Progress 10/01/01 to 06/30/05
Outputs
The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in
the central nervous system . Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of
AT acting on the CA.
Impacts
The results of this work are going to help explain the fundamental processes involved in the hormonal regulation of egg development in a devastating pest of row and vegetable crops. Additionally, this work will provide insight into the mode of action of hormones at the sub-cellular level.
Publications
- Pszczolkowski, M. A., A. Peterson, A. Srinivasan, and S. B. Ramaswamy. 2005. Pharmacological analysis of ovarial patency in Heliothis virescens. J. Insect Physiol. 51:445-453.
- Rachinsky A., A. Mizoguchi, A. Srinivasan, and S. B. Ramaswamy. 2006. Allatotropin-like peptide in Heliothis virescens: localization and quantification. Arch. Insect Physiol. Biochem. (Accepted December 2005).
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Progress 01/01/04 to 12/31/04
Outputs
The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence in H. virescens. A monoclonal antibody against Manse-AT was used to develop an enzyme-linked immunosorbent assay (ELISA) to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used to develop protocols for whole-mount immunocytochemistry to localize in the central
nervous system cells that contain Manse-AT-immunoreactivity. Several groups of Manse-AT-immunoreactive cells were discovered in brains, subesophageal ganglia, thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in numerous locations in the central nervous system indicates that these peptides have other additional, unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females, or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by
changes in the amounts of AT acting on the CA.
Impacts
The results of this work are going to help explain the fundamental processes involved in the hormonal regulation of egg development in a devastating pest of row and vegetable crops. Additionally, this work will provide insight into the mode of action of hormones at the sub-cellular level.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Our long- term goal is to isolate and identify juvenile hormone (JH) receptors on the membrane of follicular cells of Heliothis virescens ovaries. This task requires prior establishment of a rapid bioassay for patency and pharmacological studies on the pathways that signal from putative JH receptors to the cellular machinery that ultimately causes shrinkage of follicle cells. Also, JH receptor could be isolated if the response of follicle cells to JH could be enhanced. To that end we (1) established a rapid and sensitive bioassay for measuring the degree of patency in this species, (2) examined the response of follicle cells to juvenile hormones and triiodothyronine (T3), (3) pharmacologically identified pathways of follicle cells responses to juvenile hormones and T3 , (4) verified whether or not patency could be autoinduced by juvenile hormone. Incubations with both JH homologs produced stereotypic dose-dependent responses. Maximum patency indexes were reached at
concentrations of 10-9 mole. JH I caused shrinkage of follicle cells even at the lowest tested concentration (10-13 mole). JH III affected follicle cells at concentrations of 10-11 mole and higher. The response to triiodothyronine followed the general pattern that has been produced by JHs, however, higher concentrations were needed to initiate follicle cells shrinkage (10-9 mole) and produce maximum response (10-7 mole). Vitamin E, alpha-tocopherol, had no effects on follicle cells at any of tested concentrations. Actions of JHs and T3 were suppressed by Na+, K+ ATPase inhibitor, ouabain, in a dose-dependent manner at 10-4 and 10-3 mole. Increase of patency index was also caused by protein kinase C analogue, PDBU. Significant increase of patency index was observed at concentrations as low as 10-11 mole, and maximum response at 10-7 mole. Stimulatory actions of JHs were suppressed by proteine kinase C inhibitor, H-7. The pathway utilized by JH III in the process of patency was
particularly sensitive to H-7; this inhibitor was already effective at concentration as low as 10-6 mole, and at 10-3 mole JH III- evoked patency index dropped to the values characteristic for oocytes incubated in the absence of juvenile hormones. The action of juvenile hormone I was inhibited at concentrations of 10-4 and 10-3 mole. H-7 had no effects on triiiodothyronine- evoked patency. The dose-dependent responses to JH I in incubation medium differed between induced oocytes (that had been excised from JH I- injected females), and non-induced oocytes (those that had been excised from oil- injected females). Generally, the response curves revealed similar pattern, but maximum responses to JH I in incubation medium occurred in induced oocytes at lower concentration than in non-induced oocytes.
Impacts
The results of this work are going to help explain the fundamental processes involved in the hormonal regulation of egg development in a devastating pest of row and vegetable crops. Additionally, this work will provide insight into the mode of action of hormones at the sub-cellular level.
Publications
- Rachinsky A., A. Srinivasan, and S. B. Ramaswamy. 2003. Regulation of juvenile hormone biosynthesis by Manduca sexta allatotropin-like peptide in Heliothis virescens. Arch. Insect Biochem. & Physiol. 54:121-133.
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Progress 01/01/02 to 12/31/02
Outputs
The concerted action of hormones such as juvenile hormone and allatotropin is necessary for egg development in Heliothis virescens, and the activity of hormones is mediated by various second messengers. Allatotropins likely act on corpora allata (CA) by binding to membrane receptors of CA cells, thereby stimulating intracellular signal transduction pathways. Calcium regulates JH biosynthesis in H. virescens. A calcium ionophore, A23187, stimulated JH production, as did Manduca sexta allatotropin (Mas-AT). The stimulatory effects of A23187 on CA activity are likely the result of a direct increase of calcium concentrations in CA cells. Thapsigargin, a drug that increases intracellular calcium concentrations, significantly stimulated CA activity. The membrane-permeable calcium chelator, BAPTA/AM, antagonized the stimulatory effects of thapsigargin and those of Mas-AT. These results suggest that Mas-AT may indeed affect CA activity by increasing intracellular calcium
concentration. The involvement of calcium and other components of the second messenger cascades has been further corroborated with the use of specific antagonists and antagonists. Future studies will investigate in more detail the involvement of the inositoltrisphosphate/ diacylglycerol signaling pathway, and address the potential involvement of the cyclic AMP signaling pathway, the cyclic GMP signaling pathway, of calcium/calmodulin dependent protein kinases and of protein phosphatases.
Impacts
The results of this work are going to help explain the fundamental processes involved in the hormonal regulation of egg development in a devastating pest of row and vegetable crops. Additionally, this work will provide insight into the mode of action of hormones at the sub-cellular level.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
We have demonstrated the necessity of hormones such as juvenile hormone and allatotropin in egg development in the tobacco budworm, Heliothis virescens, and that the effect of these hormones is apparently mediated by various intermediaries, the second messengers. We have Results from research in our laboratory and data from some recently published studies provide evidence that H. virescens allatotropins may be structurally similar or even identical to that of Manduca sexta (Mas-AT). We have previously shown that Mas-AT-immunoreactive material is present in the H. virescens central nervous system, and also in axons projecting into the neuroendocrine corpora allata (CA). Allatostatins presumably act on the CA by binding to membrane receptors of CA cells, thereby stimulating intracellular signal transduction pathways. In order to identify second messengers involved in regulation of JH biosynthesis in H. virescens, we are investigating the role of calcium (Ca2+), using
agents that increase [Ca2+]i, and using the [Ca2+]i-increasing agents combined with a membrane-permeable Ca2+ chelator. Ca2+ clearly is an important regulator of JH biosynthesis in H. virescens. Drugs that are known to increase intracellular Ca2+ concentrations through different mechanisms (Ca2+ ionophore A23187, endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin), significantly stimulated JH production, as did Mas-AT. The membrane-permeable Ca2+ chelator BAPTA/AM antagonized the stimulatory effects of Mas-AT. This suggests that Mas-AT may stimulate CA activity by increasing intracellular Ca2+ concentration. However, BAPTA/AM did not antagonize thapsigargin(10-7M)-induced stimulation of JH biosynthesis. At an optimum concentration of 10-7M, thapsigargin may release more Ca2+ from intracellular stores than BAPTA is able to bind. BAPTA may well antagonize thapsigargin effects at sub-optimum concentrations of the stimulant. The corpora allata of H. virescens seemed to be rather
insensitive to changes in extraglandular Ca2+ concentrations, suggesting efficient regulation of Ca2+ homeostasis in CA cells. Data from experiments in which we investigated the effects on CA activity of varying Ca2+ concentrations and of the Ca2+ ionophore A23187 in one-day-old virgin H. virescens females, indicated that an increase in intracellular Ca2+ may be correlated with stimulation of JH biosynthesis. Further experiments will address the potential involvement of the inositoltrisphosphate/diacylglycerol signaling pathway, the cyclic AMP signaling pathway, the cyclic GMP signaling pathway, of Ca2+/CaM dependent protein kinases and of protein phosphatases.
Impacts
The results of this work are going to help explain the fundamental processes involved in the hormonal regulation of egg development in a devastating pest of row and vegetable crops. Additionally, this work will provide insight into the mode of action of hormones at the sub-cellular level.
Publications
- No publications reported this period
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