Identification of methyl farnesoate from in vitro culture of the retrocerebral complex of adult females of the moth Heliothis virescens LepidopteraNoctuidae and its conversion to juvenile hormone III.код для вставкиСкачать
98 Teal and Proveaux Archives of Insect Biochemistry and Physiology 61:98105 (2006) Identification of Methyl Farnesoate From In Vitro Culture of the Retrocerebral Complex of Adult Females of the Moth, Heliothis virescens (Lepidoptera: Noctuidae) and Its Conversion to Juvenile Hormone III P.E.A. Teal* and A.T. Proveaux Gas chromatographic-mass spectral analysis of extracts obtained from in vitro culture of isolated retrocerebral complexes obtained from adult females of the moth Heliothis virescens resulted in identification of methyl farnesoate as well as juvenile hormone III (JH III) but not JH III acid. Inhibition of JH biosynthesis by incubation of tissue in synthetic Manduca sexta allatostatin (Manse-AST, p Glu-Val-Arg-Phe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-COOH) reduced production of these chemicals to negligible levels. However, incubation of tissue in the presence of Manse-AST plus farnesol resulted in production of significant amounts of both methyl farnesoate and JH III. Tissue incubated in the presence of Manse-AST plus methyl farnesoate produced only JH III. The results indicated that methyl farnesoate is naturally produced by the corpora allata of adult females of Heliothis virescens÷ However, tissue incubated in the presence of Manse-AST plus JH III acid also produced JH III in amounts equivalent to that produced by tissue incubated with methyl farnesoate. Thus, both methyl farnesoate and JH III acid could serve as a precursor for biosynthesis of JH III. Arch. Insect Biochem. Physiol. 61:98105, 2006. Wiley-Liss, Inc. Published 2006 KEYWORDS : juvenile hormone biosynthesis; methyl farnesoate; Lepidoptera INTRODUCTION coordination of reproductive competence with sexual behavior in female moths. All insects produce the JH homolog methyl Juvenile hormones (JH) are required for vitel- E,6E)-10,11-epoxy-3,7,11-trimethyl-2,6-dodeca- logenesis in adult female Lepidoptera (Cusson et (2 al., 1994; Satyanarayana et al., 1992; Zeng et al., dienoate (JH III). Biosynthesis of the sesquiterpene 1997). Consequently, female reproductive compe- skeleton of JH III has been studied in detail and is tence depends on production of JH. Indeed, among apparently common to all insects (see Schooley some species, pheromone biosynthesis, mating, and Baker, 1985, and references therein). Interest- and ovarian development are delayed until critical ingly, Lepidoptera are unique in that they also syn- levels of JH are produced (Cusson et al., 1994; thesize other homologs of JH formed by variable Gadenne, 1993; Picimbon et al., 1994). Thus, JH substitution of the methyl moieties at positions 3, acts as one of the critical hormones regulating the 7, and/or 11 of the carbon skeleton with ethyl Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida Use of a trade, firm, or corporation name in this publication is for information and convenience of the reader. Such use does not constitute official endorsement or approval by the United States Department of Agriculture or the Agriculture Research Service of any product or service to the exclusion of others that may be suitable. *Correspondence to: Peter E.A . Teal, CMAVE-USDA-ARS, 1700 SW 23 DR., PO BOX 14565, Gainesville, FL, 32604. E-mail: firstname.lastname@example.org Received 30 June 2005; Accepted 20 August 2005 Published 2006 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America. DOI: 10.1002/arch.20104 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Production of Methyl Farnesoate by Moths 99 groups. The most common of these higher forms following reports the identification of MF from ex- E,6E,10cis)-(10R,11S)-10,11-epoxy- tracts of incubations of isolated CA-CC complexes 3,7,11-trimethyl-2,6-tridecadienoate (JH II) and and adult females of the tobacco budworm moth, (2 E ,6 E ,10 cis )-(10 R ,11 S )-10,11-epoxy-7- Heliothis virescens and the conversion of MF to JH of JH, methyl (2 methyl ethyl-3,11-dimethyl-2,6-tridecadienoate (JH I) are III by the retrocerebral complexes (RCs). produced by adult female Lepidoptera (Baker et al., 1985; Edwards et al., 1995; Lessman et al., 1989; Roller et al., 1967, Shu et al., 1997). The terminal steps in JH biosynthesis, which result in epoxidization and esterification of the acid analogs, are well documented in Orthoptera and Dictyoptera (Schooley and Baker, 1985, and references therein). In these orders, farnesoic acid is first acted upon by methyl transferase in the presence of S-adenosyl-methionine (SAM) to form methyl farnesoate (MF), which is then acted upon by an epoxidase in the presence of O2 to form JH III. These terminal steps are less well understood in Lepidoptera. Studies using homogenates of the corpora cardiaca (CC)-corpora allata (CA) from adult females of the tobacco hornworm moth, Manduca sexta, showed that MF was formed when homogenates were incubated with farnesoic acid in the absence of NADPH (Reibstein et al., 1976). However, when NADPH was included no detectable amounts of MF were found, instead JH III was produced (Reibstein et al., 1976). Indeed, when tissue homogenates were incubated with labeled MF plus NADPH no label was incorporated into JH III. These results suggest strongly that epoxidation of farnesoic acid, homofarnesoate, and dihomofarnesoate precedes esterification in Lepidoptera. However, liquid chromatographic analysis of extracts from in vitro incubations of isolated CA from adult females of Pseudaletia unipuncta resulted in recovery of a radiolabeled product(s) that eluted prior to that of MF (Cusson et al., 1991). These authors suggested that the unidentified labeled product(s) were methyl homofarnesoate (MHF) METHODS AND MATERIALS Chemicals Capillary GC\GC-MS grade ethyl acetate, hexane, and methanol were from Burdick and Jackson and 18 megohm water was obtained from a Milli Q UVplus® water purification system. Tissue culture medium 199 containing Hanks salts and glutamine was obtained from Gibco (Gaithersburg, MD). Manse-AST was custom synthesized at the Interdisciplinary Center for Biotechnology Research, Protein Core Facility (University of Florida). The peptide was purified by reversed phase liquid chromatography as described elsewhere (Abernathy et al., 1996) and assessed to be ~97% pure by analytical reversed phase liquid chromatography, mass spectroscopy, and amino acid analysis. (E,E)-3,7,11-trimethyl,2,6,10-dodecatrien-1-ol acetate (farnesyl acetate, FA) and (E,E)-farnesol were purchased from Aldrich (Milwaukee, WI). Synthetic JH I, II, and III were gifts from D. A. Schooley (University of Nevada, Reno, NV) and MF was a gift from S. Tobe (University of Toronto, Toronto, ON). These synthetics were purified by liquid chromatography using a Rheodyne 7125® injector, a Kratos Spectraflow 400® pump, and a Waters 410® differential refractometer using an Adsorbosil® silica column (250 ´ 4.6 mm, 5- mm particles) eluted with 5% ethyl acetate in hexane (flow = 1.5 ml/min). Mass spectral analysis of purified sesquiterpenes indicated that all were at least 98% pure and that MF and FA did not contain any and methyl dihomofarnesoate (MDHF), the non- of the JH homologs. JH III acid was synthesized epoxidized methyl ester analogs of JH II and JH I, by saponification of JH III. JH III, dissolved in which are the major JH homologs produced by methanol, was added drop-wise to an equal vol- these female moths. If this were so, then Lepidoptera ume of 2M KOH and the mixture was stirred over- could employ MF, MDHF, and MHF as precursors night at 25 C. The reaction mixture was neutralized for the epoxidase and, thus, JH biosynthesis could with 1M HCl and extracted with hexane. The aque- proceed as in the Orthoptera and Dictyoptera. The ous phase, containing JH III acid, was applied to a Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. ° 100 Teal and Proveaux solid phase extraction column (Alltech C18 Extract- to minimize tissue damage. In some studies, RCs ml Clean, 500 mg packing) that had been precondi- were placed in 30 tioned acetonitrile 2% Ficoll 400, 72 mg/ml CaCl2, and 0.6 mM so- followed by 8 ml of water. After application of the dium acetate and 0.6 mM sodium propionate with JH III acid extract, the column was washed with 4 or without 10 nM Manse-AST in a conical amber ml of water and the JH III acid eluted with 1 ml of vial (Teal, 2001). In other studies, some RCs were 60% acetonitrile in water. The JH III acid fraction incubated for 24 h in media containing 10 nM was diluted with an equal volume of water and Manse-AST and 0.1 further purified by liquid chromatography under acid. In these cases, farnesol or JH III acid was first gradient conditions using an Adsorbosil® C18 col- dissolved in a solution of medium 199 containing umn (250 by application ´ of 4.6 mm, 5- 4 mm ml of of medium 199 containing mM of either farnesol or JH III particles) and detec- 10% acetone and appropriate amounts of this so- tion with a Kratos Spectra Flow 757® variable lution were added to the incubation medium prior wavelength detector set at 210 nm. The column was to adding tissues. Tissue was incubated at 25 C in eluted using a linear gradient of 30% acetonitrile the dark on a rotary shaker (80 rpm) for 24 h. to 70% acetonitrile in water over 40 min at 1 ml/ Incubations were stopped by the addition of 50 min using a Kratos Spectra Flow 430® gradient each of first methanol and then hexane contain- former. Under these conditions, JH III acid eluted ing 10 pg/ at 24 min and JH III at 33 min. The fraction con- vortexed at 3,200 rpm for 2 min, centrifuged at taining JH III acid was diluted by addition of an 18,000 equal volume of water and extracted with an equal organic layer removed. The aqueous layer was ex- volume of hexane, to remove any residual apolar tracted two additional times with 50 contaminants, prior to extraction three times with The organic fractions were combined in a clean equal volumes of dichloromethane to extract the vial and concentrated under N2 to approximately JH III acid. Analysis of the dichloromethane frac- 50 ° ml ml FA as internal standard. Samples were g for 5 min, to break the emulsion, and the ml of hexane. ml prior to mass spectral analysis. tion by GC-Mass spectroscopy indicated that the To determine if JH III acid was produced by tis- acid was free of JH III. An aliquot of the acid was sue incubated with FOH, we incubated and extracted dissolved in methanol and derivatized to the me- tissues as above. However, we reserved the metha- thyl ester by addition of six times the volume of nol/water extract, which would contain the JH III hexanes containing 2M trimethylsilyldiazomethane acid, and diluted it by addition of 100 (Aldrich) and stirring for 1 h. Analysis of the This was then subjected to reversed phase liquid derivatized sample indicated that 98 % had been chromatography using a YMC ODS-AQ column esterified to JH III. (2 ´ mm 150 mm, 3- ml of water. particles). The column was eluted using a linear gradient from 2070% methanol in water over 40 min at a flow rate of 200 Insects ml/ min. We collected the fraction corresponding to the Female TBW moths were obtained as pupae elution volume of JH III acid and subjected this to from North Carolina State University. Adults, trans- esterification with trimethylsilyldiazomethane. The ferred to cages upon eclosion and before the wings esterified sample was analyzed by GC-MS for the had expanded, were provided with a 5% sucrose presence of JH III. solution soaked onto commercial cotton balls and held under a normal photoperiod of 12:12 (L:D) h at 26 Mass Spectral Analysis ± 2°C and 60 ± 5% relative humidity until use. The RCs, containing the CA-CC, of females Extracts of incubations of RCs were analyzed were dissected from the head under tissue culture by chemical ionization mass spectroscopy (MS) medium 199. No attempt was made to separate using a Finnigan-Matt ITS 40® ion trap mass spec- the corpora allata from the corpora cardiaca so as trometer (MS) interfaced to a Varian Star 3400® Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Production of Methyl Farnesoate by Moths gas chromatograph having a cool-on-column injector as described elsewhere (Teal et al., 2000). The analytical column, a 30 m ´ 0.25 mm (id) mm film thickness) (J&W) was interfaced to a 10 m ´ 0.25 mm (id) uncoated, de- TABLE 1. Description of Cleavage Assignments Resulting in Generation of Diagnostic Ions Used for Quantitation of MF When Analyzed by Chemical Ionization Mass Spectroscopy Using and Ion Trap Mass Spectrometer DB5-MS® (0.1 activated fused silica retention gap, and a 10 cm ´ 0.5 mm (id) length of uncoated, deactivated fused silica in the injector. Conditions of chro- Ion no. Ion description Mass to Relative charge intensity 10 1 M+1 251 2 Ion 1 CH 3OH (from ester) 219 32 3 Ion 2 CO (from ester) 191 100 4 C 10H17 O2 (ester portion after scission 169 18 45 matography were: initial injector temperature = ° 101 5 Ion 4 CH 3OH 137 C 9H13 (common terpene fragment) 121 20 at 170 C/min to 270 C; initial column tempera- 7 Ion 5 CO 109 45 ture = 40 C for 5 min; column temperature in- a ° ° ° ° ° creased at 5 C/min to 210 C; He carrier gas linear flow velocity = 24 cm/sec; GC-MS transfer line b between C7 and C8) 6 40 C for 30 sec; injector temperature increased a Mean ion intensity as a percentage of the base peak (n = 5 replicates at 50 pg/ sample). b Base peak. ° temperature = 230 C. Under these conditions, farnesyl acetate eluted at 32.3, JH III at 33.8, JH RESULTS AND DISCUSSION II at 35.4, and JH I at 37.3 min, respectively. The MS was operated in the chemical ionization (CI) In an earlier work (Teal et al., 2001), we identi- mode using isobutane as reagent gas (mass ac- fied JH I, II, and III from extracts of incubations quisition range = 60350 amu; scan rate = 1 sec). of the RC from female TBWs and employed both Identification of JH homologs was based on com- Manse-AST and allatotropin (Manduca sexta form) parison of fragmentation patterns (60300 amu) and retention indexes of compounds eluting during analysis of natural product samples with those of synthetic standards. Quantification of amounts of JH III and MF was based on ion intensities of six diagnostic ions for each compound (JH III m/e = 235, 217, 189, 147, 125, 111) and was accomplished as described by Teal et al. (2000). For MF, we used the following ions: m/e 251 = M+1; m/e 219 = M+1-CH3OH (ester); m/e 191 = M+1CH3OH-CO (ester); m/e 169 = scission between C7 and C8 yielding C10H17O2 (ester end of molecule); m/e 137 = scission between C7 and C8 to study the biosynthesis of JH in 3-day-old females. In the present study, we assessed initially the capacity of isolated CA-CC complexes from 0-, 1-, and 2-day-old TBW females to produce JH III in the presence or absence of Manse-AST. Very little JH III was produced by retrocerebral complexes from 0-day-old females and incubation of tissue from females of this age in Manse-AST had no effect on JH III production relative to controls (Fig. 1). However, incubation of CA-CC complexes from 1- or 2-day-old females in media containing Manse-AST resulted in significant inhibition of JH III production compared to controls in which no Manse-AST was added to the incubation medium yielding C 10H 17O2 - CH3OH (ester); m/e 109 = (Fig. 1). The amount of JH III produced by tissue scission between C7 and C8 yielding C10H17O2 - from 2-day-old females incubated in Manse-AST CH3OH - CO (ester) (Table 1). These ions plus was about fourfold lower than the controls. How- m/e = 121 (C9H13), a common fragment ion for ever, 43-fold less JH III was produced by tissues terpenes, have been described from analysis of from 1-day-old females incubated in Manse-AST electron impact spectra (Liedtke and Djerassi, with respect to controls. In fact, the amount of JH 1972) although the intensities of the fragments III produced by tissue from 1-day-old females in- found in our studies were different because we cubated in Manse-AST was no different from that used chemical ionization and an ion trap ms produced by day-0 females. Rescue studies by rather than double-focusing and quadrupole in- Kramer et al. (1991) on Manduca sexta and Teal struments used by Liedtke and Djerassi (1972). (2001) on TBW provided strong evidence that Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. 102 Teal and Proveaux In addition to JH III, we also found another compound in all extracts from tissue incubated with FOH. This compound was not present in extracts from control tissues incubated in Manse-AST alone and had a chromatographic retention index and mass spectrum identical to synthetic MF (Fig. 2). Amounts of JH III and MF from extracts of tissues incubated in Masne-AST plus FOH are shown in Figure 3. To determine if MF was produced without addition of precursor (FOH) or Manse-AST to the medium, we incubated RC complexes from 2day-old females in just medium 199 for 24 h. Ex- Fig. 1. Comparison of mean amounts ( ±SE) tracts of these tissues were found to contain MF of JH III present in extracts obtained from cultures of RCs of 0-, 1-, and 2-day-old females incubated in the presence or absence of Manse-AST. Means capped by the same letters (Fig. 4) although the amounts were very small compared to those of JH III. Nonetheless, the data proved conclusively that MF was naturally pro- are not significantly different in a Fishers Least Significant Difference test ( P = 0.05) (n = 5/treatment). Manse-AST acts prior to formation of FOH because in both studies the inhibitory effects of Manse-AST on JH biosynthesis could be overcome by addition of exogenous FOH to the incubation medium. Additionally, studies have provided evidence that Manse-AST acts after the production of acetyl- and propionyl-CoA (Teal et al., 2001). Thus, it may be that the difference in amounts of JH III synthesized by tissues from 1- and 2-day-old females incubated in Manse-AST reflects differences in the amount of stored precursors like mevalonate or isopentenyl pyrophosphate required for production of FOH. We chose to use 1-day-old females for studies in which we tracked the fate of exogenous precursors of JH III because tissue from these females was capable of de novo synthesis of JH III in the absence of Manse-AST but could be significantly inhibited from producing significant amounts of JH III by addition of Manse-AST to the incubation medium. Extraction of RC complexes and media from 1-day-old females incubated in the presence of Manse-AST plus FOH resulted in recovery of significantly more JH III (28.9 ± 1.2 fmol/h, n = 5) than was recov- Fig. 2. Comparison of chemical ionization mass spectra (isobutane reagent gas) of naturally produced (upper spectrum) and synthetic (lower spectrum) methyl farnesoate. ered from extracts of control preparations in which Diagnostic ions used for identification and quantification only Manse-AST was added to the medium (mean are shown above ions of interest and fragments yielding = 4.7 ± 1.1 fmol/h, n = 5; t = 3.29, 8 df). the ions are given in Table 1. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Production of Methyl Farnesoate by Moths 103 cubated tissue from 1-day-old females in the presence of Manse-AST plus MF. This resulted in production of significant amounts of JH III but no farnesal nor FOH was identified (Fig. 5). Interestingly, performing the same experiment but substituting JH III acid for MF resulted in a similar increase in JH III production relative to controls (Fig. 5) and no MF was found in these samples. These results suggest strongly that the methyl transferase involved in the esterification step in JH III biosynthesis has no specificity for either farnesoic acid or JH III acid, although it apparently does have Fig. 3. Comparison of mean amounts ( ±SE) specificity for the sesquiterpene acid analogs of the of methyl farnesoate and JH III produced by RCs from 1-day-old females incubated with Manse-AST plus farnesol (n = 6 replicates). JH homologs (Reibstein et al., 1976). To determine if JH acid was produced in detectable amounts, we incubated glands in Manse-AST plus FOH as usual but we reserved the methanolic extract that would contain JH III acid. The diluted duced by the corporal allata of females incubated methanolic extract was then chromatographed us- in culture medium. The production of MF by ing a narrow bore reversed phase LC column and homogenates of the CA complexes from adult fe- the fraction corresponding to JH acid was collected males of the tobacco hornworm moth has been and derivatized to JH III with trimethylsylil diazo- documented (Reibstein et al., 1976). However, no methane. Mass spectral analysis of this fraction 3 measurable JH III was found when [ H]-MF was did not result in our detection of JH III although incubated in Graces medium with 5 mM NADPH we injected amounts equivalent to 350500 h of plus the homogenate (Reibstein et al., 1976). To incubation. determine if MF was converted to JH III by iso- Our results show conclusively that MF is pro- lated, but otherwise intact, RC complexes, we in- Fig. 5. ±SE) of JH III pro- Comparison of mean amounts ( duced by RCs of 1-day-old females incubated in the presFig. 4. Comparison of mean amounts ( ±SE) of methyl ence of only Manse-AST or Manse-AST + FOH or Manse-AST farnesoate and JH III extracted from incubations of RCs + MF or Manse-AST + JH III acid. Means capped by the of 2-day-old females incubated in only culture medium same letter are not different in a Fishers Least Significant (n = 6 replicates). Difference test ( Archives of Insect Biochemistry and Physiology P = 0.05) (n = 6 replicates per treatment). February 2006 doi: 10.1002/arch. 104 Teal and Proveaux duced during the conversion of FOH to JH III by application of a pseudopeptide mimic of a pheromon- isolated RCs of adult female TBW moths. Addition- otropic neuropeptide. Proc Natl Acad Sci USA 93:12621 ally, our inability to identify JH III acid from incu- 12625. bations suggests that the acid is not produced by these females. It is possible that epoxidization of farnesol to JH III acid followed by esterification is Baker FC, Jamieson GC, morallo-Rejesus B, Schooley DA. 1985. Identification of the juvenile hormones from adult Attacus atlas. Insect Biochem 15:321324. the preferred route in JH III biosynthesis by these moths and that production of MF is a secondary and less effective route. However, if this were the case we would expect to recover JH III acid from incubations in which we saturated the system with Cusson M, Tobe SS, McNeil JN. 1994. Juvenile hormones: Their role in the regulation of the pheromonal communication system of the armyworm moth, Pseudaletia uni- puncta. Arch Insect Biochem Physiol 25:329345. FOH, because conversion of both JH III acid and Cusson M, Yagi KJ, Ding Q, Duve H, Thorpe A, Mcheil JN, MF to JH III occurs at an approximately equal rate Tobe SS. 1991. Biosynthesis and release of juvenile hor- (Fig. 5). This was not the case because, although mone and its precursors in insects and crustaceans: the amounts of MF increased when we saturated the search for a unifying arthropod endocrinology. Insect system by addition of FOH, we were unable to identify JH III acid from extracts incubated with FOH. Consequently, we contend that the princi- Biochem 21:16. Edwards JP, Corbitt TS, McArdle HF, Short JE, Weaver RJ. 1995. Endogenous levels of insect juvenile hormones in larval, pal method of JH III production by female TBW pupal and adult stages of the tomato moth, Lacanobia moths is via the conversion of FOH to MF, which oleracea. J Insect Physiol 41:641651. is then epoxidized to JH III in the same manner as other insect orders. Indeed, it is likely that all homologs of JH are produced in a similar fashion and that females of other noctiud moth species Gadenne C. 1993. Effects of fenoxycarb, juvenile hormone mimic, on female sexual behavior of the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). J Insect Physiol 29:2529. employ the same biosynthetic pathway. This generalization is supported by Cusson et al. (1991) Kramer SJ, Toschi A, Miller CA, Kataoka H, Quistad GB, Li who have found radio-labelled fractions from liq- JP, Carney RL, Schooley DA. 1991. Identification of an uid chromatographic separations of extracts of incubations of CA-CC complexes of the moth Pseudaletia unipuncta, which have elution characteristics that allatostatin from the tobacco hornworm, Manduca sexta. Proc Natl Acad Sci USA 88:94589462. Lessman CA, Herman WS, Schooley DA, Tsai LW, Bergor BJ, would be expected of methyl homofarnesoate and Baker FC. 1989. Detection of juvenile hormones I, II, and methyl dihomofarnesoate, the non-epoxidized acid III in adult monarch butterflies (Danus plexippus). Insect analogs of JH II and JH I. Biochem 19:431433. Liedtke RJ, Djerassi C. 1972. Mass spectral and steriochemical ACKNOWLEDGMENTS problems. CCXVIII. The electron impact induced behavior of terpenoid esters of the juvenile hormone class. J The authors thank Drs. M. Cusson, Centre de Org Chem 37:21112119. Foresterie des Laurentides, Sainte Foy, Quebec, Canada, and S. B. Ramaswamy, Department of Entomology, Kansas State University, Manhattan, KS, for helpful reviews of the manuscript. Picimbon JF, Becard JM, Sreng L, Clement JL, Gadenne C. 1994. Juvenile hormone stimulates pheromonotropic brain factor release in the female black cutworm, Agrotis ipsilon. J Insect Physiol 41:377382. LITERATURE CITED Reibstein D, Law JH, Bowlus SB, Katzenellenbogen JH. 1976. Enzymatic synthesis of juvenile hormone in Manduca sexta. Abernathy RL, Teal PEA, Meredith JA, Nachman RJ. 1996. Induction of pheromone production in a moth by topical In: Gilbert, L.I. editor. The juvenile hormones. New York, Plenum Press. p 131146. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Production of Methyl Farnesoate by Moths Roller H, Dahm KH, Sweeley CC, Trost BM. 1967. The structure of the juvenile hormone. Agnew Chem Int 6:179180. 105 idae)] and relationship with egg development. J Insect Physiol 43:719726. Satyanarayana K, Yu JH, Bhaskaran G, Dahm KH, Meola R. Teal PEA. 2001. Effects of allatotropin and allatostatin on in 1992. Regulation of vitellogeninsynthesis by juvenile hor- vitro production of juvenile hormone by the corpora al- mone in the corn earworm, Helicoverpa zea. Invert Reprod lata of virgin females of the moths of Heliothis virescens Dev 21:169178. and Manduca sexta. Peptides 23:663669. Schooley DA, Baker FC. Juvenile hormone biosynthesis. 1985. Teal PEA, Proveaux AT, Heath RR. 2000. Analysis and In: Kerkut GA, Gilbert LI, eds. Comprehensive insect physi- quantitation of insect juvenile hormones using chemical ology biochemistry and pharmacology, Vol. 8. Elmsford, ionization ion-trap mass spectrometry. Anal Biochem NY: Pergamon Press. p 551564. 277:206213. Shu S, Park YI, Ramaswamy SB, Srinivasan A. 1997. Hemo- Zeng F,Shu S, Ramaswamy SB. 1997. Vitellogenin and egg lymph juvenile hormone titers in pupal and adult stages production in the moth Heliothis virescens . Arch Insect of southwestern corn borer [Diatraea grandiosella (Pyral- Biochem Physiol 34:287300. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch.