Progress 07/01/05 to 09/30/08
Outputs
OUTPUTS: We published the results of our collaboration with Dr. Yoonseong Park, a young faculty member in the Department of Entomology at Kansas State. This project, mentione in last year's report, involved proving that the beetle Tribolium castaneum produces a diuretic hormone similar to the vertebrate peptide arginine vasopressin, AVP, that the beetle peptide has the same primary sequence as an AVP-like peptide we previously identified from Locusta migratoria, and moreover that this peptide does not act directly on the Malpighian tubule, but causes the release of other peptides from nervous tissue. The latter is an unexpected finding. This work was published in Insect Biochem. Mol. Biol. Derek Jensen, a third year graduate student in my lab, greatly improved the ELISA assay we use for isolating calcitonin-like DH. He successfully isolated this peptide to homogeneity from 16000 heads of Tenebrio molitor, and determined the primary sequence. It is a 31 amino acid peptide of sequence GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide. All that remains to be done prior to publishing this work is to show that it elevates cAMP levels in Malpighian tubules of Tenebrio molitor. PARTICIPANTS: Derek A. Jensen is a graduate student in this department, who was in his second year at the start of this year, and is now starting his third year of study. TARGET AUDIENCES: Scientists interested in peptide hormones of insects, scientists interested in control of water balance in insects at the physiological level, and scientists specifically interested in insect diuretic hormones. PROJECT MODIFICATIONS: An original goal of this project was to isolate kinins from Tenebrio molitor. As stated in last year's report, we abandoned our plan of isolating kinins from Tenebrio. These peptides are almost ubiquitous diuretic peptides in other Genera of insects. However, the genome of Tribolium castaneum, a species closely related to Tenebrio, contains neither a precursor for the kinins nor the kinin receptor exist in this species. It seems reasonable to expect, therefore, that these peptides are not found in Tenebrionid beetles, and perhaps in all beetles.
Impacts
As stated in last year's report, we abandoned our original plan of isolating kinins from Tenebrio. These peptides are almost ubiquitous diuretic peptides in other Genera of insects. However, the genome of Tribolium castaneum, a species closely related to Tenebrio, contains neither a precursor for the kinins nor the kinin receptor exist in this species. This project has shown some fundamental differences in the control of water balance in these two species of beetles vs. other genera of insects.
Publications
- Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle, M.J. Aikins, D.A. Schooley, K. Begum, M. Detheux, R.W. Beeman, and Y. Park, Insect Biochem. Mol. Biol. 38, 740-748 (2008).
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We concluded a very successful collaboration with Dr. Yoonseong Park, a young faculty member in the Department of Entomology at Kansas State. We supplied him with a peptide, the AVP-like diuretic hormone of the locust, Locusta migratoria, which we isolated and identified in 1987 in collaboration with Dr. Jacques Proux of Bordeaux. The role of this peptide as a bona fide diuretic hormone of the locust is controversial. Park's students have cloned and expressed a receptor for this peptide from the red flour beetle, Tribolium castaneum, whose genome has been sequenced. They find that the monomeric form of this simple nonapeptide binds with extremely high affinity to this receptor. We synthesized both the antiparallel and parallel dimers of this peptide and supplied them to Dr. Park for assay. The parallel dimer does not bind the receptor. The antiparallel dimer, reported by Proux to be diuretic in the locust, appears to bind to the receptor with reduced affinity compared with
the monomer. The monomer has in vivo diuretic activity in Tribolium. A functional assay was run on Tenebrio molitor Malpighian tubules; the AVP-like DH has no effect on the tubules, unless they are coincubated with the corpora cardiaca corpora allata, and the nerve cord. This strongly suggests that the role of the AVP-like DH is to trigger release of another peptide, likely the CRF-like DH, which is the actual stimulator of the tubules. A paper has recently been submitted to Proc. Natl. Acad. Sci. USA. I gave samples of the calcitonin-like DH of Diploptera punctata (cockroach), Bombyx mori (silkworm) and Anopheles gambiae (mosquito) to a collaborator, Sue Nicolson at the University of Pretoria. Her student found the cockroach peptide to be inactive on beetle Malpighian tubules, the mosquito peptide to be fairly active, and the silkworm peptide to be quite diuretic (EC50 ~ 0.6 nM). Interestingly, she also found that thapsigargin, a drug which increases intracellular Ca2+ levels, had no
diuretic effect on beetle tubules. Ca2+ is the usual second messenger of the kinins; a family of diuretics that we reported last year are absent from the genome of Tribolium. Thus, not only is the family of peptides missing in this beetle, but stimulation of the second messenger of these peptides has no diuretic effect. Regretably, she published a paper using our samples, and did not include me as a coauthor: the study would not have been possible without these samples. I am nevertheless reporting it as a publication arising from this grant. Derek Jensen, second year graduate student in my lab, has greatly improved the ELISA assay we use for isolating calcitonin-like DH. He appeared to have isolated this peptide to homogeneity from Tenebrio molitor, one of the chief aims of this grant, but the amount was too small for sequence analysis. He is now repeating the isolation with ten times as much beetle tissue (20,000 heads).
PARTICIPANTS: Derek Jensen, second year graduate student, has worked very hard and made some key improvements in our ELISA assay used to isolate the calcitonin-like diuretic hormone of Tenebrio molitor. I also had a very successful collaboration with Dr. Yoonseong Park of the Department of Entomology, Kansas State University, which has resulted in a publication recently submitted to Proc. Natl. Acad. Sci. USA. I gave samples of the calcitonin-like DH of Diploptera punctata (cockroach), Bombyx mori (silkworm) and Anopheles gambiae (mosquito) to a collaborator, Sue Nicolson at the University of Pretoria. She and a student did a nice study on the effects of these diuretic hormones on two different species of beetles. Regretably, they chose to not include me as a coauthor on the paper.
TARGET AUDIENCES: Our target audience is the international community of scientists interested in insect physiology. More specifically, we target those interested in the biological effects of peptide hormones. We also hope to interest scientists involved in insecticide discovery in this research. Evidence of interest in our work is supported by the awarding to David A. Schooley of the 2007 Kenneth Spencer Award in Food and Agricultural Chemistry by the Kansas City ACS Section.
PROJECT MODIFICATIONS: We are not trying to isolate kinins from Tenebrio because evidence from the genome of the closely related insect Tribolium castaneum shows that neither the kinins nor the kinin receptor exist in this species. In addition, Dr. Nicolson showed that elevating intracellular calcium levels in tissues (the action of the kinins in other species) has no effect on urine production by two species of beetles.
Impacts
These studies have found some fundamental differences in the control of diuresis (urine production) between beetles and other species of insects. Many beetles, including those studied here and those which feed on trees (bark beetles and 4 or 5 families of borers) have extremely dry diets and have evolved very sophisticated physiological mechanisms for minimizing their water loss. Chemical disruption of their water balance should be an excellent means for controlling these pests.
Publications
- Holtzhausen, W.D., Nicolson, S.W. (2007) Beetle diuretic peptides: The response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella). J. Insect Physiol. 53, 361-369.
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Progress 01/01/06 to 12/31/06
Outputs
Isolation of a calcitonin-like DH from 5,000 heads of pupal yellow mealworms, Tenebrio molitor, was begun last year by an undergraduate student, who had great problems with the ELISA assay used for detecting this peptide. This year a graduate student has greatly improved the assay, but found that with the alkaline phosphatase-peptide conjugate being used, the assay is extremely specific for the cockroach calcitonin-like DH for which it was developed. It cross reacts poorly with samples of the calcitonin-like DH from Drosophila melanogaster, Anopheles gambiae, and Bombyx mori. We are now synthesizing a different conjugate which targets a different epitope on the peptide. A successful collaborative project of a similar nature was begun with Dr. Yoonseong Park, a young faculty member in the Department of Entomology at Kansas State University. We have supplied him with a peptide known as the AVP-like diuretic hormone of the locust, Locusta migratoria. We isolated and
identified this peptide in 1987 in collaboration with Dr. Jacques Proux of Bordeaux. The role of this peptide as a bona fide diuretic hormone of the locust is controversial. Park has now cloned and expressed a receptor for this peptide from the red flour beetle, Tribolium castaneum, whose genome has been sequenced, but not yet published. He finds that the monomeric form of this simple nonapeptide binds with extremely high affinity to this receptor. We have now synthesized both the antiparallel and parallel dimers of this peptide and supplied them to Dr. Park for assay. The parallel dimer does not bind the receptor. The antiparallel dimer, reported by Proux to be diuretic in the locust, appears to bind to the receptor with about the same affinity as the monomer. A search of the Tribolium genome for a protein precursor of the kinins was unsuccessful. I used a variety of different kinin sequences as queries for a BLAST search without success. Dr. Park informs me that he searched the
Tribolium genome for the kinin receptor, and was also unable to find a kinin receptor. We had proposed to isolate kinins from Tenebrio molitor, because in most species they are responsible for controlling chloride excretion. However, in the blood sucking bug Rhodnius prolixus kinins have been found to have no biological activity. Thus, kinins would appear to not be involved in diuresis in beetles. This is no yet a definitive statement, because the Tribolium genome is only about 95% complete, but it seems likely that we should have found either the receptor or the peptide.
Impacts
We expect the elucidation of the structure of this diuretic hormone to eventually allow the synthesis of smaller molecules which will have the same activity. This will likely be useful for controlling insect populations by causing them to dehydrate and die.
Publications
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Progress 07/01/05 to 12/31/05
Outputs
We began the isolation of a calcitonin-like DH from 5,000 heads of pupal yellow mealworms, Tenebrio molitor. My student is monitoring the isolation using an enzyme immunoassay using an affinity purified rabbit antiserum to the calcitonin-like DH of the cockroach Diploptera punctata, which was the first of these peptides to be identified. Head extracts have been purified using solid-phase extraction with a C18 silica support eluted with acetonitrile/0.1% aqueous trifluoroacetic acid. The active fraction from this step has been purified using a semi-preparative strong cation exchange HPLC column eluted at pH 5 with a 0- 1 M sodium chloride gradient. She is currently determining which fraction contains the immunoreactive factor. Once purified to homogeneity, the peptide will be analyzed by Edman degradation and a synthetic sample prepared for biological testing.
Impacts
We expect the elucidation of the structure of this diuretic hormone to eventually allow the synthesis of smaller molecules which will have the same activity. This will likely be useful for controlling insect populations by causing them to dehydrate and die.
Publications
- none yet- only six months research in 2005
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