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

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

Systems-Scale Analysis Reveals Pathways Involved in Cellular Response to Methamphetamine

Background: Methamphetamine (METH), an abused illicit drug, disrupts many cellular processes, including energy metabolism, spermatogenesis, and maintenance of oxidative status. However, many components of the molecular underpinnings of METH toxicity have yet to be established. Network analyses of integrated proteomic, transcriptomic and metabolomic data are particularly well suited for identifying cellular responses to toxins, such as METH, which might otherwise be obscured by the numerous and dynamic changes that are induced. Methodology/Results: We used network analyses of proteomic and transcriptomic data to evaluate pathways in Drosophila melanogaster that are affected by acute METH toxicity. METH exposure caused changes in the expression of genes involved with energy metabolism, suggesting a Warburg-like effect (aerobic glycolysis), which is normally associated with cancerous cells. Therefore, we tested the hypothesis that carbohydrate metabolism plays an important role in METH toxicity. In agreement with our hypothesis, we observed that increased dietary sugars partially alleviated the toxic effects of METH. Our systems analysis also showed that METH impacted genes and proteins known to be associated with muscular homeostasis/ contraction, maintenance of oxidative status, oxidative phosphorylation, spermatogenesis, iron and calcium homeostasis. Our results also provide numerous candidate genes for the METH-induced dysfunction of spermatogenesis, which have not been previously characterized at the molecular level. Conclusion: Our results support our overall hypothesis that METH causes a toxic syndrome that is characterized by the altered carbohydrate metabolism, dysregulation of calcium and iron homeostasis, increased oxidative stress, and disruption of mitochondrial functions

Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)+-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized

Molecular tools for characterization of the legume pod borer Maruca vitrata fabricius (lepidoptera: pyraloidea: crambidae); mode of action of hermetic storage of cowpea grain

Cowpea [Vigna unguiculata subsp. unguiculata (L.) Walp] is an important food legume cultivated widely in the arid and semi-arid tropics of the world. Insects are the major challenge to cowpea production and storage in West Africa, where nearly two thirds of the world\u27s cowpea is produced and consumed. In the introductory chapter, I discuss the importance of cowpea, its major field and storage problems, and recent advances in biotechnology that offer hope for solving these problems. In the first chapter, I present results from a field survey looking for wild host plants of Maruca vitrata to assess if these can serve as natural refuges when Bt-cowpea is deployed. In the second, third and fourth chapters I describe (i) applications of the molecular barcoding approach for studying global populations of M. vitrata; (ii) results from expressed sequence tag library sequencing of M. vitrata and molecular marker discovery; and (iii) the mitochondrial genome of M. vitrata. In the fifth chapter, I discuss the mode of action of hermetic storage of cowpea grain. In the first major section of the work, two aspects of insect resistance management - needed for M. vitrata resistant Bt-cowpea deployment — have been investigated. The first is the status of wild alternate host plants of M. vitrata in the cowpea growing regions of northern Nigeria (Kano and Zaria) and southern Niger (Maradi). Results from field surveys done in these regions suggest that wild hosts are not abundantly present and thus aren\u27t available to serve as refugia for M. vitrata. Lack of molecular markers for M. vitrata was one of the constraints to characterizing its populations. I sought to fill this gap by generating genomic DNA- as well as EST-based molecular markers. I also demonstrated that molecular barcoding using cytochrome c oxidase I can be applied to distinguish global populations of M. vitrata. Data from the barcoding study strongly suggests that the genus Maruca may be a species complex or has undergone a recent genetic bottle-neck. In the second major section of the dissertation — dealing with the mode of action of hermetic storage of cowpea grain — I was able to estimate that each cowpea bruchid requires approximately 8 -10 ml of pure O2 during its development to adult hood. Feeding behavior studies, done to investigate the cause for feeding cessation in storage, show that it is the level of O2, not CO2, that influences the bruchid feeding behavior. More importantly the larval susceptibility, visible evidence in the form of desiccated larvae, and the respiratory quotients during development suggest that inability of bruchids to make water in the absence of O2 may be a major contributing factor to the success of the hermetic method of storing cowpea grain

Simplify, simplify: Lifestyle and compact genome of the body louse provide a unique functional genomics opportunity

The body louse, with its recently sequenced genome, is now primed to serve as a powerful model organism for addressing fundamental questions relating to how insects interact with their environment. One characteristic of the body louse that facilitates this research is the size of its genome—the smallest insect genome sequenced to date. This diminutive genome must nonetheless control an organism that senses and responds to its environment, reacting to threats of corporal and genomic integrity. Additionally, the body louse transmits several important human diseases compared to its very close relative, the head louse, which does not. Therefore, these two organisms comprise an excellent model system for studying molecular mechanisms associated with vector competence. To understand more fully the development of vector/pathogen interactions, we have developed an in vitro bioassay system and determined that the body louse genome appears to contain the genes necessary for RNAi. The body louse will therefore be useful for determining the set of conditions permissive to the evolution of vector competence

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.This article is from PLOS ONE; 6 (2011); e 16444; doi:10.1371/journal.pone.0016444</p