RESUMEN
Cowpea is a widely cultivated and major nutritional source of protein for many people that live in West Africa. Annual yields and longevity of grain storage is greatly reduced by feeding damage caused by a complex of insect pests that include the pod sucking bugs, Anoplocnemis curvipes Fabricius (Hemiptera: Coreidae) and Clavigralla tomentosicollis Stål (Hemiptera: Coreidae); as well as phloem-feeding cowpea aphids, Aphis craccivora Koch (Hemiptera: Aphididae) and flower thrips, Megalurothrips sjostedti Trybom (Thysanoptera: Thripidae). Efforts to control these pests remain a challenge and there is a need to understand the structure and movement of these pest populations in order to facilitate the development of integrated pest management strategies (IPM). Molecular tools have the potential to help facilitate a better understanding of pest populations. Towards this goal, we used 454 pyrosequencing technology to generate 319,126, 176,262, 320,722 and 227,882 raw reads from A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti, respectively. The reads were de novo assembled into 11,687, 7,647, 10,652 and 7,348 transcripts for A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti, respectively. Functional annotation of the resulting transcripts identified genes putatively involved in insecticide resistance, pathogen defense and immunity. Additionally, sequences that matched the primary aphid endosymbiont, Buchnera aphidicola, were identified among A. craccivora transcripts. Furthermore, 742, 97, 607 and 180 single nucleotide polymorphisms (SNPs) were respectively predicted among A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti transcripts, and will likely be valuable tools for future molecular genetic marker development. These results demonstrate that Roche 454-based transcriptome sequencing could be useful for the development of genomic resources for cowpea pest insects in West Africa.
Asunto(s)
Fabaceae/parasitología , Insectos/fisiología , Control de Plagas , Transcriptoma , África Occidental , Animales , Genes de Insecto , Insectos/genética , Metagenoma , Polimorfismo de Nucleótido SimpleRESUMEN
Cowpea (Vigna unguiculata spp unguiculata) is adapted to the drier agro-ecological zones of West Africa where it is a major source of dietary protein and widely used as a fodder crop. Improving the productivity of cowpea can enhance food availability and security in West Africa. Insect predation--predominately from the legume pod borer (Maruca vitrata), flower thrips (Megalurothrips sjostedti) and a complex of pod-sucking bugs (e.g., Clavigralla spp)--is a major yield-limiting factor in West African cowpea production. Dramatic increases in yield are shown when M. vitrata is controlled with insecticides. However, availability, costs, and safety considerations limit pesticides as a viable option for boosting cowpea production. Development of Bt-cowpea through genetic modification (GM) to control the legume pod borer is a promising approach to cowpea improvement. Cowpea expressing the lepidopteran-active Cry1Ab protein from Bacillus thuringiensis is being developed as a first generation Bt-cowpea crop for West Africa. Appropriate stewardship of Bt-cowpea to assure its sustainability under West African conditions is critical to its successful development. A first step in this process is an environmental risk assessment to determine the likelihood and magnitude of adverse effects of the Cry1Ab protein on key environmental protection goals in West Africa. Here we describe the results of an expert panel convened in 2009 to develop the problem formulation phase for Bt-cowpea and to address specific issues around gene flow, non-target arthropods, and insect resistance management.
Asunto(s)
Proteínas Bacterianas/genética , Endotoxinas/genética , Fabaceae/genética , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente/genética , África Occidental , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Monitoreo del Ambiente/legislación & jurisprudencia , Monitoreo del Ambiente/métodos , Fabaceae/crecimiento & desarrollo , Fabaceae/parasitología , Expresión Génica , Flujo Génico , Interacciones Huésped-Parásitos , Hibridación Genética , Insectos/fisiología , Control Biológico de Vectores/economía , Control Biológico de Vectores/legislación & jurisprudencia , Control Biológico de Vectores/métodos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/parasitología , Medición de Riesgo/legislación & jurisprudencia , Medición de Riesgo/métodosRESUMEN
The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation.
Asunto(s)
Frutas/parasitología , Perfilación de la Expresión Génica/métodos , Lepidópteros/genética , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , África , Animales , ADN Complementario/genética , Bases de Datos Genéticas , Sistema Digestivo/metabolismo , Etiquetas de Secuencia Expresada , Genes de Insecto/genética , Marcadores Genéticos , Genética de Población , Genotipo , Geografía , Larva/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia , Análisis de Regresión , Glándulas Salivales/metabolismo , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
We report the assembly of the 14,054 bp near complete sequencing of the mitochondrial genome of the legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae), which we subsequently used to estimate divergence and relationships within the lepidopteran lineage. The arrangement and orientation of the 13 protein-coding, 2 rRNA, and 19 tRNA genes sequenced was typical of insect mitochondrial DNA sequences described to date. The sequence contained a high A+T content of 80.1% and a bias for the use of codons with A or T nucleotides in the 3rd position. Transcript mapping with midgut and salivary gland ESTs for mitochondrial genome annotation showed that translation from protein-coding genes initiates and terminates at standard mitochondrial codons, except for the coxI gene, which may start from an arginine CGA codon. The genomic copy of coxII terminates at a T nucleotide, and a proposed polyadenylation mechanism for completion of the TAA stop codon was confirmed by comparisons to EST data. EST contig data further showed that mature M. vitrata mitochondrial transcripts are monocistronic, except for bicistronic transcripts for overlapping genes nd4/nd4L and nd6/cytb, and a tricistronic transcript for atp8/atp6/coxIII. This processing of polycistronic mitochondrial transcripts adheres to the tRNA punctuated cleavage mechanism, whereby mature transcripts are cleaved only at intervening tRNA gene sequences. In contrast, the tricistronic atp8/atp6/coxIII in Drosophila is present as separate atp8/atp6 and coxIII transcripts despite the lack of an intervening tRNA. Our results indicate that mitochondrial processing mechanisms vary between arthropod species, and that it is crucial to use transcriptional information to obtain full annotation of mitochondrial genomes.
Asunto(s)
Perfilación de la Expresión Génica , Genoma Mitocondrial/genética , Lepidópteros/genética , Análisis de Secuencia de ADN , Animales , Mapeo Cromosómico , Cromosomas de Insectos , Codón/análisis , Codón/genética , Etiquetas de Secuencia Expresada , Frutas/parasitología , Genoma de los Insectos/genética , Lepidópteros/fisiología , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , FilogeniaRESUMEN
Maruca vitrata Fabricius is a pantropical lepidopteran pest of legumes. Phylogenetic analysis of a mitochondrial cytochrome c oxidase-I gene (cox1) fragment indicates that three Maruca sp. mitochondrial lineages have unique geographic distributions [lineages 1 and 2: Australia, Taiwan, and West Africa (Niger, Nigeria, and Burkina Faso), and lineage 3: Puerto Rico]. The haplotype (T30, T114) is specific to lineages 1&2 and was assayed by NsiI and SacI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) within population samples; it was not observed in the Puerto Rican samples, but was nearly fixed among samples from West Africa, Australia and Taiwan (85.5-100%). Re-sequencing and phylogenetic analyses of PCR-RFLP defined cox1 haplotypes indicate that nucleotide diversity is highest among samples from West Africa. Phylogenetic reconstruction based upon ribosomal DNA (rDNA) internal transcribed spacer-2 (ITS-2) sequences provided additional evidence for three Maruca sp. clades. These data suggest that multiple unique Maruca species or subspecies are present worldwide, which has implications for the management of this pest species-complex.
Asunto(s)
Lepidópteros/genética , África , Animales , Australia , ADN Intergénico/genética , ADN Ribosómico/genética , Genética de Población , Geografía , Haplotipos , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Puerto Rico , TaiwánRESUMEN
The biopesticide Spinosad controls many insect pests of stored-food products. Laboratory and field trials were carried out to determine the efficacy of this pesticide against the cowpea weevil, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), the main storage pest of cowpea, Vigna unguiculata, Walp, in West Africa. In the laboratory, Spinosad caused high mortality of adult C. maculatus and decreased the number of eggs laid by females. Spinosad, however, was less toxic in the 24 h treatment to C. maculatus than deltamethrin, an insecticide commonly used in Burkina Faso to control this insect. In "on-farm" experiments, Spinosad was effective in controlling C. maculatus. After 6 mo of storage, the number of insects emerging from cowpeas seeds was reduced by >80% by coating seeds with Spinosad but only by 43% by coating with deltamethrin. Less than 20% of the seeds were perforated in the Spinosad treatment compared with 29% for deltamethrin. Spinosad controlled C. maculatus throughout the 6 mo of cowpea storage whereas deltamethrin failed to control C. maculatus after 3 mo of storage. Spinosad has the potential to be more effective in controlling C. maculatus than deltamethrin.