Progress 07/01/99 to 07/01/04
Outputs
Storage of large quantities of juvenile hormone (JH) in male abdomens represents an atypical phenomenon known in some moths and butterflies. Juvenile hormone, stored in male accessory sex glands may be transferred to the female during copulation, but the physiological significance of the JH transfer remains unknown. Using the moth, Heliothis virescens, as a model, we demonstrated that JH transferred from male to the promiscuous female promotes JH synthesis and egg development in the female. We propose this explains the reason for JH transfer, particularly, in species that exhibit last male sperm precedence, and that this hormone acts as a bioactive substance which the male uses for co-opting and regulating the females gonadotropic mechanisms, thereby, ensuring that his investment will benefit his own paternity, and not that of males which mate after him. Under the influence of JH, the insect oocyte undergoes patency during which it sequesters nutritive proteins as a
result of intercellular spaces opening between follicular epithelial cells. Juvenile hormone-stimulated patency was analyzed in follicular epithelial cells of H. virescens, using chemicals that in vertebrates specifically target elements of two second messenger pathways: cAMP-dependent and inositol triphosphate/diacylglycerol signaling pathways. Juvenile hormone I and JH III cause patency that is suppressed by the Na/K-ATPase inhibitor, ouabain. Both protein kinase C activator, phorbol 12,13-dibutyrate, PDBu, and protein kinase A activator, 8-Br-cAMP, stimulated patency in follicular epithelial cells. The protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, H-7, predominantly suppressed patency caused by JH III, whereas the protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide, H89, predominantly suppressed patency caused by JH I. Moreover, patency was caused by the adenylate cyclase activator,
N,N-dimethyl-(3R,4aR,5S,6aS,10S,10aR,10bS)-5-(acetyloxy)-3-ethenyldod ecahydro-10,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-1H-naphtho[2 ,1-b]pyran-6-yl ester b-alanine hydrochloride, NKH 477, and peptide Gs-protein activator, cholera toxin. On the other hand, the JH I-evoked patency was suppressed either by adenylate cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, SQ 22,536, or GPAnt-2 (GlP-Glu-D-Trp-Phe-D-Trp-D-Trp-Met-NH2), a peptide antagonistic to Gs proteins that activate adenylate cyclase. Neither of these inhibitors affected JH III-evoked patency. These results suggest that, in the process of patency in H. virescens, JH I predominantly signals via the cAMP-dependent second messenger system, whereas JH III acts via the inositol triphosphate/diacylglycerol signaling pathway. Moreover, by providing the first pharmacological evidence that JH I acts on the follicular epithelial cells via activation of G-protein, we speculate that the JH I membrane receptor shares
similarities with G-protein-coupled receptors of vertebrates. Current and future studies will focus on deciphering the role of G-protein coupled receptors in activating egg development and we will attempt to characterize the hormone receptor.
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
We study the relationship between various hormones, egg development, and mating in a cosmopolitan pest, Heliothis virescens, and other moths and butterflies. We investigated the role of juvenile hormone (JH) transferred during copulation. Egg output by females mated to chemically allatectomized males was equal to that of virgin females, and significantly less than that in females mated to oil-injected males. Gonadotropic activity of female corpora allata did not increase immediately after copulation. On average, isolated abdomens implanted with corpora allata derived from just uncoupled females produced slightly less eggs than those implanted with corpora allata from virgin females. JH production in vitro was lower in mated than in virgin females during three h after copulation. Injections of 0.005 and 0.01 microgram JH/insect were significantly more effective than oil only. Injection of JH II and JH III, at similar doses, did not increase egg output. Injections of JH
I did not stimulate egg maturation in ovaries directly; this hormone exerts its effects only in the presence of an intact retrocerebral complex. Injections of JH I stimulated gonadotropic and synthetic activities of corpora allata. Upon implantation to isolated female abdomens, corpora allata derived from JH-injected females increased egg production almost twice in comparison to corpora allata from oil- treated females. Also, corpora allata derived from JH I- injected females produced more juvenile hormone in vitro than glands obtained from oil- injected females. Re-implantation of the moth's own corpora allata to isolated abdomen and concurrent injection of JH I did not stimulate egg output by direct action on corpus allatum. Similarly, egg output was not elevated by JH I injections in females with severed VNC.
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
We are determining the relationship between various hormones, egg development, and mating in a cosmopolitan pest, Heliothis virescens, and other moths and butterflies. Egg production is dependent on juvenile hormone (JH). JH biosynthesis is stimulated by mating-induced production/release of stimulatory brain hormones, such as allatotropins (AT). H. virescens AT may be structurally similar to that of the Manduca sexta AT (Mas-AT). We demonstrated a role for inositol triphosphate and intracellular calcium as second messenger in the transduction of the neuropeptide signal. A role for inositoltrisphosphate/diacylglycerol (IP3/DAG) signal transduction pathway in CA activity has been tested. The drug U73122 inhibits phospholipase C (PLC) in vertebrate systems, but did not inhibit CA activity and also did not suppress Mas-AT-induced stimulation of CA activity in Heliothis. It may simply mean that U73122 is not an efficient inhibitor of insect PLCs. The DAG kinase inhibitor,
R59022, which increases DAG levels by inhibiting its degradation had no effect on CA activity by itself, but potentiated the stimulatory effects of Mas-AT. Taken together, DAG may act as a second messenger in Mas-AT-induced stimulation of CA. The phorbolester activator of PKC PDBu did not affect CA activity when applied alone, and it failed to amplify Mas-AT effects when applied together with the peptide. To find out if PKC is indeed involved in CA regulation in H. virescens, we will need to test other phorbolesters, PKC inhibitors, and measure tissue-specific PKC concentrations. Future studies will attempt to tease the details of these components.
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 are determining the relationship between various hormones, egg development, and mating in a cosmopolitan pest of many crops, the tobacco budworm, Heliothis virescens, and other moths and butterflies. Egg production in moths is dependent on juvenile hormone (JH). JH biosynthesis is stimulated by mating-induced production/release of stimulatory brain hormones, such as allatotropins (AT). H. virescens AT may be structurally similar to that of the Manduca sexta AT (Mas-AT). Synthetic Mas-AT stimulates in vitro JH biosynthesis by corpora allata (CA) of virgin H. virescens females. Mated female brains exhibits considerable Mas-AT immunoreactivity (IR), as do ventral nerve cord and accessory glands; it is very low in other tissues. Two groups of cells located at the lateral base of antennal lobes send axons into the region of CA, and may be involved in JH biosynthesis. Several cell groups occur in thoracic ganglia and single cells occur in each abdominal ganglion. Blood
JH titers, ovarian development, and fat body utilization were monitored weekly in adult Speyeria idalia females, which extended from mid-June to early October in Kansas. S. idalia females mate just once, soon after they emerge in mid- to late June. Butterflies undergo a reproductive diapause through mid-August, related to absence of or very low JH titers. Oogenesis and fat body depletion do not commence until August/September, and oviposition occurs. Onset of oogenesis coincides with a rapid rise in blood titers of JH I, JH II, and JH III. This reproductive strategy appears to be an adaptation to coordinating life cycle with that of seasonally restricted violets on which larvae feed. In vitro catabolism of JH in blood of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titer.
Maximal JHE activity was recorded at 24 h, and preceded the peak in JH titres at 48 h. Topical application of JH II or fenoxycarb enhanced JHE activity up to 640 and 56%, respectively. Oviposition was sensitive to exogenous JH and declined significantly with dosages >100 pg. Insect parasitoids co-opt the host1s physiological systems to assure their development at the host1s expense. In the parasitic wasp, Cotesia congregata- tobacco hornworm, Manduca sexta association, host development is suspended prior to pupation, suggestive of endocrine disruption. A critical titer of JH is required for successful metamorphosis, but abnormally high JH levels arrest parasitized larval host developmental. JH III, present in negligible concentrations in non-parasitized larvae, predominates in blood of parasitized hosts. The parasitoid secretes JH III that in concert with stimulation of host JH biosynthesis and inhibition of host JH degradation contributes to high circulating titer in host hemocoel.
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.
Publications
- Kopper, B. J., S. Shu, R. E. Charlton, and S. B. Ramaswamy. 2001. Do regal fritillary butterflies (Lepidoptera : Nymphalidae) have a summer reproductive diapause? Ovarian and fat body development correlations with juvenile hormone titers. Ann. Entomol. Soc. Am. 94:427-432.
- Webb, T. J., S. B. Ramaswamy, A. Srinivasan, and S. Dorn. 2001. Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella as functions of age, mating status, and hormone treatment. Arch. Insect Biochem. Physiol. 49:10-21.
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Progress 01/01/00 to 12/31/00
Outputs
In an effort to continue explaining the role of hormones in egg development in Heliothis virescens, we are undertaking various physiological and immunological experiments. An enzyme-lined immunosorbent assay (ELISA) was used to measure concentrations of Manduca sexta allatotropin-immunoreactive material in various tissues of mated and virgin H. virescens females. Higher levels of Mas-AT-like immunoreactivity were observed only in the ventral nerve cord of mated females compared with that of virgin females. There was no difference in immunoreactivity in brain, accessory glands, fat body, and spermatheca of mated and virgin females. Using two different immunocytochemical protocols, several groups of immunoreactive cells have been localized in brains and suboesophageal ganglia of H. virescens females. Two groups of cells that are located at the lateral base of the antennal lobes appear to send axons into the region of the corpora allata and thus, may be involved in
regulation of JH biosynthesis. In addition, several immunoreactive cell groups occur in the thoracic ganglia (one in the prothoracic ganglion and four in the pterothoracic ganglion) and single immunoreactive cells occur in each of the abdominal ganglia. The cell groups in the thoracic and the abdominal ganglia may be involved in functions other than JH biosynthesis regulation, which we will undertake in future experiments. We are currently using the immunocytochemical method to determine if there are axonal connections present between Mas-AT immunoreactive cells and the corpora allata. Additionally, we have just initiated research on determining the role of the different JH homologs in the reproductive physiology of moths.
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.
Publications
- Zeng, F., Shu, S., Ramaswamy, S.B. and Srinivasan, A. 2000. Vitellogenin in hemolymph of pupal Diatraea grandiosella (Lepidoptera : Pyralidae). Ann. Entomol. Soc. Am. 93:291 294.
- Ramaswamy, S. B., Shu, S., Mbata, G.N., Rachinsky, A., Park, Y.I, Crigler, L., Donald, S., and Srinivasan, A. 2000. Role of Juvenile Hormone-Esterase in Mating-Stimulated Egg Development in the Moth, Heliothis virescens. Insect Biochem. Mol. Biol. 30:785-791
- Palli, S. R., Ladd, T.R., Tomkins, W.L., Shu, S., Ramaswamy, S.B., Tanaka, Y. and Retnakaran, A. 2000. Choristoneura fumiferana entomopoxvirus prevents metamorphosis by modulating juvenile hormone and ecdysteroid titers. Insect Biochem. Mol. Biol. 30: 863 868.
- Zeng, F., Shu, S., Ramaswamy, S.B. and Srinivasan, A. 2000. Vitellogenin in hemolymph of pupal Diatraea grandiosella (Lepidoptera : Pyralidae). Ann. Entomol. Soc. Am. 93:291 294.
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Progress 01/01/99 to 12/31/99
Outputs
The focus of this project is to study the physiological regulation of reproduction in tobacco budworm moths, regal fritillaries, monarch butterflies, and other lepidopterans. In Heliothis virescens, egg production is strictly dependent on juvenile hormone (JH). Mating induces a sharp increase in JH titers in mated females, resulting in higher egg production. Increase in JH titers is due to effects on JH degradation and on stimulation of JH biosynthesis. JH titer in virgin females follows a diel pattern, increasing towards the end of the light period, remaining high around the onset of the dark period, and declining during the night. In mated females, pattern of JH titers is not as pronounced. JH-esterase (JHE), an enzyme involved in degrading JH, activity in mated females is significantly lower than that of virgins during daytime. JHE activity in mated females is low during daytime and high at the onset of night. JHE inhibitors inhibit JHE activity and stimulate egg
production in virgin females. JH biosynthesis appears to be stimulated by mating-induced production and/or release of stimulatory brain neuropeptides, such as allatotropins (AT). Synthetic Manduca sexta AT (Mas-AT) stimulates in vitro JH biosynthesis by corpora allata of virgin H. virescens females in a dose-dependent manner. The brain of mated females exhibits considerable Mas-AT immunoreactivity followed by ventral nerve cord and accessory glands, it is very low or non-existent in ovaries, fat body, and flight muscle. The concentrations in the ventral nerve cord of virgin females is lower than in mated. JH is necessary for oogenesis in the codling moth, Cydia pomonelia. Rudimentary ovarian structures are recognizable in day 3-4 pupae, when hemolymph JH is still undetectable. Developing oocytes are first appear on day 8, coincident with very low JH titers. Chorionation is only evident after emergence, following an increase in JH in pharate adult. JH II is predominant in hemolymph of
virgin and mated females. Treatment of pupae or newly emerged adults with JH did not stimulate ovarian protein, but fenoxycarb depressed the number of oocytes filling > 50 pct follicular volume. Chorion deposition was stimulated by JHs 1, 11, or III, but not by fenoxycarb. Mating enhanced choriogenesis and egg laying, and, since hemolymph JH titres were concomitantly elevated (2-fold), it is postulated that the rise in JH elicited both these events. Studies have just been initiated on monarch butterflies to determine the connection between JH, reproduction, and migration. Similar studies with regal fritillaries should provide insight into the role of JH in reproduction in this endangered species. Current studies include the use of Mas-AT antibody for an immunocytochemical analysis of AT distribution in H. virescens. Future studies will include characterization of the H. virescens AT. Results should allow us to determine how neuropeptides along with other hormones control production of
eggs and, therefore, may allow us to devise better means of controlling noxious pests such as the tobacco budworm.
Impacts
Egg production in moths and butterflies is dependent on hormones such as juvenile hormone and allatotropin, which are affected by mating. Current studies include explaining distribution and characterization of these hormones. Results should allow us to determine control of egg production and, therefore, may allow us to devise better means of controlling noxious pests such as the tobacco budworm.
Publications
- Webb, T.J., Shu,S., Ramaswamy, S.B. and Dorn, S. 1999. The influence of juvenile hormone and mating on oogenesis and oviposition in Cydia pomonella L. (Lepidoptera : Tortricidae). Arch. Insect Biochem. Physiol. 41:186-200.
- Sevala, V., Shu, S., Ramaswamy, S.B. and Schal, C. 1999. Lipophorin of female Blattella germanica (L.): Characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons. J. Insect Physiol. 45:431-441.
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