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.

Effects of PICS bags on insect pests of sorghum during long-term storage in Burkina Faso.

The PICS bags, originally developed for cowpea storage, were evaluated for sorghum (Sorghum bicolor) preservation. Batches of 25 kg of sorghum grain were stored in 50 kg PICS or polypropylene (PP) bags under ambient conditions for 12 months and assessed for the presence of insect pests and their damage, seed viability and, oxygen and carbon dioxide variations. The grain was incubated for 35 days to assess whether any insects would emerge. After six months of storage, oxygen levels decreased in the PICS bags compared to polypropylene bags. After 12 months of storage, only two pests, Rhyzopertha dominica and Sitophilus zeamais were found in the PICS bags. However, in PP bags there were additional pests including Tribolium castaneum and Oryzeaphilus mercator and Xylocoris flavipes. Grain weight loss and damage caused by these insects in the PP bags were significantly higher compared to those stored in PICS bags. Germination rates of sorghum grains stored in PP bags decreased significantly while no changes were observed in grains stored in PICS bags when compared to the initial germination. After the incubation post storage period, there was a resurgence of R. dominica in sorghum grains from PICS bags but the population levels were significantly lower compared to polypropylene bags. PICS bags preserved the quality and viability of stored sorghum grains and protected it from key insect pests. The PICS technology is effective for long-term sorghum storage but the potential resurgence of insects in low-oxygen environment calls for further research.

Development of an Optimum Diet for Mass Rearing of the Rice Moth, Corcyra cephalonica (Lepidoptera: Pyralidae), and Production of the Parasitoid, Habrobracon hebetor (Hymenoptera: Braconidae), for the Control of Pearl Millet Head Miner.

The rice moth, Corcyra cephalonica Stainton, an alternate host for the production of the parasitoid, Habrobracon hebetor Say, was reared on different diets, including pearl millet [Pennisetum glaucum (L.) R. Br.] (Poales: Poaceae) flour only, and in combinations of flours of sorghum [Sorghum bicolor (L.) Moench] (Poales: Poaceae), peanut (Arachis hypogea L.) (Fabales: Fabaceae), and cowpea [Vigna unguiculata (L.) Walp.] (Fabales: Fabaceae) to identify the optimal and economical proportion to be used under the conditions of Niger. The addition of cowpea or peanut to the pearl millet diet slightly increased C. cephalonica larval development time. Likewise, the addition of cowpea or peanut to cereal diets yielded a higher C. cephalonica larval survival. Female moths emerging from larvae fed on cereal and legume mixed diets produced higher eggs compared to the ones fed on sole and mixed cereals. Among legumes, cowpea addition is most interesting in terms of cost/production of C. cephalonica larvae. However, female moths emerging from larvae fed on different millet cowpea mix (5, 25, and 50%) laid significantly more eggs than those fed on sole pearl millet. Further, individual C. cephalonica larvae fed on 75% pearl millet + 25% cowpea produced significantly more H. hebetor. With an initial 25 C. cephalonica larvae kept for a 3-mo rearing period, the number of H. hebetor parasitoids produced will reach 2.68-10.07 million. In terms of cost/production ratio, the 75% pearl millet: 25% cowpea yielded better results.

Obstacles to integrated pest management adoption in developing countries.

Despite its theoretical prominence and sound principles, integrated pest management (IPM) continues to suffer from anemic adoption rates in developing countries. To shed light on the reasons, we surveyed the opinions of a large and diverse pool of IPM professionals and practitioners from 96 countries by using structured concept mapping. The first phase of this method elicited 413 open-ended responses on perceived obstacles to IPM. Analysis of responses revealed 51 unique statements on obstacles, the most frequent of which was "insufficient training and technical support to farmers." Cluster analyses, based on participant opinions, grouped these unique statements into six themes: research weaknesses, outreach weaknesses, IPM weaknesses, farmer weaknesses, pesticide industry interference, and weak adoption incentives. Subsequently, 163 participants rated the obstacles expressed in the 51 unique statements according to importance and remediation difficulty. Respondents from developing countries and high-income countries rated the obstacles differently. As a group, developing-country respondents rated "IPM requires collective action within a farming community" as their top obstacle to IPM adoption. Respondents from high-income countries prioritized instead the "shortage of well-qualified IPM experts and extensionists." Differential prioritization was also evident among developing-country regions, and when obstacle statements were grouped into themes. Results highlighted the need to improve the participation of stakeholders from developing countries in the IPM adoption debate, and also to situate the debate within specific regional contexts.

Development of reference transcriptomes for the major field insect pests of cowpea: a toolbox for insect pest management approaches in west Africa.

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.

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.