/genética , Colinesterases/genética , Proteínas de Insetos/genética , Mariposas/enzimologia , Proteômica , Animais , Carboxilesterase/química , Carboxilesterase/metabolismo , Colinesterases/química , Colinesterases/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Dados de Sequência Molecular , Mariposas/química , Mariposas/classificação , Mariposas/genética , Mutação , Filogenia , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.
Assuntos
Amidoidrolases/metabolismo , Baculoviridae/patogenicidade , Regulação para Baixo , Isoenzimas/metabolismo , Amidoidrolases/classificação , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Quitina/metabolismo , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Isoenzimas/classificação , Isoenzimas/genética , Dados de Sequência Molecular , Mariposas/enzimologia , Mariposas/genética , Mariposas/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Spodoptera/enzimologia , Spodoptera/genética , Spodoptera/virologiaRESUMO
The Tetraviridae is a family of non-enveloped positive-stranded RNA insect viruses that is defined by the T=4 symmetry of virions. We report the complete Euprosterna elaeasa virus (EeV) genome sequence of 5698 nt with no poly(A) tail and two overlapping open reading frames, encoding the replicase and capsid precursor, with approximately 67% amino acid identity to Thosea asigna virus (TaV). The N-terminally positioned 17 kDa protein is released from the capsid precursor by a NPGP motif. EeV has 40 nm non-enveloped isometric particles composed of 58 and 7 kDa proteins. The 3'-end of TaV/EeV is predicted to form a conserved pseudoknot. Replicases of TaV and EeV include a newly delineated VPg signal mediating the protein priming of RNA synthesis in dsRNA Birnaviridae. Results of rooted phylogenetic analysis of replicase and capsid proteins are presented to implicate recombination between monopartite tetraviruses, involving autonomization of a sgRNA, in the emergence of bipartite tetraviruses. They are also used to revise the Tetraviridae taxonomy.
Assuntos
Genoma Viral , Insetos/virologia , Vírus de RNA/genética , RNA Viral/genética , Análise de Sequência de DNA , Animais , Proteínas do Capsídeo/genética , Análise por Conglomerados , Evolução Molecular , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Filogenia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestrutura , RNA Viral/química , RNA Polimerase Dependente de RNA/genética , Homologia de Sequência de Aminoácidos , Sintenia , Vírion/ultraestruturaRESUMO
The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.
Assuntos
Anti-Infecciosos/farmacologia , Mariposas/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/metabolismo , Sequência de Bases , Hemolinfa/química , Hemolinfa/metabolismo , Dados de Sequência Molecular , Mariposas/genética , Mariposas/metabolismo , Peptídeos/genética , Peptídeos/metabolismoRESUMO
RNA interference (RNAi) is an important defence against viruses and transposable elements (TEs). RNAi not only protects against viruses by degrading viral RNA, but hosts and viruses can also use RNAi to manipulate each other's gene expression, and hosts can encode microRNAs that target viral sequences. In response, viruses have evolved a myriad of adaptations to suppress and evade RNAi. RNAi can also protect cells against TEs, both by degrading TE transcripts and by preventing TE expression through heterochromatin formation. The aim of our review is to summarize and evaluate the current data on the evolution of these RNAi defence mechanisms. To this end, we also extend a previous analysis of the evolution of genes of the RNAi pathways. Strikingly, we find that antiviral RNAi genes, anti-TE RNAi genes and viral suppressors of RNAi all evolve rapidly, suggestive of an evolutionary arms race between hosts and parasites. Over longer time scales, key RNAi genes are repeatedly duplicated or lost across the metazoan phylogeny, with important implications for RNAi as an immune defence.
Assuntos
Evolução Molecular , Interferência de RNA/imunologia , Animais , Elementos de DNA Transponíveis , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , MicroRNAs/genética , MicroRNAs/imunologia , Filogenia , RNA Viral/genética , RNA Viral/imunologia , Vírus/genética , Vírus/imunologia , Vírus/patogenicidadeRESUMO
The peritrophic matrix from the midgut of the caterpillar, Helicovera armigera, was solubilized by treatment with anhydrous trifluoromethanesulfonic acid, apparently by depolymerisation of its chitin component. This allowed the efficient extraction of proteins in a technique that may be broadly applicable to the analysis of other structures containing chitin. Gel electrophoresis and mass spectrometry of tryptic peptides were used to identify the extracted proteins with gut-expressed cDNA sequences. The major proteins of this cohesive, digestion-resistant structure are chitin deacetylase-like and mucin-like proteins, the latter with multiple chitin-binding domains that may cross-link chitin fibrils to provide a barrier against abrasive food particles and parasites, one of the major functions of the matrix. Other proteins found in the H. armigera gut peritrophic matrix suggest that the matrix is a dynamic, complex structure that may participate in the immobilization of digestive enzymes, actively protect the gut from parasite invasion and intercept toxins such as lectins and Bacillus thuringiensis crystal proteins.