Progress 07/01/05 to 06/30/11
Outputs
OUTPUTS: The study was designed to study the neuroendocrine system of larvae and neuroendocrine interaction with the nervous system of reproductive female Helicoverpa zea. A method was developed to detect and quantify Hez-PBAN (Helicoverpa zea pheromone biosynthesis activating neuropeptide) in the neural ganglia of H. zea larvae and females. This method employs paramagnetic beads conjugated with polyclonal antibodies to separate Hez-PBAN and closely related peptides. The pepties are eluted, fractionated using micro- and nano-scale HPLC and detected using an ultraviolet detector with a microflow cell. Fractions may then be further examined using MALDI-TOF or electrospray mass spectrometry techniques. The method was further improved to detect sub-picogram levels of Hez-PBAN and other peptides in very tiny neuroendocrine tissue samples. Using these methods PBAN and PGNs (PBAN-gene encoded neuropeptides) were present in the subesophageal ganglion and corpora cardiaca; very small amounts of PBAN were located in the ventral connective and its ganglia. PBAN was detected in the hemolymph just before light off (beginning of dark phase of diurnal cycle) and immediately before light on (beginning of light phase of diurnal cycle). PBAN and PGNs were detectable in the ventral nervous system late in the second dark phase of adult females. These methods were also used to confirm previously published work indicating that PBAN is present in the appreciable amounts in the hemolymph but not in the terminal abdominal ganglion of reproductive females. Plans were made to further investigate the female reproductive system during the various phases of her reproductive cycle. Unfortunately this work could not be completed due to Peter W. K. Ma's lengthy illness and subsequent retirement. Dr. Ma was an invited speaker for an Entomological Society of America symposium on chemical ecology where he presented some of these new techniques. PARTICIPANTS: Peter W. K. Ma (PI) Srinidi Mohan (Post-doctoral associate) Helga Vamenta (Undergraduate student worker) Sonya M. Baird (Research associate Mississippi State University TARGET AUDIENCES: Post-doctoral associate and undergraduate student worker received training in use of micro- and nanoscale HPLC, MALDI-TOF mass spectrometry and electrospray mass spectrometry. PROJECT MODIFICATIONS: Dr. Ma suffered a lengthy illness and subsequent retirement. Portions of the work originally proposed could not be completed due to these circumstances.
Impacts
Techniques were developed for isolation and detection of sub-picogram quantities of Hez-PBAN (Helicoverpa zea pheromone biosynthesis activating neuropeptide) and PGNs (PBAN-gene encoded neuropeptides) from very tiny samples of nervous system tissues and hemolymph. These techniques employ dissection and homogenization of tissues followed by separation of the target peptides using polyclonal antibodies conjugated to paramagnetic beads. The target peptides are then eluted and fractionated using micro- and nanoscale HPLC with micro-flow cell and ultraviolet detector. This is followed by electrospray or MALDI-TOF mass spectrometry for determination of peptide mass. Further adaptation of these methods for use in the female H. zea at various stages of her reproductive cycle could not be completed due to Peter W. K. Ma's lengthy illness and subsequent retirement. Dr. Ma was an invited speaker for an Entomological Society of America symposium on chemical ecology where he presented some of these new techniques.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Peter W.K. Ma retired in June 2011 after a lengthy illness. Project was discontinued. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Peter W.K. Ma retired in June 2011 after a lengthy illness. Project was discontinued.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The research is focused on the physiology of neuropeptides. HezPBAN (Helicoverpa zea-Pheromone Biosynthesis Activating Neuropeptide) is used as a model system. HezPBAN, a thirty-three-amino acid peptide is encoded in a gene that also expressed four other peptides, PGN-24 PGN-18 PGN-8 PGN-7 (HezPBAN gene encoding neuropeptide). Previous studies have shown that they are expressed and present in other nerve tissues such as the thoracic ganglia and the abdominal ganglia. Since all the PGNs are structurally very similar to HezPBAN because they contain the FXPRL amide pentapeptide sequence that are important for turning on sex pheromone production. It is hypothesized that HezPBAN alone may not be involved in regulation of sex pheromone production in the moth. Using immunoprecipitation and nano-spray chromatography interfaced with mass spectrometry, experiments were set up to test the hypothesis by measuring the HezPBAN and PGN immunoreactivity in the hemolymph of the moth. A truncated-PBAN was used as an internal standard. Hemolymph samples were collected from pharate adults and continue until the end of the second scotophase. Samples were processed and injected onto nano-LC (liquid chromatography). Amount of neuropeptide was calculated by comparing the resulting ion intensity with that of the internal standard. Preliminary results show that HezPBAN level is not the highest at or before 2 hour into the first scotophase, time when pheromone titer is the highest. It is possible that the entire compliment of Hez-PBAN gene expression that effect the single process of sex pheromone biosynthesis. Further experiments are needed to confirm this hypothesis. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
There are no outcomes to report.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The neuropeptide, pheromone biosynthesis activating neuropeptide (PBAN) was studied by immunoprecipitation, nano-LC-mass spectrometry. PBAN and all other (PGNs) PBAN-gene encoding neuropeptide were present in the subesophageal ganglion, corpora cardiaca ; the ventral connective contains small amount of PBAN. PBAN is present in the hemolymph just after light off and before light on. PBAN and th PGNs are present in the ventral nervous system late in the second dark phase.
PARTICIPANTS: Srinidi Mohan, Mississippi State University Sonya M. Baird Mississippi State University Helga Vermenta Mississippi State University
Impacts
Studying the neuroendocrine system in pest species could lead to development of novel control strategies that aim to disrupt their reproduction.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The main objective of the project is to look at how the neuroendocrine system interacts with the reproductive processes in insects. Using Helicoverpa zea as a model, we have developed a very sensitive assay to determine the titer of a neuropeptide PBAN (pheromone biosynthesis activating neuropeptide) in very small tissue samples. We directly conjugate PBAN polyclonal antibodies to paramagnetic beads using DMP (Dimethyl pimelimidate.2HCl). Tissue samples such as a portion of the corpora cardiaca was dissected and homogenized in very small volume of buffer. The homogenate is then treated with the antibody conjugated magnetic bead. The adsorbed peptides were then eluted into a small volume of buffer. The eluate was then loaded onto a C18 guard-cartridge on the injector of a micro-high performance liquid chromatograph. After rinsing the cartridge with water, the peptides are flushed into a C18 micro-capillary column for separation. The peptides are detected by mass a
spectrometer fitted with a nano-spray source. The method proved to be very sensitive as it allows the detection of peptides into the sub-picogram levels. The method is also very useful since it detects peptides in very tiny neuroendocrine tissues. We are going to use the methods to study the neuropeptide levels in small amount of tissue.
Impacts
The ability to detect minute amount of neuropeptide in small nervous tissues samples is important and crucial for the understanding of the functions of these peptides.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Helicoverpa zea larvae were used as a model to study the physiology of the neuroendocrine system. Larvae at different developmental stages were dissected to expose their central nervous system. The central nervous system were surgically removed and separated into brain; subesophageal ganglion; connectives between subesophageal ganglion and first thoracic ganglion; thoracic ganglia 1-3, abdominal ganglion ganglia, and terminal abdominal ganglion. Tissues were homogenizd and treated with paramagnetic beads that was conjugated to an antibody that was raised against Hez-PBAN (Helicoverpa zea pheromone biosynthesis activating neuropeptide). Absorbed peptids were eluted with a elution buffer containing glycine (pH 2). The eluted peptides were fractionated on a microHPLC using a gradient of acetonitrite and water at 10 microliters per minute flowrate. Synthetic peptides representing the peptide species that were derived from post-translational modfication of preproHez-PBAN
(prepro Helicoverpa zea-pheromone biosynthesis activating neuropeptide) were used as to gain insight on the retention times of these peptide species. Peptides in the elutents was detected using ultraviolet detector using a microflow cell. Fractions corresponding to the retention times of the peptides were collected and analyzed with MALDI-MS (matrix assisted laser desorption/ionization mass spectrometry). Further analyzes of the fraction will involve MALDI-TOP-TOP-MS (matrix assisted laser desorption/ionization time of flight-time of flight mass spectrometry), which will give precise molecular masses and sequence identities of the peptides in the fraction.
Impacts
Studying the neuroendocrine system could lead to development of strategies that target the neuroendocrine system or physiologies it controls in pest insects.
Publications
- No publications reported this period
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