Efficacy of a cry1Ab Gene for Control of Maruca vitrata (Lepidoptera: Crambidae) in Cowpea (Fabales: Fabaceae).

Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20-80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

Regulatory considerations surrounding the deployment of Bt-expressing cowpea in Africa: report of the deliberations of an expert panel.

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.

Transcriptome sequencing, and rapid development and application of SNP markers for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae).

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.

Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae).

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.

Geographic distribution of phylogenetically-distinct legume pod borer, Maruca vitrata (Lepidoptera: Pyraloidea: Crambidae).

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.