RESUMO
1,3-Bis(pentafluorophenyl-imino)isoindoline (A(F)) and 3,6-di-tert-butyl-1,8-bis(pentafluorophenyl)-9H-carbazole (B(F)) have been designed as preorganized anion receptors that exploit anion-π interactions, and their ability to bind chloride and bromide in various solvents has been evaluated. Both receptors A(F) and B(F) are neutral but provide a central NH hydrogen bond that directs the halide anion into a preorganized clamp of the two electron-deficient appended arenes. Crystal structures of host-guest complexes of A(F) with DMSO, Cl(-), or Br(-) (A(F):DMSO, A(F):Cl(-), and A(2)(F):Br(-)) reveal that in all cases the guest is located in the cleft between the perfluorinated flaps, but NMR spectroscopy shows a more complex situation in solution because of E,Z/Z,Z isomerism of the host. In the case of the more rigid receptor B(F), Job plots evidence 1:1 complex formation with Cl(-) and Br(-), and association constants up to 960 M(-1) have been determined depending on the solvent. Crystal structures of B(F) and B(F):DMSO visualize the distinct preorganization of the host for anion-π interactions. The reference compounds 1,3-bis(2-pyrimidylimino)isoindoline (A(N)) and 3,6-di-tert-butyl-1,8-diphenyl-9H-carbazole (B(H)), which lack the perfluorinated flaps, do not show any indication of anion binding under the same conditions. A detailed computational analysis of the receptors A(F) and B(F) and their host-guest complexes with Cl(-) or Br(-) was carried out to quantify the interactions in play. Local correlation methods were applied, allowing for a decomposition of the ring-anion interactions. The latter were found to contribute significantly to the stabilization of these complexes (about half of the total energy). Compounds A(F) and B(F) represent rare examples of neutral receptors that are well preorganized for exploiting anion-π interactions, and rare examples of receptors for which the individual contributions to the binding energy have been quantified.
RESUMO
Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.
Assuntos
Adaptação Fisiológica/genética , Genoma de Inseto , Herbivoria , Spodoptera/genética , Animais , Produtos Agrícolas , Larva/genética , Especificidade da EspécieRESUMO
Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/toxicidade , Besouros/efeitos dos fármacos , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Animais , Toxinas de Bacillus thuringiensis , Linhagem Celular , Besouros/genética , Feminino , Resistência a Inseticidas/genética , MasculinoRESUMO
Polyphagous insect herbivores are adapted to many different secondary metabolites of their host plants. However, little is known about the role of ATP-binding cassette (ABC) transporters, a multigene family involved in detoxification processes. To study the larval response of the generalist Helicoverpa armigera (Lepidoptera) and the putative role of ABC transporters, we performed developmental assays on artificial diet supplemented with secondary metabolites from host plants (atropine-scopolamine, nicotine and tomatine) and non-host plants (taxol) in combination with a replicated RNAseq experiment. A maximum likelihood phylogeny identified the subfamily affiliations of the ABC transporter sequences. Larval performance was equal on the atropine-scopolamine diet and the tomatine diet. For the latter we could identify a treatment-specific upregulation of five ABC transporters in the gut. No significant developmental difference was detected between larvae fed on nicotine or taxol. This was also mirrored in the upregulation of five ABC transporters when fed on either of the two diets. The highest number of differentially expressed genes was recorded in the gut samples in response to feeding on secondary metabolites. Our results are consistent with the expectation of a general detoxification response in a polyphagous herbivore. This is the first study to characterize the multigene family of ABC transporters and identify gene expression changes across different developmental stages and tissues, as well as the impact of secondary metabolites in the agricultural pest H. armigera.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Mariposas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Herbivoria , Inativação Metabólica/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Paclitaxel/metabolismo , Filogenia , Metabolismo Secundário/genética , Alcaloides de Solanáceas/metabolismoRESUMO
Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.
Assuntos
Proteínas de Bactérias/farmacologia , Caderinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Mariposas/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Animais , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Caderinas/metabolismo , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/microbiologia , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células Sf9RESUMO
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
Specialist insect herbivores have evolved efficient ways to adapt to the major defenses of their host plants. Although Manduca sexta, specialized on Solanaceous plants, has become a model organism for insect molecular biology, little is known about its adaptive responses to the chemical defenses of its hosts. To study larval performance and transcriptomic responses to host and non-host plants, we conducted developmental assays and replicated RNAseq experiments with Manduca larvae fed on different Solanaceous plants as well as on a Brassicaceous non-host plant, Brassica napus. Manduca larvae developed fastest on Nicotiana attenuata, but no significant differences in performance were found on larvae fed on other Solanaceae or the non-host B. napus. The RNAseq experiments revealed that Manduca larvae display plastic responses at the gene expression level, and transcriptional signatures specific to the challenges of each host- and non-host plant. Our observations are not consistent with expectations that specialist herbivores would perform poorly on non-host plants. Instead, our findings demonstrate the ability of this specialized insect herbivore to efficiently use a larger repertoire of host plants than it utilizes in the field.