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S-adenosylmethionine:Juvenile hormone acid methyltransferase in male accessory reproductive glands of Hyalophora cecropia (L.)
ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 198, No. 1, November, pp. 175-181, 1979
S-Adenosylmethionine:Juvenile Accessory Reproductive
Hormone Acid Methyltransferase in Male Glands of Hyalophora cecropia (L.)l
GONTER F. WEIRICHl Institute
of Developmental Biology,
AND
Texas A&M
MICHAEL University,
G. CULVER3 College Station, Texas 7784.3
Received March 23, 1979; revised July 25, 1979 The accessory reproductive glands of the adult male Hyalophora cecropia contain S-adenosylmethionine:juvenile hormone acid methyltransferase. The enzyme is soluble and can be found in the gland epithelium as well as in the glandular secretion, but not in any other part of the genital tract of the unmated male. The appearance of this enzyme activity in the pharate adult precedes the formation of a measurable pool of its substrate, juvenile hormone acid, and the onset of the juvenile hormone accumulation in the accessory reproductive glands. The accessory reproductive glands of Anthrea pernyi and Manduca sexta, species which do not accumulate juvenile hormone, lack methyltransferase activity. It is concluded that the methyltransferase is an essential component of the juvenile hormone accumulation mechanism-in H. cecropia.
The males of the silkmoth Hyalophora cecropia (L.) have been known for over 20
moths (1, 2) where, beginning in the late pharate adult (3-5) or young adult (6), it years for their exceptionally high content is accumulated during most of the adult life. of JH4 (1). The function of this large amount This phenomenon has so far been in apparent of hormone remains elusive to this day, but conflict with the observation that the hemosignificant progress has been made recently lymph of the moth has high JH esterase toward understanding the physiological activity (7) and lacks a carrier protein caevents leading to this accumulation. pable of protecting the hormone against enThe JH, though originating from the cor- zymatic hydrolysis (Weirich and Culver, in pora allata, is found in the abdomen of the preparation). Shirk et al. (8) identified the ’ This work was supported by National Science male AGs as the “repository” for the hormone where it can be sequestered from the Foundation Grant PCM ‘72-01892to Drs. H. Rijller and K. H. Dahm and by Organized Research, Texas hemolymph either unaltered or in a process accompanied by the exchange of the methyl A&M University. 2 Present address: Insect Physiology Laboratory, ester group (9). Agricultural Research, SEA, USDA, Beltsville, Md. The formation of the methyl ester has 20705. been characterized as one of the final steps 3 Present address: Waters Associates, Maple Street, of JH biosynthesis in adult H. cecropia males Milford, Mass. 01757. in vivo (10) and in corpora allata of various * Abbreviations used: AG, accessory reproductive gland; JH, juvenile hormone, not differentiating be- species in vitro (11, 12). Methionine can be utilized as a methyl donor for the ester fortween the three naturally occurring hormones; JHI, racemic methyl (2E,6E,10cis)-lO,ll-epoxy-7-ethylmation by whole animals (10) and by corpora 3,11-dimethyl-2,6-tridecadienoate; JHA, juvenile allata cultured in vitro (9, 13-19) whereas hormone acid, not differentiating between acids of the corpora allata homogenates require SAM three naturally occurring hormones; JHA-I, racemic for the methyl transfer reaction (1’7,20,21). (2E,6E,10cis)-10,ll-epoxy-7-ethyl-3,1l-dimethylIn the present paper, we have examined 2,6-tridecadienoic acid; JHAMT, S-adenosylmethiothe possibility that a similar reaction may nine:juvenile hormone acid methyltransferase; SAM, be involved in the JH accumulation of the S-adenosyl-L-methionine; PPO, 2,5-diphenyloxazole; H. cecropiu male AG and found that this is dimethyl-POPOP, p-bis[2-(4methyl-&phenyloxazolyl)]benzene. indeed the case. We have demonstrated the 175
0003-9861/79/130175-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
WEIRICH
AND CULVER
presence of the enzyme, S-adenosylmethionine:juvenile hormone acid methyltransferase (EC 2.l.l.xx), in the AG of H. cecropia males and present data on its tissue and subcellular distribution. This enzyme catalyzes the reaction: JHA + SAM + JH + S-adenosylhomocysteine. For comparison, two species which do not accumulate JH, Antherea pernyi and Manduca sexta (2,9), were included in this study. MATERIALS
AND METHODS
Animals. Pupae of H. cecropia and A. pernyi were obtained from commercial suppliers and stored at 5°C for at least 5 months. Adult development was initiated by transfer to 27°C. Some animals had terminated diapause prior to this transfer and, consequently, required less time than others to complete adult development. Morphological criteria (22, 23) were therefore used to determine the stage of adult development in experiments involving pharate adults. M. se&a was reared as described previously (24). Dissection and fractionation of tissues. AGs and other tissues were dissected from carbon dioxide-narcotized males, freed from adhering fat body, and rinsed with ice-cooled Grace’s tissue culture medium (Grand Island Biological Co., Grand Island, N. Y.). Where indicated, tissue and content of AGs were separated by allowing spontaneous extrusion from short pieces of the tubular glands and subsequently rinsing them with the aid of a capillary pipet. The tissues and/or gland contents were homogenized in Grace’s medium with small all-glass homogenizers by hand and in some cases the incubations were carried out in these homogenizers. In other cases, the homogenate was fractionated by one or two centrifugations at 4°C (30 min at 12,OOOg,60 min at 100,OOOg). Chemicals. JH-I was obtained from Eco-Control, Cambridge, Massachusetts, and purified by high resolution liquid chromatography. JHA-I (reference for thin layer chromatography) was prepared by alkaline hydrolysis of JH-I. JH-I[‘7-ethyl-l,2-3H(N)] of 11.2 Ci/mmol specific activity (corrected for decay) was purchased from New England Nuclear, Boston, Massachusetts. PHIJHA-I for JHAMT assays was prepared by enzymatic hydrolysis from PHIJH-I, diluted to various specific activities with unlabeled JH-I, by a modification of the method described by Metzler et al. (10). SAM chloride (grade II, Sigma, St. Louis, MO.) was purified by ion exchange chromatography on AG l-X8 (Bio-Rad, Richmond, Calif.) (25). SAM[methyl-3H] (7.5 Ci/mmol) was obtained from Amersham/Searle, Arlington Heights, Illinois.
Enzym assay. The incubation mixtures contained 10e6to 10m5M rH)THA-I, lo-” to lo-’ M SAM or PHISAM (neutralized with 0.1 M potassium phosphate buffer, pH ‘7.5), and the tissue preparation as indicated, in a total volume of 50 to 200 ~1 of Grace’s medium. After 180 to 200 min of incubation at 25”C, the reactions were stopped by addition of ethylacetate. Unlabeled JH was added as carrier, the reaction mixtures were extracted four times with 1 ml ethylacetate, and the extracts were analyzed by thin layer chromatography on silica HF-254 (E. Merck, Darmstadt, Germany) using a solvent system of 15% ethylacetate and 1% glacial acetic acid in benzene. The plates were divided according to sidemarkers of JH and JHA and the silica was transferred to scintillation vials and thoroughly mixed with a scintillation mixture (100 g naphthalene, 7 g PPO, and 0.05 g dimethyl-POPOP/ liter dioxane). The radioactivity was determined in a Searle Mark III liquid scintillation system with a counting efficiency of 50%. The amounts of labeled methylation products were calculated from the specific activity of the added labeled precursor, with no correction for the dilution by endogenous precursors. As a consequence, the data represent underestimates of the total quantities formed.
RESULTS
Tissue Distribution Changes
and Developmental
When homogenates of H. cecropia male AG tissue or luminal content were incubated with PHIJHA-I and SAM, both tissue and content produced similar amounts of rH]JH (Table I). At a PHIJHA-I concentration of 10e6M, 32 to 52 pmol was methylated; at a PHIJHA-I concentration of 4.0 to 9.0 x lop5 M, the yield of PHIJH was 428 to ‘714pmol. Despite the higher absolute amount of PHIJH-I, the proportion of rH]JHA converted was lower at the higher pH]JHA-I concentrations. In subsequent experiments rH]JHA-I was applied in the 1O-6M range. The accumulation of JH in the AGs of H. cecropia males is known to begin shortly before or after adult eclosion (3-6). This led us to speculate that induction or activation of the JHAMT may be responsible for the initiation of the JH accumulation. To test this hypothesis, we assayed AG homogenates obtained from pharate adults at various stages of development for JHA-methylating activity. As shown in Table II, the AGs have high JHAMT activity as early as 8 days prior to eclosion. Its occurrence
JUVENILE
HORMONE ACID METHYLTRANSFERASE
IN REPRODUCTIVE
177
GLANDS
TABLE I METHYLATION OF [3H] JHA-I IN HOMOGENATESOF MALE ADULT H. cecropia AG TISSUE AND CONTENT” [3H]JH synthesis Homogenate of gland tissue
Homogenate of gland content
Percentage of applied acid
pm01
Percentage of applied acid
pm01
52 45
49
7
572
4
428
Concentration of Incubation No.
Moth No.
1 2
1
3 4 5
2
6 7
3
Age
[3H] JHA-I
SAM
(day)”
(PM)
(PM)
1 3
3
1.5 1.1
100 100
35
1.2 86 43
200 200 90
27 7
87 57
200 90
8
32 620 714
“ Incubations contained homogenate equivalent to 0.13 gland pair (incubation Nos. 1, 2) or 0.25 gland pair (incubation Nos. 3-7). Incubation time was 180 min (Nos. 1, 2) or 200 min (Nos. 3-7). Total volume was 100 ~1 for incubations containing homogenized gland tissue (Nos. l-4, 6) or 200 ~1 for incubations containing homogenized gland content (Nos. 5, 7). * Day 1 = day of eclosion.
is apparently not correlated with the onset of JH accumulation. The AGs, testes, vas deferens, seminal vesicles. and common duct were dissected from a fully formed male moth shortly before eclosion and assayed under identical conditions. Only the kGs showed JHAMT activity (Table III) whereas the other parts
of the male genital tract did not. Thus, at least in the unmated male, the enzyme is not released from the AGs. Subcellular Distribution In order to determine the subcellular localization of the JHAMT, an AG homogenate was subjected to a two-step fractionation.
TABLE II METHYLATION OF [“HI JHA-I IN AC HOMOGENATES OF PHARATE ADULT H. cecropia”
Characteristic
Day of pharate adult development*
[3H] JH-I synthesis
TABLE III METHYLATION OF [8H] JHA-I IN VARIOUS SEGMENTS OF THE MALE GENITAL TRACT OFH. cecropia”
(pmol)
[“HI JH-I synthesis (pm00
Beginning antenna1 pigmentation Complete antenna1 pigmentation Complete wing pigmentation
14
41
16
41
20
39
a Each incubation contained 1.0 x 10e6M [3H]JHA-I, 1.0 X 1O-4M SAM, and homogenate equivalent to 0.5 gland pair in a total volume of 100 ~1. Incubation time 180 min. b Adult eclosion at Days 22-23 (Refs. (22, 23)).
Testes Vas deferens Seminal vesicle Accessory glands Common duct
0 0 0 37 0
a Homogenates of the indicated organs (0.25 animal equivalent each), dissected from a fully formed pharate adult male (Day 22), were incubated for 180 min with 2.0 x 10m6M [3H]JHA-I and 1.8 x 10m4M SAM in a total volume of 54 ~1.
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TABLE IV METHYLATIONOF [SHIJHA-I BYVARIOUSSUBCELLULARFRACTIONSOFTHE H. cecropia MALE AG” [3H] JH-I synthesis
Fraction Crude homogenate 12,000g Supernate 100,OOOgSupernate 100,OOOgPellet
Concentration of [3H] JHA-I (I*@
Gland pair equivalents
1.3
0.07
2.0 2.4
0.07 0.07
1.2
1.0
pm01
nmol/gland pair equivalent
54 114 124 26
0.77 1.63 1.77 0.03
(I AG from Day 1 adult males. All incubations contained 1.0 x 10m4M SAM and the total volume was 100 ~1. Incubation time, 180 min.
Neither the 12,000g centrifugation (30 min) nor the 100,OOOgcentrifugation (60 min) eliminated the activity in the resulting supernate, and the microsomal pellet had only marginal activity (Table IV). It can be concluded, therefore, that the JHAMT is a soluble enzyme. Time Cour.se and Substrate Requirements
of PHISAM. Labeled material cochromatographing with JH was produced in quantities comparable to the quantities of JH obtained in incubations with PHIJHA-I and unlabeled SAM (Table V, lines 4 and 5). The yield of this methylation product was dependent on the concentration of PHISAM. These results suggest the existence of a pool of JHA in the AGs which can provide the precursor for JH formation, or the potential for a methyl exchange of JH stored in the glands. To test the latter possibility, we added JH-I and PHISAM to the AG homogenate assuming that an increased JH-I concentration should result in an increased methyl
As shown in Fig. 1, the JHA-I methylation proceeds nearly linearly for 120 min before it levels off. In order to identify the limitations of the assay system, we determined the effects of various components of the reaction mixture on the yield of PHIJH (Table V). The complete reaction mixture, containing lop6 M PHIJHA-I, 6.3 x 10F5M SAM, and 12,000g supernate equivalent to 0.13 gland pair in 100 ~1 of Grace’s medium, yielded 34.4 pmol JH-I after a 3-h incubation. Following a “blank” incubation without enzyme, radioactivity equivalent to 3.4 pmol PH]JH-I was found in the JH fraction. All other data were corrected for the amount 10 of rH]JH-I found in corresponding enzymefree incubation mixtures. Omission of SAM did not eliminate the production of JH-I but 120 180 60 reduced it to 18.4 pmol, suggesting that the Incubation time (mid gland homogenate either contained SAM or was capable of synthesizing it. FIG. 1. Time course of PHIJHA-I methylation. In other incubations, PHIJHA-I was Homogenate of two pairs of AGs of Day 1 adult H. omitted from the reaction mixture and the cecropia males was centrifuged for 30 min at 12,000g. formation of JH or JH-like methylation Each incubation contained supernate equivalent to 0.13 products was monitored by determining the gland pair, 1.0 x 10e6M PHIJHA-I, and 6.2 x 10e5M incorporation of the labeled methyl group SAM in a total volume of 104 ~1. Mean * SE.
JUVENILE
HORMONE
ACID
METHYLTRANSFERASE TABLE
IN REPRODUCTIVE
GLANDS
179
V
THE EFFECTS OF JHA-I, JH-I, AND SAM ADDITIONS ON THE JHAMT REACTION IN AG HOMOGENATESOFH. cecropia” [“HI JHA-I
No.
(FM)
JH-I
SAM
r3H] JH formed
(PM)
(PM)
(pmol)”
1.0
63
34.4 k 0.3
63
3.4 18.4 33.2 16.7
No enzyme
1.0 1.1
63 r3H] 8.7[3H]
1.0 1.0[3H]
8.7[3H]
zk 1.7 k 2.3 k 1.4 2 3.9
11.1 ? 2.9 13H]JH hydrolyzed 10.7 2 0.7
(’ Homogenate of two pairs of AGs of Day 1 adult males were centrifuged for 30 min at12,OOOg.Supernate equivalent to 0.13 gland pair was incubated as shown for 180 min in a total volume of 100 ~1. h Mean 2 SE. Except for No. 2, corrections were made for the amounts found in incubations without homogenate.
exchange. This, however, was not the case (Table V, line 6). Apparently, the enzyme does not facilitate the direct exchange of the methyl group in the JH-I molecule. The gland homogenate, however, had JH esterase activity (Table V, line 7). Precursor
Pool
Since the AG homogenates of Day 1 adult males apparently contain JHA which can be methylatecl under in vitro incubation conditions (Table V), it was of interest to evaluate AGs of other age groups for the availability of this precursor(s). When AG homogenates of pharate adults were incubated with 1O-5M PHISAM, they yielded only marginal amounts of labeled methylation product(s) (Table VI) whereas in glands of adult males the quantity increased to the level previously found in incubations containing 10e6M PHIJHA-I and 10e5M unlabeled SAM. These results suggest that the AGs do not contain a significant pool of JHAs prior to the time of eclosion. Other Species
We also tested two lepiclopteran species which do not accumulate JH, Antherea pemyi and Manduca sexta, for JHAMT activity. No labeled JH or JH-like material was found
after incubations of AG homogenates of young adult males either with [3H]JHA-I plus unlabeled SAM, or with PHISAM. DISCUSSION
In the present study, we have used enzymatically prepared PH]JHA-I as a specific tool to test for JHAMT activity in various tissues and tissue fractions. The methylation of JHA without distinction between the three naturally occurring homologs can also be measured by the incorporation of the labeled methyl group of [methyL3H]SAM. This approach, however, is not specific since the formation of other labeled methylation products can not be ruled out. We have shown that the male AGs of adult Hyalophora cecropia have a methyltransferase capable of methylating JHA to JH. Similar to other methyltransferases, the enzyme is soluble, most of the activity remaining in the supernate of a 60 min 100,OOOg centrifugation. In contrast to the JH formation in corpora allata homogenates ((20); M. G. Peter, unpublished), the methylation of JHA in AG homogenates is not entirely dependent on exogenous SAM, even though the yield can be substantially increased by the addition of SAM. Apparently, the glands have an enclogenous SAM pool or the capacity to produce SAM under in vitro conditions.
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TABLE VI JHA IN AG HOMOGENATES OF H. cecropiaa [3H]Methylation produ& Stage*
PA PA PA PA
A A A
Age’ (day) 14’ 16’ 20’ 22’ 1s 3h 3h
Concentration of [3H]-SAM (PM) 10
10 10
15 8.7 10 10
Gland pair equivalents 0.5 0.5 0.5 0.25 0.13 0.25 0.25
Incubation time (mm) 180 180
180 180 180 200 200
pm01
pmol/gland pair equivalent
0.03 0.52 0.12 0.35 1.0 40.9 46.8
0.06 1.04 0.24 1.40 7.69 163.6 187.2
a Total volume for Day 22 pharate adult was 54 ~1, for all others 100 ~1. b pA, pharate adult; A, adult. c for pA, see Ref. (23); for A, Day 1 = day of eclosion. d Cochromatographing with JH. ’ Data obtained from same AG homogenates as those in Table II. ’ Same AG homogenate as in Table III. g Moth No. 1 of Table I. h Moth Nos. 2 and 3 of Table I.
It is obvious from our results that the AG can perform the reaction that has been characterized as the last or one of the final steps of the JH biosynthesis in the corpora allata (11, 12, 21). Interestingly, the substrate for this reaction is also one of the primary degradation products of JH, found in many insect species including H. cecropia (26,27) as the result of enzymatic hydrolysis in the hemolymph (7). In fact, the hemolymph of adult H. cecropia has high JH esterase activity (‘7) and apparently no carrier protein capable of protecting the hormone from enzymatic hydrolysis (Weirich and Culver, in preparation). Therefore, it is very unlikely that JH could be carried intact via the hemolymph to the AGs if, indeed, it is secreted by the corpora allata. This seemingly paradoxical situation can be resolved by the assumption that the AGs are not sequestering JH proper from the hemolymph but the JHA which, in turn, is (re)methylated by the JHAMT. Some JH esterase activity was also found in the AG homogenates (Table V), probably accounting for the incorporation of the PHImethyl group of rH]methionine observed in incubations of AGs with JH (9). Because of the presence of hydrolyzing and methylat-
ing enzymes in the AG homogenate, the yields determined for either reaction are only approximations. We were actually measuring the difference between two antagonistic reactions with the one for which the higher substrate concentration was provided outweighing the other. How this relates to the in viva situation and whether or not the JH esterase of the AG has a physiological function remains a matter of conjecture. Also, the quantitation and the kinetic parameters of the JHAMT will have to be worked out in future investigations after at least partial purification of the enzyme. JHAMT activity is present at least 1 week before eclosion, but -JHA is not traceable in AG homogenates until after eclosion (Table VI). Thus, it is not the induction or activation of the enzyme that is determining the onset of the JH accumulation in the AGs but rather the availability of the substrate, JHA. Surprisingly, the JHAMT is not confined to the epithelium of the AG but can be found in the secretion as well. The function of the enzyme in the glandular secretion is obscure, but it is interesting to note that it is also the secretion that contains all of the stored JH (8, 9).
JUVENILE
HORMONE
ACID
METHYLTRANSFERASE
Finally, JHAMT is not an enzyme universally found in male AGs of adult Lepidoptera. We have demonstrated that it exists in AGs of H. cecropia and it may occur in glands of other Saturniid species accumulating JH, but it is not found in two lepidopteran species that do not accumulate measurable amounts of JH, Antherea pernyi (also Saturniidae) and Manduca sexta (Sphingidae). Therefore, the JHAMT seems to be an essential component of the JH accumulating mechanism. ACKNOWLEDGMENTS We thank Dr. H. Rijlier for his continued interest and encouragement, Dr. P. Shirk for his help in dissecting accessory glands, Dr. K. H. Dahm for purified samples of JH-I and JHA-I and for helpful suggestions during the preparation of the manuscript, and Dr. M. G. Peter for purifying SAM. REFERENCES 1. WILLIAMS, C. M. (1956) Nature (London) 178, 212-213. 2. WILLIAMS, C. M. (1963) Biol. Bull. 124,355-367. 3. WILLIAMS, C. M. (1959) Biol. Bull. 116323-338. 4. WILLIAMS, C. M. (1961) Biol. Bull. 121, 572-585. 5. GILBERT, L. I., AND SCHNEIDERMAN, H. A. (1961) Gen. Comp. Endocrinol. 1, 453-472. 6. METZLER, M., DAHM, K. H., MEYER, D., AND ROLLER, H. (1971) 2. Naturforsch. 26b, 1270- 1276. 7. WEIRICH, G., AND WREN, J. (1976) Physiol. 2001. 49, 341-350. 8. SHIRK, P. D., DAHM, K. H., AND ROLLER, H. (1976) 2. Naturjorsch. 31c, 199-200. 9. DAHM, K. H., BHASKARAN, G., PETER, M. G., SHIRK, P. D., SESHAN, K. R., AND ROLLER, H. (1976) in The Juvenile Hormones (Gilbert, L. I., ed.), pp. 19-47, Plenum, New York. 10. METZLER, M., MEYER, D., DAHM, K. H., ROLLER, H., AND SIDDALL, J. B. (1972) 2. Naturforsch. 27b, 321-322.
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GLANDS
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11. PRAY, G. E., AND TOBE, S. S. (1974) Life Sci. 14, 575- 586. 12. PRA’IT, G. E., TOBE, S. S., AND WEAVER, R. J. (1975) Ezperkmtia 31, 120- 122. 13. JUDY, K. J., SCHOOLEY, D. A., DUNHAM, L. L., HALL, M. S., BERGOT, B. J., AND SIDDALL, J. B. (1973) Proc. Nat. Acad. Sci. USA 70, 1509- 1513. 14. JUDY, K. J., SCHOOLEY, D. A., HALL, M. S., BERGOT, B. J., AND SIDDALL, J. B. (1973) Life Sci. 13, 1511-1516. 15. JUDY, K. J., SCHOOLEY, D. A., TROETSCHLER, R. G., JENNINGS, R. C., BERGOT, B. J., AND HALL, M. S. (1975) Life Sci. 16, 1059-1066. 16. SCHOOLEY, D. A., JUDY, K. J., BERGOT, B. J., HALL, M. S., AND SIDDALL, J. B. (1973) Proc. Nat. Acad. Sci. USA 70, 2921-2925. 17. SCHOOLEY, D. A., JUDY, K. J., BERGOT, B. J., HALL, M. S., AND JENNINGS, R. C. (1976) in The Juvenile Hormones (Gilbert, L. I,, ed.), pp. 101-117, Plenum, New York. 18. JENNINGS, R. C., JUDY, K. J., SCHOOLEY, D. A., HALL, M. S., AND SIDDALL, J. B. (1975) Life Sci. 16, 1033-1040. 19. TOBE, S. S., AND STAY, B. (1977) Gen. Comp. Endocrinol. 31, 138-147. 20. REIBSTEIN, D., AND LAW, J. H. (1973) Biochem. Biophys. Res. Commun. 55, 266-272. 21. REIBSTEIN, D., LAW, J. H., BOWLUS, S. B., AND KATZENELLENBOGEN, J. A. (1976) in The Juvenile Hormones (Gilbert, L. I., ed.), pp. 131-146, Plenum, New York. 22. SCHNEIDERMAN, H. A., AND WILLIAMS, C. M. (1954) Biol. Bull. 106, 238-252. 23. JUDY, K. J. (1968) Ph.D. Thesis, Northwestern University. 24. WEIRICH, G., WREN, J., AND SIDDALL, J. B. (1973) Insect Biochem. 3, 397-407. 25. SHAPIRO, S. K., AND EHNINGER, D. J. (1966) Anal. Biochem. 15, 323-333. 26. SLADE, M., AND ZIBITT, C. H. (1972) in Insect Juvenile Hormones (Menn, J. J., and Beroza, M., eds.), pp. 155-176, Academic Press, New York. 27. AJAMI, A. M., AND RIDDIFORD, L. M. (1973) J. Insect Physiol. 19, 635-645.
S-Adenosylmethionine:Juvenile Accessory Reproductive
Hormone Acid Methyltransferase in Male Glands of Hyalophora cecropia (L.)l
GONTER F. WEIRICHl Institute
of Developmental Biology,
AND
Texas A&M
MICHAEL University,
G. CULVER3 College Station, Texas 7784.3
Received March 23, 1979; revised July 25, 1979 The accessory reproductive glands of the adult male Hyalophora cecropia contain S-adenosylmethionine:juvenile hormone acid methyltransferase. The enzyme is soluble and can be found in the gland epithelium as well as in the glandular secretion, but not in any other part of the genital tract of the unmated male. The appearance of this enzyme activity in the pharate adult precedes the formation of a measurable pool of its substrate, juvenile hormone acid, and the onset of the juvenile hormone accumulation in the accessory reproductive glands. The accessory reproductive glands of Anthrea pernyi and Manduca sexta, species which do not accumulate juvenile hormone, lack methyltransferase activity. It is concluded that the methyltransferase is an essential component of the juvenile hormone accumulation mechanism-in H. cecropia.
The males of the silkmoth Hyalophora cecropia (L.) have been known for over 20
moths (1, 2) where, beginning in the late pharate adult (3-5) or young adult (6), it years for their exceptionally high content is accumulated during most of the adult life. of JH4 (1). The function of this large amount This phenomenon has so far been in apparent of hormone remains elusive to this day, but conflict with the observation that the hemosignificant progress has been made recently lymph of the moth has high JH esterase toward understanding the physiological activity (7) and lacks a carrier protein caevents leading to this accumulation. pable of protecting the hormone against enThe JH, though originating from the cor- zymatic hydrolysis (Weirich and Culver, in pora allata, is found in the abdomen of the preparation). Shirk et al. (8) identified the ’ This work was supported by National Science male AGs as the “repository” for the hormone where it can be sequestered from the Foundation Grant PCM ‘72-01892to Drs. H. Rijller and K. H. Dahm and by Organized Research, Texas hemolymph either unaltered or in a process accompanied by the exchange of the methyl A&M University. 2 Present address: Insect Physiology Laboratory, ester group (9). Agricultural Research, SEA, USDA, Beltsville, Md. The formation of the methyl ester has 20705. been characterized as one of the final steps 3 Present address: Waters Associates, Maple Street, of JH biosynthesis in adult H. cecropia males Milford, Mass. 01757. in vivo (10) and in corpora allata of various * Abbreviations used: AG, accessory reproductive gland; JH, juvenile hormone, not differentiating be- species in vitro (11, 12). Methionine can be utilized as a methyl donor for the ester fortween the three naturally occurring hormones; JHI, racemic methyl (2E,6E,10cis)-lO,ll-epoxy-7-ethylmation by whole animals (10) and by corpora 3,11-dimethyl-2,6-tridecadienoate; JHA, juvenile allata cultured in vitro (9, 13-19) whereas hormone acid, not differentiating between acids of the corpora allata homogenates require SAM three naturally occurring hormones; JHA-I, racemic for the methyl transfer reaction (1’7,20,21). (2E,6E,10cis)-10,ll-epoxy-7-ethyl-3,1l-dimethylIn the present paper, we have examined 2,6-tridecadienoic acid; JHAMT, S-adenosylmethiothe possibility that a similar reaction may nine:juvenile hormone acid methyltransferase; SAM, be involved in the JH accumulation of the S-adenosyl-L-methionine; PPO, 2,5-diphenyloxazole; H. cecropiu male AG and found that this is dimethyl-POPOP, p-bis[2-(4methyl-&phenyloxazolyl)]benzene. indeed the case. We have demonstrated the 175
0003-9861/79/130175-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
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presence of the enzyme, S-adenosylmethionine:juvenile hormone acid methyltransferase (EC 2.l.l.xx), in the AG of H. cecropia males and present data on its tissue and subcellular distribution. This enzyme catalyzes the reaction: JHA + SAM + JH + S-adenosylhomocysteine. For comparison, two species which do not accumulate JH, Antherea pernyi and Manduca sexta (2,9), were included in this study. MATERIALS
AND METHODS
Animals. Pupae of H. cecropia and A. pernyi were obtained from commercial suppliers and stored at 5°C for at least 5 months. Adult development was initiated by transfer to 27°C. Some animals had terminated diapause prior to this transfer and, consequently, required less time than others to complete adult development. Morphological criteria (22, 23) were therefore used to determine the stage of adult development in experiments involving pharate adults. M. se&a was reared as described previously (24). Dissection and fractionation of tissues. AGs and other tissues were dissected from carbon dioxide-narcotized males, freed from adhering fat body, and rinsed with ice-cooled Grace’s tissue culture medium (Grand Island Biological Co., Grand Island, N. Y.). Where indicated, tissue and content of AGs were separated by allowing spontaneous extrusion from short pieces of the tubular glands and subsequently rinsing them with the aid of a capillary pipet. The tissues and/or gland contents were homogenized in Grace’s medium with small all-glass homogenizers by hand and in some cases the incubations were carried out in these homogenizers. In other cases, the homogenate was fractionated by one or two centrifugations at 4°C (30 min at 12,OOOg,60 min at 100,OOOg). Chemicals. JH-I was obtained from Eco-Control, Cambridge, Massachusetts, and purified by high resolution liquid chromatography. JHA-I (reference for thin layer chromatography) was prepared by alkaline hydrolysis of JH-I. JH-I[‘7-ethyl-l,2-3H(N)] of 11.2 Ci/mmol specific activity (corrected for decay) was purchased from New England Nuclear, Boston, Massachusetts. PHIJHA-I for JHAMT assays was prepared by enzymatic hydrolysis from PHIJH-I, diluted to various specific activities with unlabeled JH-I, by a modification of the method described by Metzler et al. (10). SAM chloride (grade II, Sigma, St. Louis, MO.) was purified by ion exchange chromatography on AG l-X8 (Bio-Rad, Richmond, Calif.) (25). SAM[methyl-3H] (7.5 Ci/mmol) was obtained from Amersham/Searle, Arlington Heights, Illinois.
Enzym assay. The incubation mixtures contained 10e6to 10m5M rH)THA-I, lo-” to lo-’ M SAM or PHISAM (neutralized with 0.1 M potassium phosphate buffer, pH ‘7.5), and the tissue preparation as indicated, in a total volume of 50 to 200 ~1 of Grace’s medium. After 180 to 200 min of incubation at 25”C, the reactions were stopped by addition of ethylacetate. Unlabeled JH was added as carrier, the reaction mixtures were extracted four times with 1 ml ethylacetate, and the extracts were analyzed by thin layer chromatography on silica HF-254 (E. Merck, Darmstadt, Germany) using a solvent system of 15% ethylacetate and 1% glacial acetic acid in benzene. The plates were divided according to sidemarkers of JH and JHA and the silica was transferred to scintillation vials and thoroughly mixed with a scintillation mixture (100 g naphthalene, 7 g PPO, and 0.05 g dimethyl-POPOP/ liter dioxane). The radioactivity was determined in a Searle Mark III liquid scintillation system with a counting efficiency of 50%. The amounts of labeled methylation products were calculated from the specific activity of the added labeled precursor, with no correction for the dilution by endogenous precursors. As a consequence, the data represent underestimates of the total quantities formed.
RESULTS
Tissue Distribution Changes
and Developmental
When homogenates of H. cecropia male AG tissue or luminal content were incubated with PHIJHA-I and SAM, both tissue and content produced similar amounts of rH]JH (Table I). At a PHIJHA-I concentration of 10e6M, 32 to 52 pmol was methylated; at a PHIJHA-I concentration of 4.0 to 9.0 x lop5 M, the yield of PHIJH was 428 to ‘714pmol. Despite the higher absolute amount of PHIJH-I, the proportion of rH]JHA converted was lower at the higher pH]JHA-I concentrations. In subsequent experiments rH]JHA-I was applied in the 1O-6M range. The accumulation of JH in the AGs of H. cecropia males is known to begin shortly before or after adult eclosion (3-6). This led us to speculate that induction or activation of the JHAMT may be responsible for the initiation of the JH accumulation. To test this hypothesis, we assayed AG homogenates obtained from pharate adults at various stages of development for JHA-methylating activity. As shown in Table II, the AGs have high JHAMT activity as early as 8 days prior to eclosion. Its occurrence
JUVENILE
HORMONE ACID METHYLTRANSFERASE
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177
GLANDS
TABLE I METHYLATION OF [3H] JHA-I IN HOMOGENATESOF MALE ADULT H. cecropia AG TISSUE AND CONTENT” [3H]JH synthesis Homogenate of gland tissue
Homogenate of gland content
Percentage of applied acid
pm01
Percentage of applied acid
pm01
52 45
49
7
572
4
428
Concentration of Incubation No.
Moth No.
1 2
1
3 4 5
2
6 7
3
Age
[3H] JHA-I
SAM
(day)”
(PM)
(PM)
1 3
3
1.5 1.1
100 100
35
1.2 86 43
200 200 90
27 7
87 57
200 90
8
32 620 714
“ Incubations contained homogenate equivalent to 0.13 gland pair (incubation Nos. 1, 2) or 0.25 gland pair (incubation Nos. 3-7). Incubation time was 180 min (Nos. 1, 2) or 200 min (Nos. 3-7). Total volume was 100 ~1 for incubations containing homogenized gland tissue (Nos. l-4, 6) or 200 ~1 for incubations containing homogenized gland content (Nos. 5, 7). * Day 1 = day of eclosion.
is apparently not correlated with the onset of JH accumulation. The AGs, testes, vas deferens, seminal vesicles. and common duct were dissected from a fully formed male moth shortly before eclosion and assayed under identical conditions. Only the kGs showed JHAMT activity (Table III) whereas the other parts
of the male genital tract did not. Thus, at least in the unmated male, the enzyme is not released from the AGs. Subcellular Distribution In order to determine the subcellular localization of the JHAMT, an AG homogenate was subjected to a two-step fractionation.
TABLE II METHYLATION OF [“HI JHA-I IN AC HOMOGENATES OF PHARATE ADULT H. cecropia”
Characteristic
Day of pharate adult development*
[3H] JH-I synthesis
TABLE III METHYLATION OF [8H] JHA-I IN VARIOUS SEGMENTS OF THE MALE GENITAL TRACT OFH. cecropia”
(pmol)
[“HI JH-I synthesis (pm00
Beginning antenna1 pigmentation Complete antenna1 pigmentation Complete wing pigmentation
14
41
16
41
20
39
a Each incubation contained 1.0 x 10e6M [3H]JHA-I, 1.0 X 1O-4M SAM, and homogenate equivalent to 0.5 gland pair in a total volume of 100 ~1. Incubation time 180 min. b Adult eclosion at Days 22-23 (Refs. (22, 23)).
Testes Vas deferens Seminal vesicle Accessory glands Common duct
0 0 0 37 0
a Homogenates of the indicated organs (0.25 animal equivalent each), dissected from a fully formed pharate adult male (Day 22), were incubated for 180 min with 2.0 x 10m6M [3H]JHA-I and 1.8 x 10m4M SAM in a total volume of 54 ~1.
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TABLE IV METHYLATIONOF [SHIJHA-I BYVARIOUSSUBCELLULARFRACTIONSOFTHE H. cecropia MALE AG” [3H] JH-I synthesis
Fraction Crude homogenate 12,000g Supernate 100,OOOgSupernate 100,OOOgPellet
Concentration of [3H] JHA-I (I*@
Gland pair equivalents
1.3
0.07
2.0 2.4
0.07 0.07
1.2
1.0
pm01
nmol/gland pair equivalent
54 114 124 26
0.77 1.63 1.77 0.03
(I AG from Day 1 adult males. All incubations contained 1.0 x 10m4M SAM and the total volume was 100 ~1. Incubation time, 180 min.
Neither the 12,000g centrifugation (30 min) nor the 100,OOOgcentrifugation (60 min) eliminated the activity in the resulting supernate, and the microsomal pellet had only marginal activity (Table IV). It can be concluded, therefore, that the JHAMT is a soluble enzyme. Time Cour.se and Substrate Requirements
of PHISAM. Labeled material cochromatographing with JH was produced in quantities comparable to the quantities of JH obtained in incubations with PHIJHA-I and unlabeled SAM (Table V, lines 4 and 5). The yield of this methylation product was dependent on the concentration of PHISAM. These results suggest the existence of a pool of JHA in the AGs which can provide the precursor for JH formation, or the potential for a methyl exchange of JH stored in the glands. To test the latter possibility, we added JH-I and PHISAM to the AG homogenate assuming that an increased JH-I concentration should result in an increased methyl
As shown in Fig. 1, the JHA-I methylation proceeds nearly linearly for 120 min before it levels off. In order to identify the limitations of the assay system, we determined the effects of various components of the reaction mixture on the yield of PHIJH (Table V). The complete reaction mixture, containing lop6 M PHIJHA-I, 6.3 x 10F5M SAM, and 12,000g supernate equivalent to 0.13 gland pair in 100 ~1 of Grace’s medium, yielded 34.4 pmol JH-I after a 3-h incubation. Following a “blank” incubation without enzyme, radioactivity equivalent to 3.4 pmol PH]JH-I was found in the JH fraction. All other data were corrected for the amount 10 of rH]JH-I found in corresponding enzymefree incubation mixtures. Omission of SAM did not eliminate the production of JH-I but 120 180 60 reduced it to 18.4 pmol, suggesting that the Incubation time (mid gland homogenate either contained SAM or was capable of synthesizing it. FIG. 1. Time course of PHIJHA-I methylation. In other incubations, PHIJHA-I was Homogenate of two pairs of AGs of Day 1 adult H. omitted from the reaction mixture and the cecropia males was centrifuged for 30 min at 12,000g. formation of JH or JH-like methylation Each incubation contained supernate equivalent to 0.13 products was monitored by determining the gland pair, 1.0 x 10e6M PHIJHA-I, and 6.2 x 10e5M incorporation of the labeled methyl group SAM in a total volume of 104 ~1. Mean * SE.
JUVENILE
HORMONE
ACID
METHYLTRANSFERASE TABLE
IN REPRODUCTIVE
GLANDS
179
V
THE EFFECTS OF JHA-I, JH-I, AND SAM ADDITIONS ON THE JHAMT REACTION IN AG HOMOGENATESOFH. cecropia” [“HI JHA-I
No.
(FM)
JH-I
SAM
r3H] JH formed
(PM)
(PM)
(pmol)”
1.0
63
34.4 k 0.3
63
3.4 18.4 33.2 16.7
No enzyme
1.0 1.1
63 r3H] 8.7[3H]
1.0 1.0[3H]
8.7[3H]
zk 1.7 k 2.3 k 1.4 2 3.9
11.1 ? 2.9 13H]JH hydrolyzed 10.7 2 0.7
(’ Homogenate of two pairs of AGs of Day 1 adult males were centrifuged for 30 min at12,OOOg.Supernate equivalent to 0.13 gland pair was incubated as shown for 180 min in a total volume of 100 ~1. h Mean 2 SE. Except for No. 2, corrections were made for the amounts found in incubations without homogenate.
exchange. This, however, was not the case (Table V, line 6). Apparently, the enzyme does not facilitate the direct exchange of the methyl group in the JH-I molecule. The gland homogenate, however, had JH esterase activity (Table V, line 7). Precursor
Pool
Since the AG homogenates of Day 1 adult males apparently contain JHA which can be methylatecl under in vitro incubation conditions (Table V), it was of interest to evaluate AGs of other age groups for the availability of this precursor(s). When AG homogenates of pharate adults were incubated with 1O-5M PHISAM, they yielded only marginal amounts of labeled methylation product(s) (Table VI) whereas in glands of adult males the quantity increased to the level previously found in incubations containing 10e6M PHIJHA-I and 10e5M unlabeled SAM. These results suggest that the AGs do not contain a significant pool of JHAs prior to the time of eclosion. Other Species
We also tested two lepiclopteran species which do not accumulate JH, Antherea pemyi and Manduca sexta, for JHAMT activity. No labeled JH or JH-like material was found
after incubations of AG homogenates of young adult males either with [3H]JHA-I plus unlabeled SAM, or with PHISAM. DISCUSSION
In the present study, we have used enzymatically prepared PH]JHA-I as a specific tool to test for JHAMT activity in various tissues and tissue fractions. The methylation of JHA without distinction between the three naturally occurring homologs can also be measured by the incorporation of the labeled methyl group of [methyL3H]SAM. This approach, however, is not specific since the formation of other labeled methylation products can not be ruled out. We have shown that the male AGs of adult Hyalophora cecropia have a methyltransferase capable of methylating JHA to JH. Similar to other methyltransferases, the enzyme is soluble, most of the activity remaining in the supernate of a 60 min 100,OOOg centrifugation. In contrast to the JH formation in corpora allata homogenates ((20); M. G. Peter, unpublished), the methylation of JHA in AG homogenates is not entirely dependent on exogenous SAM, even though the yield can be substantially increased by the addition of SAM. Apparently, the glands have an enclogenous SAM pool or the capacity to produce SAM under in vitro conditions.
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TABLE VI JHA IN AG HOMOGENATES OF H. cecropiaa [3H]Methylation produ& Stage*
PA PA PA PA
A A A
Age’ (day) 14’ 16’ 20’ 22’ 1s 3h 3h
Concentration of [3H]-SAM (PM) 10
10 10
15 8.7 10 10
Gland pair equivalents 0.5 0.5 0.5 0.25 0.13 0.25 0.25
Incubation time (mm) 180 180
180 180 180 200 200
pm01
pmol/gland pair equivalent
0.03 0.52 0.12 0.35 1.0 40.9 46.8
0.06 1.04 0.24 1.40 7.69 163.6 187.2
a Total volume for Day 22 pharate adult was 54 ~1, for all others 100 ~1. b pA, pharate adult; A, adult. c for pA, see Ref. (23); for A, Day 1 = day of eclosion. d Cochromatographing with JH. ’ Data obtained from same AG homogenates as those in Table II. ’ Same AG homogenate as in Table III. g Moth No. 1 of Table I. h Moth Nos. 2 and 3 of Table I.
It is obvious from our results that the AG can perform the reaction that has been characterized as the last or one of the final steps of the JH biosynthesis in the corpora allata (11, 12, 21). Interestingly, the substrate for this reaction is also one of the primary degradation products of JH, found in many insect species including H. cecropia (26,27) as the result of enzymatic hydrolysis in the hemolymph (7). In fact, the hemolymph of adult H. cecropia has high JH esterase activity (‘7) and apparently no carrier protein capable of protecting the hormone from enzymatic hydrolysis (Weirich and Culver, in preparation). Therefore, it is very unlikely that JH could be carried intact via the hemolymph to the AGs if, indeed, it is secreted by the corpora allata. This seemingly paradoxical situation can be resolved by the assumption that the AGs are not sequestering JH proper from the hemolymph but the JHA which, in turn, is (re)methylated by the JHAMT. Some JH esterase activity was also found in the AG homogenates (Table V), probably accounting for the incorporation of the PHImethyl group of rH]methionine observed in incubations of AGs with JH (9). Because of the presence of hydrolyzing and methylat-
ing enzymes in the AG homogenate, the yields determined for either reaction are only approximations. We were actually measuring the difference between two antagonistic reactions with the one for which the higher substrate concentration was provided outweighing the other. How this relates to the in viva situation and whether or not the JH esterase of the AG has a physiological function remains a matter of conjecture. Also, the quantitation and the kinetic parameters of the JHAMT will have to be worked out in future investigations after at least partial purification of the enzyme. JHAMT activity is present at least 1 week before eclosion, but -JHA is not traceable in AG homogenates until after eclosion (Table VI). Thus, it is not the induction or activation of the enzyme that is determining the onset of the JH accumulation in the AGs but rather the availability of the substrate, JHA. Surprisingly, the JHAMT is not confined to the epithelium of the AG but can be found in the secretion as well. The function of the enzyme in the glandular secretion is obscure, but it is interesting to note that it is also the secretion that contains all of the stored JH (8, 9).
JUVENILE
HORMONE
ACID
METHYLTRANSFERASE
Finally, JHAMT is not an enzyme universally found in male AGs of adult Lepidoptera. We have demonstrated that it exists in AGs of H. cecropia and it may occur in glands of other Saturniid species accumulating JH, but it is not found in two lepidopteran species that do not accumulate measurable amounts of JH, Antherea pernyi (also Saturniidae) and Manduca sexta (Sphingidae). Therefore, the JHAMT seems to be an essential component of the JH accumulating mechanism. ACKNOWLEDGMENTS We thank Dr. H. Rijlier for his continued interest and encouragement, Dr. P. Shirk for his help in dissecting accessory glands, Dr. K. H. Dahm for purified samples of JH-I and JHA-I and for helpful suggestions during the preparation of the manuscript, and Dr. M. G. Peter for purifying SAM. REFERENCES 1. WILLIAMS, C. M. (1956) Nature (London) 178, 212-213. 2. WILLIAMS, C. M. (1963) Biol. Bull. 124,355-367. 3. WILLIAMS, C. M. (1959) Biol. Bull. 116323-338. 4. WILLIAMS, C. M. (1961) Biol. Bull. 121, 572-585. 5. GILBERT, L. I., AND SCHNEIDERMAN, H. A. (1961) Gen. Comp. Endocrinol. 1, 453-472. 6. METZLER, M., DAHM, K. H., MEYER, D., AND ROLLER, H. (1971) 2. Naturforsch. 26b, 1270- 1276. 7. WEIRICH, G., AND WREN, J. (1976) Physiol. 2001. 49, 341-350. 8. SHIRK, P. D., DAHM, K. H., AND ROLLER, H. (1976) 2. Naturjorsch. 31c, 199-200. 9. DAHM, K. H., BHASKARAN, G., PETER, M. G., SHIRK, P. D., SESHAN, K. R., AND ROLLER, H. (1976) in The Juvenile Hormones (Gilbert, L. I., ed.), pp. 19-47, Plenum, New York. 10. METZLER, M., MEYER, D., DAHM, K. H., ROLLER, H., AND SIDDALL, J. B. (1972) 2. Naturforsch. 27b, 321-322.
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11. PRAY, G. E., AND TOBE, S. S. (1974) Life Sci. 14, 575- 586. 12. PRA’IT, G. E., TOBE, S. S., AND WEAVER, R. J. (1975) Ezperkmtia 31, 120- 122. 13. JUDY, K. J., SCHOOLEY, D. A., DUNHAM, L. L., HALL, M. S., BERGOT, B. J., AND SIDDALL, J. B. (1973) Proc. Nat. Acad. Sci. USA 70, 1509- 1513. 14. JUDY, K. J., SCHOOLEY, D. A., HALL, M. S., BERGOT, B. J., AND SIDDALL, J. B. (1973) Life Sci. 13, 1511-1516. 15. JUDY, K. J., SCHOOLEY, D. A., TROETSCHLER, R. G., JENNINGS, R. C., BERGOT, B. J., AND HALL, M. S. (1975) Life Sci. 16, 1059-1066. 16. SCHOOLEY, D. A., JUDY, K. J., BERGOT, B. J., HALL, M. S., AND SIDDALL, J. B. (1973) Proc. Nat. Acad. Sci. USA 70, 2921-2925. 17. SCHOOLEY, D. A., JUDY, K. J., BERGOT, B. J., HALL, M. S., AND JENNINGS, R. C. (1976) in The Juvenile Hormones (Gilbert, L. I,, ed.), pp. 101-117, Plenum, New York. 18. JENNINGS, R. C., JUDY, K. J., SCHOOLEY, D. A., HALL, M. S., AND SIDDALL, J. B. (1975) Life Sci. 16, 1033-1040. 19. TOBE, S. S., AND STAY, B. (1977) Gen. Comp. Endocrinol. 31, 138-147. 20. REIBSTEIN, D., AND LAW, J. H. (1973) Biochem. Biophys. Res. Commun. 55, 266-272. 21. REIBSTEIN, D., LAW, J. H., BOWLUS, S. B., AND KATZENELLENBOGEN, J. A. (1976) in The Juvenile Hormones (Gilbert, L. I., ed.), pp. 131-146, Plenum, New York. 22. SCHNEIDERMAN, H. A., AND WILLIAMS, C. M. (1954) Biol. Bull. 106, 238-252. 23. JUDY, K. J. (1968) Ph.D. Thesis, Northwestern University. 24. WEIRICH, G., WREN, J., AND SIDDALL, J. B. (1973) Insect Biochem. 3, 397-407. 25. SHAPIRO, S. K., AND EHNINGER, D. J. (1966) Anal. Biochem. 15, 323-333. 26. SLADE, M., AND ZIBITT, C. H. (1972) in Insect Juvenile Hormones (Menn, J. J., and Beroza, M., eds.), pp. 155-176, Academic Press, New York. 27. AJAMI, A. M., AND RIDDIFORD, L. M. (1973) J. Insect Physiol. 19, 635-645.