ABSTRACT
The complete mitochondrial genome of the tarnished plant bug, Lygus lineolaris, comprised 17,027 bp. The genome contained 13 protein coding regions, 22 tRNA genes and 2 ribosomal RNA genes. The gene arrangement corresponded to the common order found among insect mtDNAs which was considered to be the ancestral arrangement. The protein coding genes started with ATN and stopped with TAA or TAG. The nucleotide distribution was 76.0% A + T. The control region contained two repeat regions, one was 24 bp and the other was 161 bp. The Genbank accession for the complete L. lineolaris mt genome is EU401991.
Subject(s)
Genome, Insect , Genome, Mitochondrial , Heteroptera/genetics , Animals , Base Pairing/genetics , Base Sequence , DNA, Mitochondrial/genetics , Molecular Sequence AnnotationABSTRACT
Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.
Subject(s)
Bacterial Proteins/toxicity , Biosynthetic Pathways/drug effects , Endotoxins/toxicity , Energy Metabolism/drug effects , Hemolysin Proteins/toxicity , Tenebrio/drug effects , Tenebrio/metabolism , Transcriptome/drug effects , Administration, Oral , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/administration & dosage , Base Sequence , DNA, Complementary/genetics , Endotoxins/administration & dosage , Gene Expression Profiling , Hemolysin Proteins/administration & dosage , Larva/drug effects , Larva/metabolism , Microarray Analysis , Molecular Sequence Data , Sequence Analysis, DNA , Tenebrio/genetics , Time FactorsABSTRACT
We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene.
Subject(s)
Embryonic Development , Houseflies/enzymology , Insect Proteins/genetics , Ornithine Decarboxylase/metabolism , Amino Acid Sequence , Animals , Blotting, Southern , Cloning, Molecular , DNA, Complementary/isolation & purification , Houseflies/genetics , Houseflies/growth & development , Molecular Sequence Data , Ornithine Decarboxylase/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
The transcriptome response of Tribolium castaneum larvae to dietary protease inhibitors was evaluated by whole-genome microarray analysis. RNA was isolated from guts of larvae fed control diet (no inhibitor), or diets containing 0.1% E-64 (cysteine protease inhibitor), 5.0% soybean trypsin inhibitor (STI, serine protease inhibitor), or a combination of 0.1% E-64 and 5.0% STI. Data were analyzed by pairwise analysis, in which each inhibitor treatment group was compared to control, or ANOVA of all treatment groups. In pairwise analysis, the expression of only 253 genes was significantly altered (p<0.05) in response to STI treatment, whereas E-64 and combination treatments resulted in 1574 and 1584 differentially regulated genes. The data indicate that treatments containing E-64, whether alone or in combination, significantly impacts gene expression in T. castaneum larvae. ANOVA analysis revealed 2175 genes differentially expressed in inhibitor-treated larvae compared to control (p<0.05), including genes related to proteases that were mostly up-regulated, namely cathepsins B and L, chymotrypsins, and nonproteolytic cysteine cathepsin or serine protease homologs. Inhibitor treatments induced the differential expression of other gut-related genes, as well as genes encoding proteins of unknown function. These data suggest that T. castaneum larvae compensate for dietary cysteine protease inhibitors by altering large-scale gene expression patterns.
Subject(s)
Cysteine Proteinase Inhibitors/pharmacology , Microarray Analysis , Serine Proteinase Inhibitors/pharmacology , Tribolium/genetics , Tribolium/metabolism , Animals , Chymotrypsin/genetics , Chymotrypsin/metabolism , Cysteine Proteinase Inhibitors/metabolism , Drug Combinations , Gene Expression Profiling , Larva/metabolism , Leucine/analogs & derivatives , Leucine/metabolism , Leucine/pharmacology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Glycine max/genetics , Glycine max/metabolism , Trypsin Inhibitors/pharmacologyABSTRACT
The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability as normalizers in comparisons across tissues and/or developmental stages. In less diverse samples, such as comparisons within developmental stages or tissue only, normalizers for mRNA were more stable and consistent. Overall, the genes for ribosomal proteins rps6, rpl13a, rps3, and rps18 were the most stable normalizers for broad scale gene expression analysis in T. castaneum. However, their stability ranking was dependent upon the instrument as well as the analysis program. These data emphasize the need to optimize normalizers used in all real-time polymerase chain reaction experiments specifically for the experimental conditions and thermocycler and to carefully evaluate data generated by computational algorithms.
Subject(s)
Genes, Insect , Polymerase Chain Reaction/methods , Tribolium/genetics , Animals , Base Sequence , DNA Primers , Life Cycle Stages , Tribolium/physiologyABSTRACT
To investigate gene expression in Tribolium castaneum exposed to Beauveria bassiana, reference genes for qPCR were evaluated. Of these, beta-actin, alpha-tubulin, and RPS6 were not stable. The most stable were ribosomal protein genes, RPS3, RPS18, and RPL13a. Syntaxin1, syntaxin6, and E-cadherin may be appropriate for some experimental systems.
