Isolation of l (+)-methionine sulfoxide related to diapause induction in the silkworm, Bombyx mori

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Insect Biochem. Molec. Biol. Vol. 25, No. 9, pp. 975-980, 1995

Pergamon

0965-1748(95)00027-5

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/95 $9.50 + 0.00

Isolation of L(+)-Methionine Sulfoxide Related to Diapause Induction in the Silkworm, Bombyx mori JUN YANG,* MAKOTO KIMURA,t EIICHI KUWANO,I" KOICHI SUZUKI*$ Received 15 July 1994; revised and accepted 27 February 1995

L( -~- )-Methionine sulfoxide [L( + )-Met(O)] was isolated from pupal hemolymph of the silkworm, Bombyx mori, by means of thin layer chromatography and high performance liquid chromatography. Its stucture was determined by FD-and FAB-mass spectrometry and by comparison with authentic L( + )-Met(O). The concentration of natural L( + )-Met(O) was very low in the fifth larval instar, but increased rapidly during the progression of the wandering stage and throughout the pupal and adult stages. The concentration of L( + )-Met(O) was higher in the hemolymph of diapause egg producers than in non-diapause egg producers, suggesting a relationship with the regulation of diapause and/or development. L( + )-methionine sulfoxide Diapause induction Silkworm

INTRODUCTION In many insects, free amino acids in the hemolymph have been analyzed throughout developmental periods, and diapause. The occurrence of free amino acids in hemolymph is especially notable in insects. Their hemolymph concentrations are 6-to 50-fold higher than in vertebrates (Wyatt, 1961) and 100 to 300 times higher than in human blood (Chen, 1985). Amino acid pools are very substantial and are important for protein metabolism during insect development (Chen, 1966, 1985; Bodnaryk, 1978). Lucas and Levenbook (1966) isolated L( + )-methionine sulfoxide [L(+ )-Met(O)] for the first time from a natural organism, the blowfly Phormia regina. This amino acid is found only rarely in nature and does not occur after the acid hydrolysis of most proteins. However, it is found as a constituent of the natural protein in the resiliums of surf clams (Kikuchi and Tamiya, 1981) and certain other specific proteins (Brot and Weissbach, 1988). In a previous study with the silkworm Bombyx mori (Yang et al., 1992), we found that the concentration of an unidentified amino acid, which is eluted between aspartic acid and threonine, is higher in the whole bodies of newly hatched larvae and the brains of newly ecdysed pupae that have experienced an environmental regimen of diapause induction than in those reared under a non-diapause *Faculty of Agriculture, Iwate University, Morioka 020, Japan. tFaculty of Agriculture, Kyushu University, Fukuoka 812, Japan. SAuthor for correspondence. 975

inducing condition. The concentrations of all other amino acids were the same in the newly hatched larvae and pupal brains irrespective of rearing conditions. This result stimulated us to analyze the relationship between diapause induction and environmental conditions, since embryonic diapause induction is determined under the environmental regimen experienced during maternal embryonic development in the bivoltine race of the silkworm (Kogure, 1933). Our present study demonstrates that this amino acid, isolated from the silkworm hemolymph, is L(+ )-Met(O) and discusses a possible role of this amino acid in diapause induction. MATERIALS AND METHODS Insects

Larvae of the silkworm, a bivoltine race (Daizo) of B. mori, were fed on mulberry leaves and semi-artificial

diet at 24°C under the condition of light 12/dark 12 h. Diapause eggs 12 h after oviposition were treated with HCI (S.G., 1.075) for 5 min to inhibit diapause initiation (Yamashita and Suzuki, 1991). According to the procedure of Kogure (1933), one group of eggs was incubated at 27°C under constant light throughout embryonic development. This group can be anticipated to produce diapause eggs in the next generation (HL-condition). The other group, was incubated at 15°C in complete darkness for 24 h, will produce non-diapause eggs (LD-condition) in the next generation.

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J U N Y A N G et al.

Thin layer chromatographic (TLC) plates (20 × 20 cm, 0.25 mm thick) of silica gel 60 was obtained from E. Merck Co. All chemicals were of analytical grade.

Instrumental analysis F D - a n d FAB-mass spectrometry were conducted on a JEOL DX300 and JMS-SX102 mass spectrometer, respectively. ~H-NMR were determined with JEOL JNM-FX 100 spectrometer in D20, using 2,2-dimethyl-2silapentane-5-sulfonate as an internal standard. Optical rotation was measured on a Union automatic polarimeter PM-101.

Preparation and analysis ojfree amino acids

Chemicals L(+)-Methionine sulfoxide [Met(O)] was purchased from Sigma Chemical Co. The two diastereomers, L(+)-and L( -- )-Met(O), were resolved by fractionation of the picrates according to the procedure of Lavine (1947). L(+)-Met(O). [~]~ +97.0°C (synthetic) (c. 1.0 in water) [Lit. (Lucas and Levenbook, 1966), [~]~ +99.0 _+ 2°C (synthetic), [~]~ +99.7 ___2°C (natural) (c. 1.0 in water)]. ~H-NMR 6:2.1-2.5 (2H, m), 2.72 (3H, s), 2.8-3.2 (2H, m), 3.86 (IH, t, J 6 Hz). Anal. Found: C, 36.49; H, 6.74; N, 8.44%. Calcd for CsH,NO3S: C, 36.36; H, 6.67; N, 8.48%. The absolute configuration at the sulfur atom of L(+)-Met(O) has been established to be (S)-configuration (Christensen and Kjar, 1965). L(--)-Met(O). [~]~ -82.0°C (synthetic) (c. 1.0 in water) [Lit. (Lucas and Levenbook, 1966), [~]~ - 7 7 . 0 _+ 2°C (synthetic) (c. 1.0 in water)]. ~H-NMR 6:2.1 2.5 (2H, m), 2.72 (3H, s), 2.8 3.3 (2H, m), 3.84 (1H, t, J = 6 Hz). Anal. Found: C, 36.45; H, 6.76; N, 8.39%. =

Larval hemolymph was collected by cutting the abdominal legs and pupal hemolymph was collected by puncturing the abdominal cuticle under cold anesthesia. Hemolymph samples were collected in 1.5 ml Eppendorf tubes containing a small amount of phenylthiourea and centrifuged at 10,000 rpm for 5 min to remove hemocytes. Supernatants were stored at - 8 0 ° C until used. Preparations were rapidly thawed, mixed with an aliquot of 5% (w/v) sulfosalicylic acid and the mixtures centrifuged at 10,000 rpm for 5 min. After passage through a membrane filter (cellulose nitrate, 0.2 pm) the clear supernatant was injected into a high speed amino acid analyzer (MLC-203, ATTO Co.), described previously (Suzuki et al., 1984; Yang et al., 1992). Authentic t.(+)-Met(O) and L( -- )-Met(O) were also subjected to the same analysis. We found that the free amino acid concentration and the composition of the supernatant fluid did not change after storage and thawing when compared to fresh samples.

Extract of unident~'ed am&o acid from pupal hemolymph on TLC The clear supernatant from the hemolymph preparations was applied to the lane on the plate with a microsyringe. Chromatograms were monodimensionally

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Retention time F I G U R E 1. A n unidentified amino acid derivative extracted from pupal hemolymph of the silkworm. The insert photograph shows that the T L C chromatogram, developed from the pupal hemolymph extract, was visualized by ninhydrin reagent. After extraction of the ninhydrin-positive c o m p o u n d s corresponding to each band visualized on both edges of the T L C plate, they were analyzed by a high speed amino acid analyzer. An unidentified amino acid (X-AA) was eluted between aspartic acid and threonine, and its retention time was 26.8 rain. The arrows indicate X - A A on the T L C plate and on the amino acid analyzer.

M E T H I O N I N E SULFOXIDE A N D DIAPAUSE INDUCTION

X-AA fraction fromTLC CA) X-AA 50

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TSK gel ODS-80Ts (Tosoh Co., Tokyo). The column was eluted with a linear gradient of 16-50% acetonitrile in 0.05% trifluoroacetic acid (TFA) over 65 min at a flow rate of 1 ml/min. The elution profile was monitored by UV absorbance at 210 nm, and each peak was collected and analyzed on the amino acid analyzer. Unknown amino acid fractions were rechromatographed on a second H P L C under the same conditions. A third H P L C was performed using an isocratic elution with 16% acetonitrile, using the same column, but without T F A at a flow rate of 0.5 ml/min.

16

Bioassay of L( + )-Met(O)

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The amino acid was injected into 10 individuals of non-diapause egg producers within 24 h after larval-pupal ecdysis, using a fine microglass tube (10 #l/individual). Eggs produced subsequently by the treated individuals were evaluated for the presence or absence of diapause.

(B) 16% C H 3 C N

RESULTS

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In this study we isolated X-AA from pupal hemolymph by TLC. After developing 0.5 ml of hemolymph extract, thirteen spots were observed on both edges of the plate by spraying with ninhydrin solution (Fig. 1). All bands were scraped off, and the amino acids were extracted and analyzed on the amino acid analyzer• The third band

Retention time (min) FIGURE 2. The first and third reversed phase HPLC on a TSK gel ODS-80Ts column of X-AA fraction from the TLC plate. (A) On the first HPLC, the X-AA fraction was chromatographed with a linear gradient of 16-50% CH3CNin the presenceof 0.05% TFA at a flowrate of 1 ml/min. Each peak was examined by the amino acid analyzer and the double peak representing X-AA (arrow) was collected and rechromatographed with the same conditions. (B) The X-AA fraction recovered from the second HPLC was rechromatographed with 16% CH3CN in the absence of TFA at a flow rate of 0.5 ml/min.

developed with the solvent system of 1-butanol-acetic acid-water (4:1:2, v/v). After development, plates were completely air-dried and repeatedly developed 4 times with the same solvent to obtain a favorable separation (each chromatogram required for about 6 h). It was necessary for identification to run reference lanes of standard amino acids parallel to the experimental lanes and to spray them with a solution of 0.1% ninhydrin in butanol. Each area on the gel corresponding to the Rf value of standards or unknown spots, was scraped off and the amino acids extracted with water. Extracts were centrifuged at 10,000 rpm for 5 min, passed through a membrane filter (cellulose nitrate, 0.2 #m) and finally lyophilyzed. The components of these extracts were identified on the high speed amino acid analyzer.

Isolation of an unknown amino acidfrom TLC extract by high performance liquid chromatography (HPLC) TLC extracts were subjected to reverse phase high performance liquid chromatography ( R P - H P L C ) on

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FIGURE 3. FD-mass spectrum of the compound isolated from pupal hemolymph (A) and authentic L(_+)-Met(O) (B).

978

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J U N Y A N G et al.

X-AA occurred in the position of c(+)-Met(O), when both X-AA and L(+)-Met(O) were injected simultaneously into the column of the analyzer. X-AA also exhibited a circular dichroism (CD) curve (water) with a positive maximum at 210 nm which is identical with that of c(+)-Met(O). From these results, X-AA was determined to be h(+)-Met(O). From the results of the amino acid analyzer (Yang et al., 1992), we believe that X-AA in hatched larvae and pupal brains is also identical with c( + )-Met(O). We also examined the changes of c(+)-Met(O) in the hemolymphs of final instar larvae, pupae and emerged adults. As shown in Fig. 5, the hemolymph of the larvae, pupae and adults that experienced HL-condition during embryonic development, contained a higher concentration of L( + )-Met(O) than those which experienced the

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Retention time (min) F I G U R E 4. C h r o m a t o g r a m of L(4-)-Met(O), L(+)-Met(O), L(-- )Met(O) and the purified X - A A by the high speed amino acid analyzer. Authentic L( _+)-Met(O) revealed two peaks (A) and it was resolved to I.( - )-Met(O) (B) and L( + )-Met(O) (C). L( + )-Met(O) and the purified X-AA preparations were run on the high speed amino acid analyzer following simultaneous injection (D).

contained X-AA which was detected as a single peak on the amino acid analyzer (Fig. 1). TLC separation was used for further preparative purification of X-AA, since most of the other amino acids could be easily removed. X-AA first eluted as a double peak [Fig. 2(A)] and therefore was reapplied and eluted under the same conditions. The fraction which now eluted in a single peak (not shown as data) was reapplied to the same column using the second elution schedule and X-AA was collected as a single peak [Fig. 2(B)]. The high resolution FAB mass spectrum of X-AA indicated a molecular formula CsH~2NO3S (m/z 166.0537, [ M + H]+), consistent with that of c-Met(O). The FD-mass spectrum of X-AA showed a [M + H] + ion at m/z 166 and the fragment at m/z 121 arising from the loss of a carboxyl group. Its fragmentation pattern was identical with that of authentic c(+)-Met(O) (Fig. 3). X-AA was compared with authentic c( _+)-Met(O), L(+)-Met(O) and L( -- )-Met(O), using a high speed amino acid analyzer. Figure 4 shows that the peak of

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F I G U R E 5. Changes in the content of L( + )-Met(O) in the hemolymph during the periods of the final instar larvae, pupae and the new adults. The silkworms experiencing the regimen of high temperature (27c C) and continuous light throughout the period of embryonic development (H L, ©), and of low temperature (15'C) and continuous darkness throughout the period of embryonic development (LD, • ) . After the fourth larval ecdysis they were divided into female and male groups. The inset figures in the female and male groups show the expanded data from day 0 to wandering stage in the final instar larvae. W, wandering larva; P, pharate pupa; NP, new pupa; A, new adult. Dashed lines indicate that the period of the final instar larvae (females and males) LD-condition experienced is short (2 days) and the wandering stage is equal to day 5. The data express mean values + SD of 3~4 replicates of different preparations.

METHIONINE SULFOXIDE AND DIAPAUSE INDUCTION

979

T A B L E 1. Changes in the content of methionine in the hemolymph during the periods

of the finalinstar larva, pupa and adult. Methioninecontents(/tmoles/ml) Female Stages

Final instar

larva

Pupa

Days

HL*

Male LD**

HL

LD

0 1 2 3 4 5 6 W

0.31 0.32 0.42 0,56 0.60 0.43 0.55 0.73

___+0.04t __+0.07 + 0.03 + 0.05 + 0.05 + 0.03 + 0.02 -I- 0.06

0.34 0.40 0.47 0.50 0.56

+ 0.02 __+0.12 __+0.08 + 0.06 + 0.08 --4j -0.71 __+0.04

0.27 0.37 0.44 0.39 0.37 0.32 0.50 0.56

+ 0.01 -t- 0.04 + 0.16 + 0.08 + 0.05 + 0.02 + 0.07 + 0.05

0.25 0.43 0.53 0.41 0.42

P 0 1 2 3 4 5 7

0.83 0.84 1.05 1.07 1.20 0.82 0.74 0,72

+ 0.07 + 0.09 + 0.19 + 0,12 __+0.04 + 0.04 _+ 0.09 + 0.09

0.74 0.79 0.99 1.01 1.12 0.87 0.70 0.62

0,71 0.79 0.95 1.12 1.27 1.02 0.90 0,86

__+0.07 + 0.16 + 0.14 _ 0.15 _+ 0.14 + 0.09 + 0,08 + 0.04

0.77 0.89 1.02 1.12 1.22 1.10 0.91 0.81

Adult

0,55 + 0.05

_+ 0.12 + 0.04 + 0.17 -I- 0.16 _+ 0.10 + 0,07 + 0.11 _+ 0.16

0.55 _ 0.05

0.69 + 0.06

+ 0.03 + 0.06 + 0.10 + 0.07 + 0.07 --0.50 ___+0.03 + 0.04 +_ 0.08 + 0.15 ± 0.07 + 0.16 _+ 0.08 + 0.04 + 0.05

0.67 + 0.09

The silkworms experienced the regimen of high temperature (27°C) and continuous light throughout the period of embryonic development (HL*), and of low temperature (15°C) and continuous darkness thoughout the period of embryonic development (LD**). After the 4th larval ecdysis, they were divided to female and male groups. t E a c h data expresses mean values + SD of three to four replicates of different preparations. §The period of the final instar larvae (females and males) experienced LD-condition is short for 2 days and the wandering stage is equal to day 5. W, wandering larvae; P, pharate pupae.

LD-condition. In each stage, that concentration in insects which experienced HL-condition was 1.5 times higher than those of the LD-condition and particularly, the maximum difference was observed in pharate pupae (pre-pupae) of each sex. Female pharate pupae which experienced HL-condition, had 2.6-fold higher concentrations of L(+)-Met(O) in the hemolymph, compared with those that experienced LD-condition. Methionine concentrations increased from pharate pupal stage to pharate adult stage but did not show noticeable difference between insects that experienced HL-condition and LD-condition at any stage (Table 1). Concentrations of other amino acids also showed the same pattern as in the case of methionine (unpublished data). The effects of authentic L(+ )-Met(O) on non-diapause egg producers investigated in this experiment, indicated that injections of about 3-or 4-fold equivalents of this amino acid in vivo (25 ~tmol/individual injected) did not convert non-diapause induced insects to diapause producer at all following evaluation of the oviposited eggs. DISCUSSION L-Met(O) has so far been found in several insects. In the silkworm, B. mori, L-Met(O) was found in protein free extracts of larval and adult stages according to analysis by the amino acid analyzer (Osanai and Kikuta, 1981;

Osanai and Yonezawa, 1984). These reports have not mentioned the configuration at the sulfur atom of L-Met(O), although Lucas and Levenbook (1966) reported for the first time the isolation of L(+)-Met(O) from the blowfly, P. regina. Recently, Laurema and Varis (1991) indicated that Met(O) is one of the most abundant components of the free amino acids in the salivary glands of Lygus species. In addition, the diastereoisomeric forms of L-Met(O) were separated by Laurema (1989) and according to retention times the form in insects including Lygus was determined to be L(+)-Met(O). If L-Met(O) is involved in important physiological functions of Bombyx insect, determination of the configuration around the asymmetric sulfur atom of L-Met(O) is indispensable. Commercial L(+)-Met(O), a mixture of the diastereomers, shows a double peak characteristic on a high speed amino acid analyzer (Fig. 4). The amino acid isolated from pupal hemolymph of the silkworm was found to be coincident with one of diastereomers of L(_ )-Met(O) by the amino acid analyzer. Consequently, the structure of this amino acid was determined by means of FD-and FAB-mass spectrometers and comparison with authentic L(+)-Met(O) which is prepared by resolution of L(+ )-Met(O) by using picric acid (Lavine, 1947). We have now concluded that one of the ninhydrin-positive compounds in the silkworm brains and newly hatched larvae is L(+)-Met(O) (Yang et al.,

980

JUN YANG et al.

1992). The present isolation of L(+)-Met(O) from hemolymph is the second report from an insect source following that of the blowfly, P. regina (Lucas and Levenbook, 1966), and therefore, we suggest that this amino acid is relatively more common in insects and perhaps may be one of the predominant free amino acids. Since Met(O) is rarely found in animal proteins, the significance and function of Met(O) have been something of a mystery in insects as well as in other animals, yeasts and plants (Brot and Weissbach, 1988). In the blowfly and the silkworm, L(+ )-Met(O) is one of the most abundant amino acids and its content varies with different stages of development. In the blowfly L(+)-Met(O) occurs in low concentrations in the larval and early pupal stages but increases rapidly during the later pupal stages and continues at a high level (6-8/~mol/g) during the adult stage (Lucas and Levenbook, 1966). In the silkworm, this amino acid is present in new fifth instar larvae, and its content increases markedly just after the wandering stage (Fig. 5) and remained at the same level (4-7/~mol/ml) as in the blowfly adult. Since Hasegawa (1957) isolated diapause hormone (DH) from pupal suboesophageal ganglia of the silkworm, the amino acid sequence of this hormone has been determined by Imai et al. (1991) and the function of DH investigated in detail after its secretion during the middle stage of diapause egg producers (Yamashita and Hasegawa, 1985). According to the finding of Kogure (1933), whether egg producers oviposit diapause eggs or non-diapause eggs, is determined by HL-and LDconditions during their embryonic development periods. This finding has been explained by inhibiting and stimulating actions of the brain to suboesophageal ganglion during the early pupal stage (Fukuda, 1952). Additionally, there may be GABA-ergic control in pupal brains for the release of DH from suboesophageal ganglion (Hasegawa and Shimizu, 1990). These data, however, do not explain the biochemical mechanism involved in diapause induction found by Kogure (1933). In our previous paper (Yang et al., 1992), we indicated that the concentration of an unidentified amino acid derivative is high in pupal brains and hatched larvae which have experienced HL-condition. The present study demonstrates that this amino acid derivative is L(+)-Met(O). Moreover, we show that the titer of this amino acid depends upon the HL- and LD-conditions experienced during the embryonic development periods (Fig. 5). In contrast, such a difference was not found in the concentration of methionine in the hemolymph during the same stages (Table 1). We suggest that L(+)-Met(O) is likely a product of the environmentally induced metabolism regulating diapause, since the exogenous administration of this amino acid does not function to induce diapause. Investigations of the metabolism of this amino acid may eventually establish a relationship between the environmental stimuli regulating diapause induction.

RREFERENCES Bodnaryk R. P. (1978) Stucture and function of insect peptides. Adt. Insect Physiol. 13, 69 70. Brot N. and Weissbach H. (1988) Methionine sulfoxide: chemistry and biochemistry. In The Chemistry of Sulphones and Sulphoxides (Eds Patai S., Rappoport Z. and Stirling C. J. M.), pp. 851 872. Wiley, Chichester. Chen P. S. (1966) Amino acid and protein metabolism in insect development. Ade. Insect Physiol. 3, 53 114. Chen P. S. (1985) Amino acid and protein metabolism. In Comprehensive Insect Physiology. Biochemist O, and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 10, pp. 177 217. Pergamon Press, Oxford. Christensen B. W. and Kjar A. (19653 The absolute configuration of methionine sulphoxide. Chem. Commun. 225-226. Fukuda S. (1952) Factors determining the production of non-diapause egg in the silkworm. Proc. Jap. Acad. 27, 582 586. Hasegawa K. (1957) The diapause hormone of the silkworm, Bomhvx mori. Nature 179, 1300 1301. Hasegawa K. and Shimizu I. (1990) GABAergic control of the release of diapause hormone from the suboesophageal ganglion of the silkworm, Bombyx mori. J. Insect Physiol. 36, 909 915. Imai K., Konno T., Nakazawa Y., Komiya T., Isobe M., Koga K., Goto T., Yaginuma T., Sakakibara K., Hasegawa K. and Yamashita O. (1991) Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proe. Jap. Acad. Ser. B. 67, 98-101. Kikuchi Y. and Tamiya N. (1981) Methionine sulfoxide in the resilium protein of surf clams. J. Biochem. 89, 1975 1976. Kogure M. (1933) The influence of light and temperature on certain characters of the silkworm, Bornbyx mori. J. Dept. Agri. Kyushu Imp. Unit,. 4, 1 93. Laurema S. (1989) Free amino acids in the psyllid Trio~_aapicalis Forest. (Homopt. Triozidae) and in carrot leaves. Ann. Agric. k~,nn. 28, 113 120. Laurema S. and Varis A.-L. (1991) Salivary amino acids in Lygus species (Heteroptera: Miridae). Insect Biochem. 21,759-765. Lavine T. F. (1947) The formation, resolution, and optical properties of the diastereoisomeric sulfoxides derived from t,-methionine. J. Biol. Chem. 169, 477~,91. Lucas F. and Levenbook L. (19663 The isolation of L(+ )-methionine sulphoxide from the blowfly Phormia regina Meigen. Biochem. J. 100, 473~478. Osanai M. and Kikuta S. (19813 Age-related changes in amino acid pool sizes in the adult silkworm, Bombyx mori. Exp. Geront. 16, 445459. Osanai M. and Yonezawa Y. (1984) Age-related changes in amino acid pool sizes in the adult silkworm, Bombyx mori, reared at low and high temperature; a biochemical examination of the rate-of-living theory and urea accumulation when reared at high temperature. Exp. Geront. 19, 37 51. Suzuki K., Hosaka M. and Miya K. (1984) The amino acid pool of Bombyx mori eggs during diapause. Insect Biochem., 14, 557 56 I. Wyatt G. R. (1961) The biochemistry of insect hemolymph. A. Ret'. Ent. 6, 75 102. Yamashita O. and Hasegawa K. (19853 Embryonic diapause. In Comprehensive Insect Physiology. Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 407--434. Pergamon Press, Oxford. Yamashita O. and Suzuki K. (1991) Roles of morphogenic hormone in embryonic diapause. In Morphogenic Hormones in Arthropods (Eds Gupta A. P.), Vol. 3, pp. 82-128. Rutgers University Press, New Brunswick. Yang J., Shimizu Y. and Suzuki K. (1992) A difference in free amino acids in the pupal brains between diapause and non-diapause of the silkworm, Bombvx mori. J. Seric. Sci. Jpn 61, 28 31.

Acknowledgements We thank Professor W. S. Bowers, the University of Arizona, for his critical reading of the manuscript. The research was supported by a grant (No. 93107) for Scientific Research from the Ministry of Education, Science and Culture, Japan.