Progress 07/01/02 to 06/30/07
Outputs
OUTPUTS: The chemical senses of insects are intimately involved in the selection of host plants and in the regulation of feeding behavior. The caterpillar of Manduca sexta has been widely used as a model for phytophagous caterpillars exhibiting some selectivity in their host plants. The chemosensory basis for feeding decisions remains unknown, but we have shown that for Manduca, the plant chemical rosmarinic acid activates the medial deterrent cell, which interacts peripherally with the medial inositol sensitive cell and vice versa, so that combinations of these two chemicals are much less deterrent than would be predicted from additive inputs of these two cells. This is the first documented peripheral interaction of medial taste cells in Manduca caterpillars, and indicates that peripheral interactions of chemosensory taste cells contribute to feeding decisions and that they are not independent channels of information for brain processing. Initial efforts to isolate the genes for
taste receptors in Heliothis virescens and Pieris brassicae maxillary styloconic enriched tissue using RT-PCR met with partial success. Some gene segments of 300-800 base pairs showed some sequence homology to Drosophila G-protein coupled taste receptors, and G protein specific reagents showed disruption of inositol taste cell functioning in styloconic cells in preliminary studies. Using the Novagten T-7 select system, a cDNA library was prepared from Pieris brassicae chemosensory enriched tissues, providing three times ten to the tenth pfu/ml. Plans to biopan this library using affinity columns for tastants like sinigrin, and sucrose, were not completed. Multiple efforts to obtain outside funding for this initial work were unsuccessful during this period, even though willing collaborators were identified.
PARTICIPANTS: Dr. Joseph Stitt, collaborator, Applied Research Labs, Penn State; Dr. Roger Gaumond, collaborator, Department of Bioengineering, Penn State; Dr. Frank Hanson, collaborator on behavior and electrophysiology, Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, MD.; and Dr. Sylvester Chyb, collaborator and sabbatical year host (2003) on electrophysiology and molecular biology, Department of Agricultural Sciences, University of London, Imperial College at Wye, Wye, Kent, England.
TARGET AUDIENCES: Research scientists in the chemical senses and the larger scientific community interested in new approaches in integrated pest management.
Impacts
Detailed analyses of chemosensory responses and feeding behavior for a weak stimulant such as inositol, and a strong deterrent, such as rosmarinic acid revealed significant peripheral interactions among medial taste cells, indicates that the widely held hypothesis that caterpillar feeding decisions are based solely on central processing of independent taste cell inputs is no longer valid. Determining the genes involved in caterpillar taste receptors will allow us to understand the molecular mechanisms by which taste receptors operate. This will in turn provide us with new molecular targets for developing chemicals that modify taste cell functioning and thereby control feeding of pest insects on their host plants. The information will also allow us to understand the mode of action of naturally occurring plant defensive chemicals and perhaps allow us to manipulate these in crop plants for a new approach to controlling pests that would not be affected by current
resistance mechanisms in pest populations.
Publications
- Stitt, J.P., J.L. Frazier, R.P. Gaumond, and F.E. Hanson. 2007. Correlation of Feeding Behavior with Chemosensory Responses to Opposing Stimuli in the Tobacco Hornworm, Manduca sexta. Submitted to J. Comp. Physiol. A. (In Press).
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Progress 01/01/06 to 12/31/06
Outputs
The chemical senses of insects are intimately involved in the selection of host plants and in the regulation of feeding behavior. Manduca sexta caterpillars feeding on glass fiber discs treated with a standard feeding stimulant mixture plus candidate feeding inhibitors exhibit differential effects depending on dose and the compound tested. Previous work has shown that compounds that activate specific taste cells, or inhibit the function of others can lead to immediate changes in feeding behavior that is dose dependent and reversible within an hour. Caffeine shows a reversible, dose-dependent reduction in the first phase of continuous biting, on the amount eaten, and on the frequency of biting that correlates with the frequency of the lateral deterrent cell firing. Quinine shows a similar dose dependent reduction in the same parameters, but has a longer lasting effect on subsequent feeding, and does not correlate with any specific deterrent cell firing. Quinine
inhibits the medial glucose cell firing at concentrations of 10 mM or above and causes an erratic firing of other cells in the lateral styloconica. Tetramethrin at 10-5 M. shows a rapid reduction in feeding that does not accumulate over a 30 minute exposure, and the effect does not readily reverse as does caffeine. Both quinine and tetramethrin are known to affect potassium and sodium ion channel functions respectively in other kinds of nerve cells and can be used as probes for understanding the kinds of ion channels involved in taste cell functioning in insects.
Impacts
Determining how taste cells function at the molecular level can provide new targets for taste cell disruption of normal functioning that in turn can reduce or prevent feeding of pest insects on crop plants. Control of pests by feeding behavior manipulation would be free from the effects of current pesticide resistance mechanisms in pest species since it would represent a new mode of action.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The chemical senses of insects are intimately involved in the selection of host plants and in the regulation of feeding behavior. Our previous work has shown that feeding behavior is closely regulated by taste cell inputs from the mouthpart receptors of the caterpillar 'Manduca sexta', particularly those of the maxillary styloconica, and thus an understanding of how these cells operate at the molecular level is needed. Earlier efforts utilizing RT-PCR on isolated chemosensory rich tissues of the caterpillars of 'Heliothis virescens' and 'Pieris brassicae' resulted in isolation of gene segments of 300-800 base pairs that show some sequence homology to known G protein coupled receptors in Drosophila. Preliminary electrophysiological evidence that G-protein specific reagents will block normal reception by sugar sensitive taste cells in both of these caterpillars further implicates G protein receptors as important in regulating feeding behavior. Our initial efforts to
obtain additional funding for this approach were not successful, and we are continuing to seek outside support to continue this work. One publication is in the final stages of acceptance.
Impacts
Determining the genes involved in caterpillar taste cell functioning will allow us to understand the molecular mechanisms by which taste receptors operate. This will provide us with new molecular targets for developing chemicals that modify taste cell functioning, and thereby deter feeding of pest insects on crop plants. This information will allow us to understand the mode of action of naturally- evolved plant defensive chemicals and perhaps allow us to manipulate these in crop plants for another new approach to controlling pest insects. Such an approach would be free from the effects of resistance mechanisms due to pesticide use in current pest populations, and would augment our IPM toolbox.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The chemical senses of insects are intimately involved in feeding, reproduction, and predator avoidance but understanding at the molecular level of how these cells operate remains unknown. Recent progress in the functional genomics of Drosophila taste cells has been used to guide our approaches to understanding genes encoding taste receptors in two species of caterpillars. Using the Novagen T-7 select 1 system, we have prepared a cDNA library from chemosensory enriched tissues of 'Pieris brassicae' larvae. Messenger RNA was used as a template for randomly primed first strand synthesis followed by second strand synthesis to yield double stranded cDNA, This was in turn digested with 'EcoR'1 and 'Hind' III restriction endonucleases and size-fractionaed by agarose gel electrophoresis. Purified cDNA was directionally ligated into the T7 select1-1b vector and subsequently packaged into phagemids using T7 packaging extracts. Initial transformants were amplified through
inoculation and subsequent culture of 'E. coli' BLT 5403 cells providing an insect gustatory cDNA library with an overall titre of 3-4 x 1010 pfu/ml. The library is stored at -80 C as 8 percent glycerol stocks for biopanning using affinity columns constructed for specific tastants like sinigrin and sucrose.
Impacts
Determining the genes involved in caterpillar taste receptors will allow us to understand the molecular mechanisms by which taste receptors operate. This will provide us with new molecular targets for developing chemicals that modify taste cell functioning and thereby control feeding of pest insects on their host plants. This information will allow us to understand the mode of action of naturally-evolved plant defensive chemicals and perhaps allow us to manipulate these in crop plants for a new approach to controlling pests that would not be affected by current resistance mechanisms in pest populations.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The chemical senses of insects are intimately involved in their ability to select host plants. Recent progress at the molecular level indicates a high investment in chemosensory genes among all animals and for insects like Drosophila as much as four percent of the genome may be dedicated to chemical sensing. We have initiated efforts to utilize rapid progress in Drosophila as a model organism as a guide to isolating and identifying genes involved in gustation in caterpillar pests. Genomic DNA as well as tRNA from chemical sense rich tissue from 'Heliothis virescens' and 'Pieris brassicae' larvae has been isolated. The purified mRNA as been used in reverse transcription coupled with polymerase chain reaction (RT-PCR) to amplify potential genes involved in gustation. We have used primers based on candidate gustatory receptor sequences from 'Heliothis virescens' adult antennae and on known gustatory receptor sequences in Drosophila. Both specific and degenerate primers
have been used in progressively increasing specificity PCR conditions to produce gene segments of 300-800 base pairs. Several of these have been cloned and sequenced and several show some homology to known G protein coupled receptors in Drosophila.
Impacts
Determining the genes involved in caterpillar taste receptors will allow us to understand the molecular mechanisms by which taste receptors operate. This will provide us with new molecular targets for developing chemicals that modify taste cell functioning and thereby control feeding of pest insects on their host plants. This information will also allow us to understand the mode of action of naturally evolved plant defensive chemicals and perhaps allow us to manipulate these in crop plants for a new approach to controlling pests that would not be affected by current resistance mechanisms in pest populations.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The chemical senses of insects have been comparatively well studied and offer both good model systems for learning more about how they work at the level of individual sensory cells and their interactions with individual molecules. A further understanding of the sensory codes in the nervous system and of the mode of action of specific chemicals at the molecular level have been undertaken in the caterpillar Manduca sexta. In this insect it has been a longheld hypothesis that the styloconic chemosensory cells were the most important sources of information for the feeding decision-making process. We have shown that this is not the case for feeding decisions involving mixtures of the stimulant, myo-inositol and the deterrent, rosmarinic acid. Measuring the responses of individual cells to each of these compounds and to mixtures of them, as well as the corresponding feeding behavior has allowed us to predict feeding behavior based solely on these cell imputs, vs. the
observed behavior where other cells from the palp tips and labral papillae are also functioning. While rosmarinic acid interacts peripherally to inhibit the inositol cell in the medial styloconica, the observed feeding is much less deterred by rosmarinic acid than would be predicted by the individual cell responses. This indicates that inositol cell responses from the other sites have a much larger effect on the feeding decision than would have been anticipated under the former hypothesis.
Impacts
Detailed analysis of chemosensory responses and feeding behavior to mixtures of a weak feeding stimulant and a strong feeding deterrent in the caterpillar `Manduca sexta' reveals that the feeding decision is affected by inositol sensitive cells from other mouthpart receptors far more than predicted by current hypotheses. The strong deterrent rosmarinic acid is far less deterrent than additive effects of the medial inositol and rosmarinic acid cells would predict, especially with their peripheral level interactions considered. This indicates that caterpillar feeding decisions may be more widely dependent on mouthpart chemosensory inputs than previously believed.
Publications
- No publications reported this period
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