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In JoVE (1)
Other Publications (19)
- Insect Biochemistry and Molecular Biology
- Insect Biochemistry and Molecular Biology
- Molecular Plant-microbe Interactions : MPMI
- Journal of Insect Physiology
- Archives of Insect Biochemistry and Physiology
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Insect Science (Online)
- Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
- Journal of Insect Physiology
- PloS One
- Journal of Insect Physiology
- Proceedings of the National Academy of Sciences of the United States of America
- PloS One
- PloS One
- PloS One
- PloS One
- Journal of Insect Physiology
- Journal of Medical Entomology
- BMC Genomics
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Articles by Omprakash Mittapalli in JoVE
JoVE General
ניתוח של ביטוי גנים בורר אש אמרלד ( Agrilus planipennis) באמצעות Real Time-PCR כמותי
Department of Entomology, The Ohio State University
כמותי בזמן אמת PCR (qRT-PCR) היא כלי יעיל לאבחון רמות ה-mRNA ברקמות חרקים שונים, שלבי התפתחות. בדוח זה אנו מציגים את השימוש qRT-PCR כדי לברר רמות ה-mRNA ברקמות הזחל שונים שלבי ההתפתחות של מינים פולשניים חרקים, דקור ברקת אפר.
Other articles by Omprakash Mittapalli on PubMed
Differential Expression of Two Cytochrome P450 Genes in Compatible and Incompatible Hessian Fly/wheat Interactions
We have recovered two Hessian fly cytochrome P450 cDNAs from an ongoing midgut EST project. CYP6AZ1 and CYP6BA1 represent two new subfamilies within the CYP6 family. The deduced amino acid sequences for CYP6AZ1 and CYP6BA1 show conserved structural and functional domains of insect P450s. Expression analysis with reverse transcription-polymerase chain reaction (RT-PCR) indicated that CYP6AZ1 is midgut specific and induced during active larval feeding, whereas CYP6BA1 was expressed in all tissues and developmental stages examined. Further expression analysis of CYP6AZ1 with RT-PCR in compatible and incompatible Hessian fly/wheat interactions suggested that CYP6AZ1 may be required for larval feeding in compatible interactions. These results should lead to a better understanding of the Hessian fly/wheat interaction with emphasis on the larval midgut as a critical interface with its host plant.
Characterization of a Serine Carboxypeptidase in the Salivary Glands and Fat Body of the Orange Wheat Blossom Midge, Sitodiplosis Mosellana (Diptera: Cecidomyiidae)
A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.
Gene-for-gene Defense of Wheat Against the Hessian Fly Lacks a Classical Oxidative Burst
Genetic similarities between plant interactions with microbial pathogens and wheat interactions with Hessian fly larvae prompted us to investigate defense and counterdefense mechanisms. Plant oxidative burst, a rapid increase in the levels of active oxygen species (AOS) within the initial 24 h of an interaction with pathogens, commonly is associated with defenses that are triggered by gene-for-gene recognition events similar to those involving wheat and Hessian fly larvae. RNAs encoded by Hessian fly superoxide dismutase (SOD) and catalase (CAT) genes, involved in detoxification of AOS, increased in first-instar larvae during both compatible and incompatible interactions. However, mRNA levels of a wheat NADPH oxidase (NOX) gene that generates superoxide (O2-) did not increase. In addition, inhibiting wheat NOX enzyme with diphenyleneiodonium did not result in increased survival of avirulent larvae. However, nitro blue tetrazolium staining indicated that basal levels of O2- are present in both uninfested and infested wheat tissue. mRNA encoded by wheat genes involved in detoxification of the cellular environment, SOD, CAT, and glutathione-S-transferase did not increase in abundance. Histochemical staining with 3,3-diaminobenzidine revealed no increases in wheat hydrogen peroxide (H2O2) during infestation that were correlated with the changes in larval SOD and CAT mRNA. However, treatment with 2',7'-dichlorofluorescin demonstrated the presence of basal levels of H2O2 in the elongation zone of both infested and uninfested plants. The accumulation of a wheat flavanone 3-hydroxylase mRNA did show some parallels with larval gene mRNA profiles. These results suggested that larvae encounter stresses imposed by mechanisms other than an oxidative burst in wheat seedlings.
Expression Patterns of Antibacterial Genes in the Hessian Fly
We report on the transcriptional patterns of three antibacterial genes, a defensin (MdesDEF-1), a diptericin (MdesDIP-1) and a lysozyme (MdesLYS-1), during development in Hessian fly, Mayetiola destructor. Quantitative analysis by real-time PCR of mRNA levels in different tissues revealed a predominance of the transcripts for all three genes in the midgut, while analysis during development revealed greatest abundance in mRNA during the 3rd-instar. An evaluation of the midgut lumen revealed the presence of a diverse bacterial flora in larvae maintained on susceptible wheat. Further, the titer of bacteria in the midgut increased approximately 250-fold from the 1st-instar through the 2nd-instar. However, no detectable titer of bacteria was observed from the midgut lumen of larvae maintained on resistant plants. PCR amplicons produced using primers designed to conserved regions of the Pseudomonas 16S rRNA gene supported taxonomic identification for some of the bacteria comprising the midgut flora as belonging to the genus Pseudomonas. Analysis of mRNA for the Hessian fly antibacterial genes in larvae feeding on susceptible and resistant plants revealed an increase in the transcript level for MdesDEF-1 in 1st-instar larvae on susceptible plants, while the transcript levels for MdesDIP-1 and MdesLYS-1 were constant. Results suggest the transcriptional patterns of the Hessian fly antibacterial genes observed could be associated with the developing midgut bacterial flora present in larvae feeding on susceptible wheat as well as microbial challenge encountered at other stages in development.
CDNA Cloning and Transcriptional Expression of a Peritrophin-like Gene in the Hessian Fly, Mayetiola Destructor [Say]
One of the well-studied components of the insect gut is the peritrophic matrix (PM). This semipermeable structure primarily functions in digestion, and protection against invasive microorganisms and mechanical damage. We report the cDNA cloning and transcription profiles of a peritrophin-A like gene (designated MdesPERI-A1) in the Hessian fly Mayetiola destructor. The predicted amino acid sequence of MdesPERI-A1 revealed a putative secretion signal peptide at its amino terminus, similarity to peritrophins from other insects including dipterans, and the presence of two chitin binding domains each containing six cysteine residues. Quantitative expression analysis of MdesPERI-A1 mRNA in different larval tissues revealed the transcript to be predominantly present in the midgut (597.9-fold) compared to other tissues assayed including salivary glands and fat bodies. Spatial expression patterns during development showed a peak expression of MdesPERI-A1 in the feeding second-instars (146-fold) and a decline in expression in the pupal and adult stages. Transcription profiling of MdesPERI-A1 during compatible (larvae on susceptible plants) and incompatible (larvae on resistant plants) interactions with wheat revealed a greater level (1.7-fold) of MdesPERI-A1 transcript in larvae on resistant plants in the initial time point examined. However, MdesPERI-A1 expression declined in larvae on resistant plants at the later time points.
Antioxidant Defense Response in a Galling Insect
Herbivorous insect species are constantly challenged with reactive oxygen species (ROS) generated from endogenous and exogenous sources. ROS produced within insects because of stress and prooxidant allelochemicals produced by host plants in response to herbivory require a complex mode of antioxidant defense during insect/plant interactions. Some insect herbivores have a midgut-based defense against the suite of ROS encountered. Because the Hessian fly (Mayetiola destructor) is the major insect pest of wheat worldwide, and an emerging model for all gall midges, we investigated its antioxidant responses during interaction with its host plant. Quantitative data for two phospholipid glutathione peroxidases (MdesPHGPX-1 and MdesPHGPX-2), two catalases (MdesCAT-1 and MdesCAT-2), and two superoxide dismutases (MdesSOD-1 and MdesSOD-2) revealed high levels of all of the mRNAs in the midgut of larvae on susceptible wheat (compatible interaction). During development of the Hessian fly on susceptible wheat, a differential expression pattern was observed for all six genes. Analysis of larvae on resistant wheat (incompatible interaction) compared with larvae on susceptible wheat showed increased levels of mRNAs in larvae on resistant wheat for all of the antioxidant genes except MdesSOD-1 and MdesSOD-2. We postulate that the increased mRNA levels of MdesPHGPX-1, MdesPHGPX-2, MdesCAT-1, and MdesCAT-2 reflect responses to ROS encountered by larvae while feeding on resistant wheat seedlings and/or ROS generated endogenously in larvae because of stress/starvation. These results provide an opportunity to understand the cooperative antioxidant defense responses in the Hessian fly/wheat interaction and may be applicable to other insect/plant interactions.
Tissue and Life Stage Specificity of Glutathione S-transferase Expression in the Hessian Fly, Mayetiola Destructor: Implications for Resistance to Host Allelochemicals
Two new Delta and Sigma glutathione S-transferases (GSTs) in the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), were characterized and transcription profiles described. The deduced amino acid sequences for the two M. destructor Delta GSTs (MdesGST-1 and MdesGST-3) showed high similarity with other insect Delta GSTs including the conserved catalytic serine residue. The deduced amino acid sequence for the M. destructor Sigma GST (MdesGST-2) showed high similarity with other insect Sigma GSTs including the conserved glutathione and substrate binding sites. Quantitative tissue expression analysis showed that mRNA levels for MdesGST-1 were predominant in fat body, whereas for MdesGST-2 and MdesGST-3 expression was predominant in the midgut. Temporal expression during development showed peak mRNA levels for MdesGST-1 during larval development, but in the pupal stage for MdesGST-2. MdesGST-3 showed a constitutive expression pattern throughout development. M. destructor feeds on wheat, and expression analysis after feeding indicated that mRNA levels for MdesGST-1 were significantly higher in incompatible interactions in which larvae fed on resistant wheat, while MdesGST-3 was significantly higher in compatible interactions when larvae fed on susceptible wheat. MdesGST-2 showed an equivalent expression pattern during both interactions. These results suggest that the M. destructor Delta GSTs are important in detoxifying wheat allelochemicals during feeding, while Sigma GST participates in metabolism of endogenous substrates.
Molecular Characterization and Responsive Expression of a Defender Against Apoptotic Cell Death Homologue from the Hessian Fly, Mayetiola Destructor
Apoptosis or programmed cell death is an active process occurring in multicellular organisms to maintain growth and development. The Hessian fly, Mayetiola destructor, is rapidly emerging as a model insect species to study insect-plant interactions and to decipher some exceptional physiological phenomena. In this study, we report the characterization and expression profiles of a putative Hessian fly defender against apoptotic cell death (DAD1) homologue designated MdesDAD1. The deduced amino acid sequence of MdesDAD1 revealed significant similarity (75% identity, 9e-42) to other insect and non-insect DAD1 sequences. Phylogenetic analysis grouped MdesDAD1 within a sub-clade consisting of other insect DAD1 homologues. Quantitative analysis indicated constitutive levels of MdesDAD1 mRNA in all the tissues examined but an altered expression pattern during development, wherein the highest mRNA levels observed were prior to pupation. Most interestingly, MdesDAD1 transcript was found to be up-regulated during incompatible (larvae reared on resistant wheat) Hessian fly/wheat interactions compared to compatible (larvae reared on susceptible wheat) interactions. These results suggest MdesDAD1 to have a putative role in the inhibition of unwanted apoptosis triggered during development and in incompatible Hessian fly/wheat interactions. The results obtained provide clues to plausible insect and host-plant factors that could be responsible for the induction of MdesDAD1.
Characterization and Expression Analysis of a Gene Encoding a Secreted Lipase-like Protein Expressed in the Salivary Glands of the Larval Hessian Fly, Mayetiola Destructor (Say)
In a salivary gland transcriptomics study we identified a cDNA with a full-length open reading frame for a gene (MdesL1) encoding a lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say). Fluorescent in situ hybridization on salivary polytenes positioned MdesL1 on the long arm of Autosome 1. BLASTp and conserved domain searches revealed the deduced amino acid sequence contained a lipase superfamily domain with similarity to lipases and phospholipases from other insects. A secretion signal peptide was identified at the amino terminus of the deduced amino acid sequence. Analysis of the transcript of MdesL1 in larval Hessian fly tissues by quantitative real-time PCR (qPCR) revealed the greatest abundance was in salivary glands. Analysis of transcript levels during development showed the greatest level was detected in feeding 1st-instar and early 2nd-instar larvae. Transcript levels increased dramatically over time in larvae feeding on susceptible wheat but were detected at low levels in larvae feeding on resistant wheat. These data suggest the protein encoded by MdesL1 is likely secreted into host-plant cells during larval feeding and could be involved in extra-oral digestion and changes in host-cell permeability or in generating a second messenger in a host-cell-signaling cascade.
Unbiased Transcriptional Comparisons of Generalist and Specialist Herbivores Feeding on Progressively Defenseless Nicotiana Attenuata Plants
Herbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking.
The Gut Transcriptome of a Gall Midge, Mayetiola Destructor
The Hessian fly, Mayetiola destructor, is a serious pest of wheat and an experimental organism for the study of gall midge-plant interactions. In addition to food digestion and detoxification, the gut of Hessian fly larvae is also an important interface for insect-host interactions. Analysis of the genes expressed in the Hessian fly larval gut will enhance our understanding of the overall gut physiology and may also lead to the identification of critical molecules for Hessian fly-host plant interactions. Over 10,000 Expressed Sequence Tags (ESTs) were generated and assembled into 2007 clusters. The most striking feature of the Hessian fly larval transcriptome is the existence of a large number of transcripts coding for so-called small secretory proteins (SSP) with amino acids less than 250. Eleven of the 30 largest clusters were SSP transcripts with the largest cluster containing 11.3% of total ESTs. Transcripts coding for diverse digestive enzymes and detoxification proteins were also identified. Putative digestive enzymes included trypsins, chymotrypsins, cysteine proteases, aspartic protease, endo-oligopeptidase, aminopeptidases, carboxypeptidases, and alpha-amylases. Putative detoxification proteins included cytochrome P450s, glutathione S-transferases, peroxidases, ferritins, a catalase, peroxiredoxins, and others. This study represents the first global analysis of gut transcripts from a gall midge. The identification of a large number of transcripts coding for SSPs, digestive enzymes, detoxification proteins in the Hessian fly larval gut provides a foundation for future studies on the functions of these genes.
Genome Sequences of the Human Body Louse and Its Primary Endosymbiont Provide Insights into the Permanent Parasitic Lifestyle
As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.
Combining Next-generation Sequencing Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species, Aphis Glycines
Aphids are one of the most important insect taxa in terms of ecology, evolutionary biology, genetics and genomics, and interactions with endosymbionts. Additionally, many aphids are serious pest species of agricultural and horticultural plants. Recent genetic and genomic research has expanded molecular resources for many aphid species, including the whole genome sequencing of the pea aphid, Acrythosiphon pisum. However, the invasive soybean aphid, Aphis glycines, lacks in any significant molecular resources.
Tissue-specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus Planipennis)
The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level.
Transcriptomic Signatures of Ash (Fraxinus Spp.) Phloem
Ash (Fraxinus spp.) is a dominant tree species throughout urban and forested landscapes of North America (NA). The rapid invasion of NA by emerald ash borer (Agrilus planipennis), a wood-boring beetle endemic to Eastern Asia, has resulted in the death of millions of ash trees and threatens billions more. Larvae feed primarily on phloem tissue, which girdles and kills the tree. While NA ash species including black (F. nigra), green (F. pennsylvannica) and white (F. americana) are highly susceptible, the Asian species Manchurian ash (F. mandshurica) is resistant to A. planipennis perhaps due to their co-evolutionary history. Little is known about the molecular genetics of ash. Hence, we undertook a functional genomics approach to identify the repertoire of genes expressed in ash phloem.
Transcriptomics of the Bed Bug (Cimex Lectularius)
Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance.
Antioxidant Genes of the Emerald Ash Borer (Agrilus Planipennis): Gene Characterization and Expression Profiles
Phytophagous insects frequently encounter reactive oxygen species (ROS) from exogenous and endogenous sources. To overcome the effect of ROS, insects have evolved a suite of antioxidant defense genes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). The emerald ash borer (Agrilus planipennis Fairmaire), an exotic invasive insect pest from Asia has killed millions of ash trees and continues to invade North America at a rapid pace. From an on-going expressed sequence tag (EST) project of A. planipennis larval tissues, we identified ESTs coding for a Cu-Zn SOD (ApSOD1), a CAT (ApCAT1) and a GPX (ApGPX1). A multiple sequence alignment of the derived A. planipennis sequences revealed high homology with other insect sequences at the amino acid level. Phylogenetic analysis of ApSOD1 grouped it with Cu-Zn SODs of other insect taxa. Quantitative real time PCR (qRT-PCR) analysis in different larval tissues (midgut, fat body, Malpighian tubule and cuticle) revealed high mRNA levels of ApCAT1 in the midgut. Interestingly, high mRNA levels for both ApSOD1 and ApGPX1 were observed in the Malpighian tubules. Assay of mRNA levels in developmental stages (larva, prepupa and adults) by qRT-PCR indicated high transcript levels of ApCAT1 and ApGPX1 in larval and prepupal stages with a decline in adults. On the other hand, the transcript levels of ApSOD1 were observed to be constitutive in all the developmental stages assayed. Results obtained reflect a plausible role of these A. planipennis antioxidant genes in quenching ROS from both diet (ash allelochemicals) as well as endogenous sources. These studies further help in understanding the adaptation/invasiveness of A. planipennis.
Identification and Validation of Reference Genes for Quantitative Real-time Polymerase Chain Reaction in Cimex Lectularius
Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.
