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Comparison of methionine metabolism in symbiotic and aposymbiotic larvae of Sitophilus oryzae L. (Coleoptera: Curculionidae)—I. Evidence for a glycine N-methyltransferase-like activity in the aposymbiotic larvae
Comp. Biochem. Physiol. Vol. 85B, No. 1, pp. 245-250, 1986 Printed in Great Britain
0305-0491/86 $3.00+0.00 Pergamon Journals Ltd
COMPARISON OF METHIONINE METABOLISM IN SYMBIOTIC A N D APOSYMBIOTIC LARVAE OF SITOPHILUS O R Y Z A E L. (COLEOPTERA: CURCULIONIDAE)--I. EVIDENCE FOR A GLYCINE N-METHYLTRANSFERASE-LIKE ACTIVITY IN THE APOSYMBIOTIC LARVAE F. GASNIER-FAUCHET*,A. GHARIB~ and P. NARDON Laboratoire de Biologie, INSA 406, 69621 Villeurbanne Cedex, France; and l"Laboratoire de Chimie Biologique, INSERM U 205, INSA 406, 69621 Villeurbanne Cedex, France (Received 13 January 1986) Alam-aet--1. Sarcosine and methionine metabolisms were investigated in symbiotic and aposymbiotic strains of the weevil Sitophilus oryzae. The follow-up of these two amino acids during larval and pupal development showed that they had opposite variations and suggested that they were involved in the same metabolic pathway. 2. The index of methylation, three times higher in the aposymbiotic larvae than in the symbiotic larvae, indicated that the tissues of aposymbiotic larvae were more suitable for transmethylations than the tissues of symbiotic larvae. 3. Nutritional experiments with methionine and glycine gave evidence that these two amino acids were involved in sarcosine biosynthesis in the weevils. The addition of ethionine, an inhibitor of methyltransferases, to the diet resulted in a decrease in the levels of sarcosine in both strains. 4. It is therefore suggested that sarcosine biosynthesis in the aposymbiotic larvae of S. oryzae results probably from a glycine N-methyltransferase-like activity, though such an enzyme is at the present time unknown in insects.
INTRODUCTION During the last years, a heightened interest in studies of animal symbiosis, especially intracellular symbiosis, has been observed. More data are now available on the physiological and biochemical interactions between the symbiotic partners, but very few concern insects, despite the presence of numerous species harbouring intracellular microorganisms. This lack of data is partly due to the difficulties encountered in isolating and cultivating the endosymbionts and particularly the bacteria, Among Coleoptera, the Curculionidae are well known for intracellular symbiosis (Buchner, 1965) and especially the Calandrini (Nardon et al., 1985). The symbiotic bacteria of Sitophilus oryzae were first discovered by Pierantoni (1927). They are located in a larval bacteriome (in both sexes), in anterior mesenteric caeca of young adults, and in female germ cells (Scheinert, 1933; Nardon and Wicker, 1981). The study of the role of these bacteria in the physiology of their host has been made possible by the obtainment of viable aposymbiotic strains according to different procedures (Nardon, 1973). Physiological comparative studies of an aposymbiotic strain of S. oryzae with the symbiotic strain from which it was issued have shown that their
*To whom all correspondence should be addressed. Present address: D~partement de Bioehimie, Facult~ de M6decine, Lyon-Sud, BP 12, 69921 Oullins Cedex, France.
vitamin requirements differed, and it was therefore concluded that the intracellular bacteria provided the insect with pantothenic acid, biotin and riboflavin (Wicker, 1983). Biochemical comparative studies with the same strains have demonstrated that they also differed in their amino acid compositions: the elimination of the symbiotic bacteria resulted in a very high increase in the sarcosine levels in the larvae and a simultaneous decrease in their methionine sulfoxide levels (Gasnier et al., 1984). These differences were also found when aposymbiotic and symbiotic strains of another Sitophilus species (S. zeamais) were compared (GasnierFauchet, 1985). Sarcosine (N-methylglycine) is uncommon in insects since traces of this methylated amino acid were reported in two species only (Firling, 1977; Mayer et al., 1975). But no study was started to elucidate the problems of the origin and the role of sarcosine in this class. In order to investigate sarcosine formation in S. oryzae larvae, known data in mammals was used, where the synthesis of this amino acid may result from the activities of two enzymes involved in choline cycle (Fig. 1). One of these enzymes, dimethylglycine dehydrogenase, a mitochondrial FAD-containing dehydrogenase, oxidizes dimethylglycine to sarcosine (Frisell and Mackenzie, 1962; Wittwer and Wagner, 1980). The other one, glycine N-methyltransferase, a cytoplasmic S-adenosylmethionine-dependent methyltransferase, catalyzes the transmethylation of glycine to sarcosine (Blumenstein and Williams, 1960; Heady and Kerr, 1973; Ogawa and Fujioka, 1982).
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Nutritional experiments involving choline and dimethylglycine as well as enzymatic experiments with mitochondria failed to demonstrate any dimethylglycine dehydrogenase activity in S. oryzae larvae (Gasnier-Fauchet, 1985) which confirms the absence of the metabolic pathway involving betaine in insects noticed by several workers (Wilfis and Hodgson, 1970). We report here the data concerning the investigation o f the second pathway involving S adenosylmethionine, glycine and glycine N methyltransferase in aposymbiotic and symbiotic S. oryzae larvae. MATERIALS AND METHODS Strains and diets Two strains of S. oryzae were used in these experiments: a symbiotic one called RR and an aposymbiotic one called SS, obtained from RR strain according to Nardon (1973). Aposymbiotic larvae and adults had no bacteriome and microscope examination of the ovaries of the females showed that they were free of the symbiotic bacteria. Both strains were reared on wheat (except in nutritional studies) at 27.5°C and 75% r.h., using the procedure of Laviolette and Nardon (1963). Since Wicker (1984) had shown that the development time, the fertility and the number of the symbiotes of S.
oryzae were not modified when they were reared on whole wheat flour pellets instead of wheat, this semi-artificial diet was used for our nutritional studies. The flour was provided to the larvae in pellets form, supplemented (or not for control experiments) with the amino acids which were to be tested: 1 and 1.5% methionine, 1% glycine, 1% methionine + 1% glycine and 1% ethionine. Since larval growth was better with whole wheat flour and starch (50%-50%, w/w) than with flour only for high concentrations of dietary methionine (1.9%), the mixture wheat flour/starch was used in this experiment only. Experimental procedures Larvae were kept on wheat until the beginning of last instar. They were then taken from inside the grains and transferred on the pellets until the end of the instar, which was reached after 5-6 days of diet. Larvae were then removed from the pellets, weighed individually, rinsed and dried. Larval free amino acids were extracted and analyzed. Sarcosine catabolism was investigated by injecting this amino acid in the larvae as follows: fully-grown last-instar larvae of both strains reared on wheat were immobilized by placing them on a watch-glass laid on ice. One hundred nmoles of sarcosine in solution in 0.1/zl of Yeager serum, whose osmotic pressure and pH had been adapted to the weevils (Nardon and Grenier, 1983), were injected in the haemolymph of the larvae. Following injection the larvae were maintained in covered beakers without access to food at 27.5°C. After 4 hr incubation periods, the larval free amino acids were extracted.
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Methionine metabofism--I Amino acid and nucleoside analyses Amino acid~. Pools of 2 or 3 larvae were homogenized in cold distilled water. An internal standard (norvaline) was added. The homogenate was deproteinized with 5% tdchloroacetic acid (w/v) and centrifuged at 10,000 g (4°C) for 3 rain. The supernatant was then freed of lipids twice as follows: two volumes of chloroform were added to the last supernatant and the mixture was centrifuged at 10,000g (4°C) for 3 min. The final supernatant containing the free amino acid fraction was evaporated to dryness. Free amino acids were rehydrated in 0.06 M citrate buffer, pH 2.20, and chromatographed with a Liquimat III Kontron automatic analyzer. Nucleosides. S-adenosyl-L-methionine (SAM) and Sadenosyl-L-homocysteine (SAH) were the only nucleosides studied. Each assay was performed with a pool of three last-instar larvae. The larvae were weighed individually and each pool was homogenized in 200 #1 cold perchloric acid (0.4 N). The mixture was centrifuged at 10,000g (4°C) for 2 min. The supernatant was collected and stored at -20°C before use. The separation of nucleosides was performed by reverse-phase HPLC and paired-ion chromatography according to Gharib et al. (1982). The eluate was monitored by an absorbance detector at 254 nm. The compounds were identified by their retention times and quantified by peak area measurement.
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The presence of sarcosine in the symbiotic strain appeared quite variable. But its concentration always remained very low compared with the aposymbiotic strain (its highest value did not exceed 3.82 nmoles/insect). If no striking difference was noticed between the aposymbiotic and the symbiotic strains concerning methionine variations (Fig. 3), the concentration of this amino acid appeared to be influenced by the sex of the insect in both strains: it remained low in the larvae and pupae (often less than 2 nmoles/insect in both strains) and increased in 10-day old adults, reaching higher values in females (from 7 to 8 nmoles/insect in both strains) than in males (from 1.92 nmoles/insect in RR strain to 4.56 nmoles/insect in SS strain).
RESULTS
Methyl-donor availability As shown in Table 1, the endogenous levels of SAH did not differ in the last-instar larvae of both strains. In contrast, the level of SAM was found significantly higher (at a 0.01% probability level) in the aposymbiotic larvae than in the symbiotic larvae (+210%). Consequently, the resultant index of methylation (SAM/SAH ratio) appeared to be significantly higher (at a 0.01 probability level) in the aposymbiotic strain than in the symbiotic one (+250%).
Sarcosine and methionine variations during development Sarcosine was found at all stages of development in aposymbiotic weevils (Fig. 2) from the 2nd larval instar until the emergence of adults. First instar larvae were not investigated (they were too small to be located in the grains). The values corresponding to second and third instar larvae were not reported since their levels of sarcosine were too low to be precisely quantified. The concentration of sarcosine increased regularly during last larval instar and reached its highest value at the end of this instar: 43.49 nmoles/insect. Then it decreased in pupae and completely disappeared in 10-day old adults.
Effect of amino acid supplementation Supplementation of the diet with methionine resulted in a graded increase in the level of sarcosine in the aposymbiotic larvae (Table 2), which was proportional to the methionine content of the diet ( + 150, +340, +470% with 1, 1.5, 1.9% supplementation, respectively). The amount of sarcosine in the symbiotic larvae increased too, but the concentrations remained three times lower at least than in the aposymhiotic larvae. As shown in Table 2, the addition of glycine to the diet did not affect the levels of sarcosine in the larvae of both strains. But the addition of glycine and methionine (1%: 1%, w/w, respectively) together in
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Fig. 2. Sarcosine variations during development in symbiotic RR and aposymbiotic SS strains. L4: fourth-instar larvae; P: pupae; E: emergence; Al0:l0 day-old adults; O: larvae then male pupae and adults; O: female pupae and adults.
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Fig. 3. Methionine variations during development in symbiotic RR and aposymbiotic SS strains. L4: fourth-instar larvae; P: pupae; E: emergence; At0:10 day-old adults; O: larvae then male pupae and adults, @: female pupae and adults.
Table I. S-AdenosyI-L-methionine (SAM) and S-adenosylL-homocysteine (SAH) levels in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Nucleoside RR SS SAM 22.50 5:7.49 (9)* 69.70 + 18.09(6) SAH 1.69 +-0.73 (9) 1.44 + 0.56 (6) SAM/SAH 15.20 +- 6.51 (9)* 53.89 + 20.61 (6) The concentrations are expressed as pmoles/mg of fresh weight. Values represent means _+SD. Number in parentheses indicate the number of determinations. *Values significantlydifferent between RR and SS strains at the 1% level (Student's t-test).
Table 2. Effect of dietary methionine (Met), glycine (Gly) and ethionine (Et) supplementation on the levels of sarcosine in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Supplementation RR SS Controls 0.32 5:0.30 (3) 2.15 + 0.58 (4) 1.0% Met 1.5% Met 1.9% Met
1.62 __.0.36 (6) 1.79 __.1.07(3) 4.70 + 2.38 (5)
5.37 +- 1.25(4) 9.54 + 2.00 (4) 12.24 + 2.60 (7)
1% Gly
0.32 +- 0.04 (2)
1.95 + 0.12 (2)
1% Gly + 1% Met
2.65+-0.47(2)
12.03+-1.21(3)
1% Et 0 (2) 1.58 + 0.05 (2) The concentrations are expressed as nmoles/mg of fresh weight. Values represent means _+SD with the number of determinations in parentheses.
Table 3. Effect of the injection of sarcosine (Sarc) on the levels of free glycine (Gly) and free serine (Ser) in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Treatment
Amino acid
Controls
Ser Gly
+ Sarc
Ser Gly
RR
4.28 + 0.95 (5) 3.02+_0.86(5)
SS
4.19 + 1.68(6) 3.17+ 1.42(5)
3.96 + 0.39 (2) 3.47 _.+2.06 (2) 2.64 + 0.22 (2) 2.49 + 0.63 (2) The concentrations are expressed as nmoles/mg of fresh weight. Values represent means + SD with the number of determinations in parentheses. For more details, see Materials and Methods.
the diet resulted in a very high increase in the a m o u n t s o f sarcosine in the aposymbiotic a n d in the symbiotic larvae which was more i m p o r t a n t t h a n the increase o b t a i n e d with m e t h i o n i n e only ( + 6 3 % for R R strain a n d + 124% for SS strain c o m p a r e d with the results o b t a i n e d with 1% m e t h i o n i n e supplementation). O n the contrary, the ingestion by the larvae o f a diet s u p p l e m e n t e d with 1% ethionine (Table 2) resulted in b o t h strains in a decrease in their levels o f sarcosine ( - 2 6 % for SS larvae, sarcosine was even a b s e n t in the symbiotic larvae). Injection o f sarcosine did n o t affect the a m i n o acid c o m p o s i t i o n o f b o t h strains. N o increase in the a m o u n t s o f glycine a n d serine were n o t e d (Table 3). T h e 100 nmoles injected were fully recovered. DISCUSSION T h e differences observed previously in the sarcosine levels o f fully-grown last-instar symbiotic a n d a p o s y m b i o t i c larvae o f S. oryzae ( G a s n i e r et al., 1984) were also f o u n d d u r i n g larval a n d p u p a l develo p m e n t . In a n o t h e r work o n Sitophilus (Wicker et al., 1985), no sarcosine was detected in the f o u r t h instar larvae. This was due to the m e t h o d used, analysis being m a d e with one larva only. In these conditions, the levels o f some a m i n o acids were at the limit of detection o f the analyzer. T h e increase in the level o f sarcosine o f actively feeding a p o s y m b i o t i c larvae a n d the decrease observed d u r i n g p u p a l instar w h e n they stopped feeding lead to the conclusion t h a t sarcosine synthesis should d e p e n d on dietary precursors. T h e g r o w t h a n d m o u l t i n g of insects are u n d e r c o n t r o l o f three m a i n groups o f h o r m o n e s : b r a i n h o r m o n e s , ecdysteroids, a n d juvenile h o r m o n e s . The last-mentioned, in which activities differ in adults a n d larvae (Sehneiderman, 1972) have been s h o w n to regulate certain S A M - d e p e n d e n t methyltransferasic activities (Rojas a n d Barbier, 1982). T h e fact t h a t m e t h i o n i n e c o n c e n t r a t i o n r e m a i n e d low in the aposymbiotic larvae a n d p u p a e a n d increased in 10-day old adults a n d t h a t sarcosine showed simultaneously
Methionine metabolism--I opposite variations suggests that these two amino acids might be involved in the same metabolic pathway and that this pathway could be influenced by juvenile hormones. The principal role of SAM, also called "active methionine" (Cantoni, 1951), is to give its methyl group in transmethylation reactions. Transmethylations in insects have been little investigated and no data about the levels of SAM and SAH in insects are available. No comparison between Sitophilus weevils and other insects is therefore possible; but it can be noticed that the concentration of SAM in the SS larvae does not differ from the concentration of SAM found in mammalian liver, one of the richest organs in methylases (Baldessarini and Kopin, 1966; Hoffman et al., 1981). It is generally admitted that the index of methylation is an indication of the methylation degree of the molecules within a tissue. The higher it is, the more methylations occur, since the K m values for SAM and the /~ for SAH of most methylases do not much differ. The index of methylation, higher in the SS strain than in the RR strain, therefore indicates that the tissues of aposymbiotic larvae are more suitable for transmethylations than the tissues of symbiotic larvae and confirms our previous hypothesis that methionine metabolism differed in the presence and in the absence of the symbiotic bacteria (GasnierFauchet, 1985). Ethionine is the ethyl analogue of methionine. Its effect as inhibitor of SAM-dependent cellular methylation reactions is well-known in mammals. There is strong evidence that it exerts its effects by lowering the levels of SAM by inhibiting SAM-synthetase and competing with SAM as a substrate in transmethylation reactions (Hoffman, 1984). The fact that the levels of sarcosine decreased in the larvae in the presence of ethionine suggests that sarcosine biosynthesis may result from the activity of a methyltransferase. Moreover the nutritional experiments with methionine and glycine demonstrate the involvement of these two amino acids in sarcosine biosynthesis. All these results give evidence for a glycine Nmethyltransferase-like activity in the aposymbiotic larvae of S. oryzae. The symbiotic larvae were shown to have the enzymes required for sarcosine synthesis but they appeared to be stimulated by high dietary methionine levels only. Free methionine content of larval tissues seems to be the limiting factor for this synthesis in both strains. In mammals, sarcosine is oxidized to glycine (see Fig. 1) by a mitochondrial sarcosine dehydrogenase (Frisell and Mackenzie, 1962), but our results suggest that this enzyme should be absent in S. oryzae larvae. Moreover, data obtained with fecal material indicate that sarcosine was not excreted by the larvae and that its very low level in the symbiotic larvae was not the consequence of an increased excretion in this strain (Gasnier-Fauchet, 1985). It appears therefore that the decrease in the sarcosine levels observed in the aposymbiotic pupae was probably the consequence of its elimination with the exuvial fluid during pupal eedysis. The facts that sarcosine and methionine had opposite variations during S. oryzae development and
249
that sarcosine biosynthesis was proportional to methionine content of the diet and also that sarcosine accumulated in larval tissues without being used suggest that sarcosine could have a role in the elimination of an excess of methionine coming from a diet too rich in this amino acid to allow a good growth of the larvae. The wheat which was used in our experiments contained 0.45% methionine, which was higher than the required concentration for an optimal growth, ranged from 0.10 to 0.30% in the absence of L-cystine, these values being lower in the presence of L-cystine (Baker, 1978). It appears therefore that the presence of sarcosine in aposymbiotic S. oryzae larvae could be an adaptative response to diets high in methionine, as it is the case in mammals (Mitchell and Benevenga, 1976) and that symbiotic larvae probably get rid of this amino acid by another metabolic pathway. In conclusion, the presence of sarcosine in a p t symbiotic larvae of S. oryzae seems to result from a glycine N-methyltransferase-like activity, though such an enzyme is at the present time unknown in insects. This work provides a starting point for further /n vitro enzymatic studies aiming at purification and characterization of this transferase.
Acknowledgements--The authors
would like to t h a n k J.
Guilland for excellent assistance throughout this study. This work was supported by grants from CNRS and INSERM. REFERENCES
Baker J. E. (1978) Sulphur amino acid requirements of larvae of Sitophilus oryzae (Coleoptera: Curculionidae). Comp. Biochem. Physiol. 60A, 355-360. Baldessarini R. J. and Kopin I. J. (1966) Sadenosylmethionine in brain and other tissues. J. Neurochem. 13, 769-777. Blumenstein J. and Williams J. R. (1960) The enzymic N-methylation of giycine. Biochem. biophys. Res. Comm. 3, 259-263. Buchner P. (1965) Endosymbiosis of Animals with Plant Microorganisms. Wiley Interscience, New York. Cantoni G. L. (1951) Activation of methionine for transmethylation. J. biol. Chem. 189, 745-754. Firling C. E. (1977) Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans. J. Insect Physiol. 23, 17-22. Frisell W. R. and Mackenzie C. G. (1962) Separation and purification of sarcosine dehydrogermse and dimethylglycine dehydrogenase. J. biol. Chem. 237, 94-98. Gasnier F., Nardon P. and Guillaud J. (1984) Influence of bacterial symbionts on the amino acid composition of S. oryzae larvae (Coleoptera: Curculionidae). Endocyt. C. Res. 1, 69-79. Gasnier-Fauchet F. (1985) Etude du r61e des bacttries symbiotiques dans le mttabolisme prottique de leur h6te Sitophilus oryzae (Coltopttre: Curculionide). Th~se de 3 e cycle. Universit6 Lyon I, Ecole Nationale Vtttrinaire de Lyon. Gharib A., Sarda N., Chabannes B., Cronenberger L. and Pacheco H. (1982) The regional concentrations of Sadenosyl-L-methionine, S-adenosyl-L-homocysteine and adenosine in rat brain. J. Neurochem. 38, 810-815. Heady J. E. and Kerr S. J. (1973) Purification and characterization of glycine N-methyltransferase. J. biol. Chem. 248, 69-72. Hoffman R. M. (1984) Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis. Biochim. biophys. Acta 738, 49-87.
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Hoffman D. R., Hanning J. A. and Cornatzcr W. E. (1981) Mierosomal phosphatidylethanolamine methyltransferase: inhibition by S-adenosylhomocysteine. Lipids 16, 561-567. Laviolette P. and Nardon P. (1963) Action des rayons y du cobalt 60 sur la mortalit6 et la fertilit6 des adultes d'un charangon du riz. Bull. Biol. Fr. et Belg. 97, 305-333. Mayer R. T., Cooper J., Farr F. M. and Singer R. H. (1975) Some effects of ionizing radiation on adult horn flies, Haematobia irritans. Insect Biochem. 5, 35-42. Mitchell A. D. and Benevenga N. J. (1976) Importance of sarcosine formation in methionine methyl carbon oxidation in rat. J. Nutr. 1116, 1702-1713. Nardon P. (1973) Obtention d'une souche asymbiotique chez le charanqon Sitophilus sasakii Tak.: diff6rentes m6thodes et comparaison avec la souche symbiotique d'origine. C.r. Acad. ScL Pans 277D, 981-994. Nardon P. and Grenier A. M. (1983) Etude des divers types d'anomalies ovariennes rencontr6es chez le charangon Sitophilus oryzae (L.). Bull. Soc. ent. Ft. 88, 292-300. Nardon P., Louis C., Nicolas G. and Kermarrec A. (1985) Mise en 6vidence et &ude des bact6ries symbiotiques chez deux charangons parasites du bananier: Cosmopolites sordidus (Germar) et Metamasius hemipterus (L.) (Col6opt~res:Curculionides). Annls Soc. ent. Fr. 21, 245-258. Nardon P. and Wicker C. (1981) La symbiose chez le genre Sitophilus (Col6opt~re: Curculionide). Principaux aspects morphologiques, physiologiques et g6n&iques. Ann. Biol. 20, 327-373. Ogawa H. and Fujioka M. (1982) Purification and characterization of glycine N-methyltransferase. J. biol. Chem. 257, 3447-3452. Pierantoni U. (1927) L'organo simbiotico nello sviluppo di
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35, 244-250. Rojas M. and Barbier M. (1982) Inhibitions of biological transmethylations by juvenile hormone III (10-epoxymethylfarnesoate). Naturwissenschaften 69, 43. Scheinert W. (1933) Symbiose und embryonalentwicklung bci Russel K~ifern. Z. Morphol. Okol. Tiere 44, 555-625. Schneider H. (1956) Morphologische und experimentelle Untersuchnngen iibcr die Endosymbiose der Korn- und Reisk~fer (Calandra granaria L. und C. oryzae). Z. Morph. Okol. Tiere 44, 555-625. Schneiderman H. A. (1972) Insect hormones and insect control. In Insect Juvenile Hormones. Chemistry and Action (Edited by Menn J. J. and Beroza M.), pp. 3-27. Academic Press, New York. Wicker C. (1983) Differentialvitamin and choline requirements of symbiotic and aposymbiotic S. oryzae (Coleoptera: Curculionidae). Comp. Biochem. Physiol. 76B, 177-182. Wicker C. (1984) Etude d'interactions nutritionnelIes et enzymatiques entre Sitophilus oryzae (Col6opt6re: Curculionide) et ses bact6riessymbiotiques intracellulaires.Th~se de Doctorat. INSA, Universit6 Lyon I. Wicker C., Guillaud J. and Bonnot G. (1985) Comparative composition of free,peptide and protein amino acids in symbiotic and aposymbiotic Sitophilus oryzae (Coleoptera: Curculionidae). Insect Biochem. 15, 537-541. Willis N. P. and Hodgson E. (1970) Absence of transmethylation reactions involving choline, betaine and methionine in the Insecta. Int. J. Biochem. I, 659-662. Wittwer A. J. and Wagner C. (1980) Identification of folate-bindingprotein of mitochondria as dimethylglycine dehydrogenase. Proc. natn. Acad. Sci. U.S.A. 77, 4484-4488.
0305-0491/86 $3.00+0.00 Pergamon Journals Ltd
COMPARISON OF METHIONINE METABOLISM IN SYMBIOTIC A N D APOSYMBIOTIC LARVAE OF SITOPHILUS O R Y Z A E L. (COLEOPTERA: CURCULIONIDAE)--I. EVIDENCE FOR A GLYCINE N-METHYLTRANSFERASE-LIKE ACTIVITY IN THE APOSYMBIOTIC LARVAE F. GASNIER-FAUCHET*,A. GHARIB~ and P. NARDON Laboratoire de Biologie, INSA 406, 69621 Villeurbanne Cedex, France; and l"Laboratoire de Chimie Biologique, INSERM U 205, INSA 406, 69621 Villeurbanne Cedex, France (Received 13 January 1986) Alam-aet--1. Sarcosine and methionine metabolisms were investigated in symbiotic and aposymbiotic strains of the weevil Sitophilus oryzae. The follow-up of these two amino acids during larval and pupal development showed that they had opposite variations and suggested that they were involved in the same metabolic pathway. 2. The index of methylation, three times higher in the aposymbiotic larvae than in the symbiotic larvae, indicated that the tissues of aposymbiotic larvae were more suitable for transmethylations than the tissues of symbiotic larvae. 3. Nutritional experiments with methionine and glycine gave evidence that these two amino acids were involved in sarcosine biosynthesis in the weevils. The addition of ethionine, an inhibitor of methyltransferases, to the diet resulted in a decrease in the levels of sarcosine in both strains. 4. It is therefore suggested that sarcosine biosynthesis in the aposymbiotic larvae of S. oryzae results probably from a glycine N-methyltransferase-like activity, though such an enzyme is at the present time unknown in insects.
INTRODUCTION During the last years, a heightened interest in studies of animal symbiosis, especially intracellular symbiosis, has been observed. More data are now available on the physiological and biochemical interactions between the symbiotic partners, but very few concern insects, despite the presence of numerous species harbouring intracellular microorganisms. This lack of data is partly due to the difficulties encountered in isolating and cultivating the endosymbionts and particularly the bacteria, Among Coleoptera, the Curculionidae are well known for intracellular symbiosis (Buchner, 1965) and especially the Calandrini (Nardon et al., 1985). The symbiotic bacteria of Sitophilus oryzae were first discovered by Pierantoni (1927). They are located in a larval bacteriome (in both sexes), in anterior mesenteric caeca of young adults, and in female germ cells (Scheinert, 1933; Nardon and Wicker, 1981). The study of the role of these bacteria in the physiology of their host has been made possible by the obtainment of viable aposymbiotic strains according to different procedures (Nardon, 1973). Physiological comparative studies of an aposymbiotic strain of S. oryzae with the symbiotic strain from which it was issued have shown that their
*To whom all correspondence should be addressed. Present address: D~partement de Bioehimie, Facult~ de M6decine, Lyon-Sud, BP 12, 69921 Oullins Cedex, France.
vitamin requirements differed, and it was therefore concluded that the intracellular bacteria provided the insect with pantothenic acid, biotin and riboflavin (Wicker, 1983). Biochemical comparative studies with the same strains have demonstrated that they also differed in their amino acid compositions: the elimination of the symbiotic bacteria resulted in a very high increase in the sarcosine levels in the larvae and a simultaneous decrease in their methionine sulfoxide levels (Gasnier et al., 1984). These differences were also found when aposymbiotic and symbiotic strains of another Sitophilus species (S. zeamais) were compared (GasnierFauchet, 1985). Sarcosine (N-methylglycine) is uncommon in insects since traces of this methylated amino acid were reported in two species only (Firling, 1977; Mayer et al., 1975). But no study was started to elucidate the problems of the origin and the role of sarcosine in this class. In order to investigate sarcosine formation in S. oryzae larvae, known data in mammals was used, where the synthesis of this amino acid may result from the activities of two enzymes involved in choline cycle (Fig. 1). One of these enzymes, dimethylglycine dehydrogenase, a mitochondrial FAD-containing dehydrogenase, oxidizes dimethylglycine to sarcosine (Frisell and Mackenzie, 1962; Wittwer and Wagner, 1980). The other one, glycine N-methyltransferase, a cytoplasmic S-adenosylmethionine-dependent methyltransferase, catalyzes the transmethylation of glycine to sarcosine (Blumenstein and Williams, 1960; Heady and Kerr, 1973; Ogawa and Fujioka, 1982).
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Nutritional experiments involving choline and dimethylglycine as well as enzymatic experiments with mitochondria failed to demonstrate any dimethylglycine dehydrogenase activity in S. oryzae larvae (Gasnier-Fauchet, 1985) which confirms the absence of the metabolic pathway involving betaine in insects noticed by several workers (Wilfis and Hodgson, 1970). We report here the data concerning the investigation o f the second pathway involving S adenosylmethionine, glycine and glycine N methyltransferase in aposymbiotic and symbiotic S. oryzae larvae. MATERIALS AND METHODS Strains and diets Two strains of S. oryzae were used in these experiments: a symbiotic one called RR and an aposymbiotic one called SS, obtained from RR strain according to Nardon (1973). Aposymbiotic larvae and adults had no bacteriome and microscope examination of the ovaries of the females showed that they were free of the symbiotic bacteria. Both strains were reared on wheat (except in nutritional studies) at 27.5°C and 75% r.h., using the procedure of Laviolette and Nardon (1963). Since Wicker (1984) had shown that the development time, the fertility and the number of the symbiotes of S.
oryzae were not modified when they were reared on whole wheat flour pellets instead of wheat, this semi-artificial diet was used for our nutritional studies. The flour was provided to the larvae in pellets form, supplemented (or not for control experiments) with the amino acids which were to be tested: 1 and 1.5% methionine, 1% glycine, 1% methionine + 1% glycine and 1% ethionine. Since larval growth was better with whole wheat flour and starch (50%-50%, w/w) than with flour only for high concentrations of dietary methionine (1.9%), the mixture wheat flour/starch was used in this experiment only. Experimental procedures Larvae were kept on wheat until the beginning of last instar. They were then taken from inside the grains and transferred on the pellets until the end of the instar, which was reached after 5-6 days of diet. Larvae were then removed from the pellets, weighed individually, rinsed and dried. Larval free amino acids were extracted and analyzed. Sarcosine catabolism was investigated by injecting this amino acid in the larvae as follows: fully-grown last-instar larvae of both strains reared on wheat were immobilized by placing them on a watch-glass laid on ice. One hundred nmoles of sarcosine in solution in 0.1/zl of Yeager serum, whose osmotic pressure and pH had been adapted to the weevils (Nardon and Grenier, 1983), were injected in the haemolymph of the larvae. Following injection the larvae were maintained in covered beakers without access to food at 27.5°C. After 4 hr incubation periods, the larval free amino acids were extracted.
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Methionine metabofism--I Amino acid and nucleoside analyses Amino acid~. Pools of 2 or 3 larvae were homogenized in cold distilled water. An internal standard (norvaline) was added. The homogenate was deproteinized with 5% tdchloroacetic acid (w/v) and centrifuged at 10,000 g (4°C) for 3 rain. The supernatant was then freed of lipids twice as follows: two volumes of chloroform were added to the last supernatant and the mixture was centrifuged at 10,000g (4°C) for 3 min. The final supernatant containing the free amino acid fraction was evaporated to dryness. Free amino acids were rehydrated in 0.06 M citrate buffer, pH 2.20, and chromatographed with a Liquimat III Kontron automatic analyzer. Nucleosides. S-adenosyl-L-methionine (SAM) and Sadenosyl-L-homocysteine (SAH) were the only nucleosides studied. Each assay was performed with a pool of three last-instar larvae. The larvae were weighed individually and each pool was homogenized in 200 #1 cold perchloric acid (0.4 N). The mixture was centrifuged at 10,000g (4°C) for 2 min. The supernatant was collected and stored at -20°C before use. The separation of nucleosides was performed by reverse-phase HPLC and paired-ion chromatography according to Gharib et al. (1982). The eluate was monitored by an absorbance detector at 254 nm. The compounds were identified by their retention times and quantified by peak area measurement.
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The presence of sarcosine in the symbiotic strain appeared quite variable. But its concentration always remained very low compared with the aposymbiotic strain (its highest value did not exceed 3.82 nmoles/insect). If no striking difference was noticed between the aposymbiotic and the symbiotic strains concerning methionine variations (Fig. 3), the concentration of this amino acid appeared to be influenced by the sex of the insect in both strains: it remained low in the larvae and pupae (often less than 2 nmoles/insect in both strains) and increased in 10-day old adults, reaching higher values in females (from 7 to 8 nmoles/insect in both strains) than in males (from 1.92 nmoles/insect in RR strain to 4.56 nmoles/insect in SS strain).
RESULTS
Methyl-donor availability As shown in Table 1, the endogenous levels of SAH did not differ in the last-instar larvae of both strains. In contrast, the level of SAM was found significantly higher (at a 0.01% probability level) in the aposymbiotic larvae than in the symbiotic larvae (+210%). Consequently, the resultant index of methylation (SAM/SAH ratio) appeared to be significantly higher (at a 0.01 probability level) in the aposymbiotic strain than in the symbiotic one (+250%).
Sarcosine and methionine variations during development Sarcosine was found at all stages of development in aposymbiotic weevils (Fig. 2) from the 2nd larval instar until the emergence of adults. First instar larvae were not investigated (they were too small to be located in the grains). The values corresponding to second and third instar larvae were not reported since their levels of sarcosine were too low to be precisely quantified. The concentration of sarcosine increased regularly during last larval instar and reached its highest value at the end of this instar: 43.49 nmoles/insect. Then it decreased in pupae and completely disappeared in 10-day old adults.
Effect of amino acid supplementation Supplementation of the diet with methionine resulted in a graded increase in the level of sarcosine in the aposymbiotic larvae (Table 2), which was proportional to the methionine content of the diet ( + 150, +340, +470% with 1, 1.5, 1.9% supplementation, respectively). The amount of sarcosine in the symbiotic larvae increased too, but the concentrations remained three times lower at least than in the aposymhiotic larvae. As shown in Table 2, the addition of glycine to the diet did not affect the levels of sarcosine in the larvae of both strains. But the addition of glycine and methionine (1%: 1%, w/w, respectively) together in
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Table I. S-AdenosyI-L-methionine (SAM) and S-adenosylL-homocysteine (SAH) levels in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Nucleoside RR SS SAM 22.50 5:7.49 (9)* 69.70 + 18.09(6) SAH 1.69 +-0.73 (9) 1.44 + 0.56 (6) SAM/SAH 15.20 +- 6.51 (9)* 53.89 + 20.61 (6) The concentrations are expressed as pmoles/mg of fresh weight. Values represent means _+SD. Number in parentheses indicate the number of determinations. *Values significantlydifferent between RR and SS strains at the 1% level (Student's t-test).
Table 2. Effect of dietary methionine (Met), glycine (Gly) and ethionine (Et) supplementation on the levels of sarcosine in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Supplementation RR SS Controls 0.32 5:0.30 (3) 2.15 + 0.58 (4) 1.0% Met 1.5% Met 1.9% Met
1.62 __.0.36 (6) 1.79 __.1.07(3) 4.70 + 2.38 (5)
5.37 +- 1.25(4) 9.54 + 2.00 (4) 12.24 + 2.60 (7)
1% Gly
0.32 +- 0.04 (2)
1.95 + 0.12 (2)
1% Gly + 1% Met
2.65+-0.47(2)
12.03+-1.21(3)
1% Et 0 (2) 1.58 + 0.05 (2) The concentrations are expressed as nmoles/mg of fresh weight. Values represent means _+SD with the number of determinations in parentheses.
Table 3. Effect of the injection of sarcosine (Sarc) on the levels of free glycine (Gly) and free serine (Ser) in symbiotic (RR) and aposymbiotic (SS) larvae of S. oryzae Treatment
Amino acid
Controls
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+ Sarc
Ser Gly
RR
4.28 + 0.95 (5) 3.02+_0.86(5)
SS
4.19 + 1.68(6) 3.17+ 1.42(5)
3.96 + 0.39 (2) 3.47 _.+2.06 (2) 2.64 + 0.22 (2) 2.49 + 0.63 (2) The concentrations are expressed as nmoles/mg of fresh weight. Values represent means + SD with the number of determinations in parentheses. For more details, see Materials and Methods.
the diet resulted in a very high increase in the a m o u n t s o f sarcosine in the aposymbiotic a n d in the symbiotic larvae which was more i m p o r t a n t t h a n the increase o b t a i n e d with m e t h i o n i n e only ( + 6 3 % for R R strain a n d + 124% for SS strain c o m p a r e d with the results o b t a i n e d with 1% m e t h i o n i n e supplementation). O n the contrary, the ingestion by the larvae o f a diet s u p p l e m e n t e d with 1% ethionine (Table 2) resulted in b o t h strains in a decrease in their levels o f sarcosine ( - 2 6 % for SS larvae, sarcosine was even a b s e n t in the symbiotic larvae). Injection o f sarcosine did n o t affect the a m i n o acid c o m p o s i t i o n o f b o t h strains. N o increase in the a m o u n t s o f glycine a n d serine were n o t e d (Table 3). T h e 100 nmoles injected were fully recovered. DISCUSSION T h e differences observed previously in the sarcosine levels o f fully-grown last-instar symbiotic a n d a p o s y m b i o t i c larvae o f S. oryzae ( G a s n i e r et al., 1984) were also f o u n d d u r i n g larval a n d p u p a l develo p m e n t . In a n o t h e r work o n Sitophilus (Wicker et al., 1985), no sarcosine was detected in the f o u r t h instar larvae. This was due to the m e t h o d used, analysis being m a d e with one larva only. In these conditions, the levels o f some a m i n o acids were at the limit of detection o f the analyzer. T h e increase in the level o f sarcosine o f actively feeding a p o s y m b i o t i c larvae a n d the decrease observed d u r i n g p u p a l instar w h e n they stopped feeding lead to the conclusion t h a t sarcosine synthesis should d e p e n d on dietary precursors. T h e g r o w t h a n d m o u l t i n g of insects are u n d e r c o n t r o l o f three m a i n groups o f h o r m o n e s : b r a i n h o r m o n e s , ecdysteroids, a n d juvenile h o r m o n e s . The last-mentioned, in which activities differ in adults a n d larvae (Sehneiderman, 1972) have been s h o w n to regulate certain S A M - d e p e n d e n t methyltransferasic activities (Rojas a n d Barbier, 1982). T h e fact t h a t m e t h i o n i n e c o n c e n t r a t i o n r e m a i n e d low in the aposymbiotic larvae a n d p u p a e a n d increased in 10-day old adults a n d t h a t sarcosine showed simultaneously
Methionine metabolism--I opposite variations suggests that these two amino acids might be involved in the same metabolic pathway and that this pathway could be influenced by juvenile hormones. The principal role of SAM, also called "active methionine" (Cantoni, 1951), is to give its methyl group in transmethylation reactions. Transmethylations in insects have been little investigated and no data about the levels of SAM and SAH in insects are available. No comparison between Sitophilus weevils and other insects is therefore possible; but it can be noticed that the concentration of SAM in the SS larvae does not differ from the concentration of SAM found in mammalian liver, one of the richest organs in methylases (Baldessarini and Kopin, 1966; Hoffman et al., 1981). It is generally admitted that the index of methylation is an indication of the methylation degree of the molecules within a tissue. The higher it is, the more methylations occur, since the K m values for SAM and the /~ for SAH of most methylases do not much differ. The index of methylation, higher in the SS strain than in the RR strain, therefore indicates that the tissues of aposymbiotic larvae are more suitable for transmethylations than the tissues of symbiotic larvae and confirms our previous hypothesis that methionine metabolism differed in the presence and in the absence of the symbiotic bacteria (GasnierFauchet, 1985). Ethionine is the ethyl analogue of methionine. Its effect as inhibitor of SAM-dependent cellular methylation reactions is well-known in mammals. There is strong evidence that it exerts its effects by lowering the levels of SAM by inhibiting SAM-synthetase and competing with SAM as a substrate in transmethylation reactions (Hoffman, 1984). The fact that the levels of sarcosine decreased in the larvae in the presence of ethionine suggests that sarcosine biosynthesis may result from the activity of a methyltransferase. Moreover the nutritional experiments with methionine and glycine demonstrate the involvement of these two amino acids in sarcosine biosynthesis. All these results give evidence for a glycine Nmethyltransferase-like activity in the aposymbiotic larvae of S. oryzae. The symbiotic larvae were shown to have the enzymes required for sarcosine synthesis but they appeared to be stimulated by high dietary methionine levels only. Free methionine content of larval tissues seems to be the limiting factor for this synthesis in both strains. In mammals, sarcosine is oxidized to glycine (see Fig. 1) by a mitochondrial sarcosine dehydrogenase (Frisell and Mackenzie, 1962), but our results suggest that this enzyme should be absent in S. oryzae larvae. Moreover, data obtained with fecal material indicate that sarcosine was not excreted by the larvae and that its very low level in the symbiotic larvae was not the consequence of an increased excretion in this strain (Gasnier-Fauchet, 1985). It appears therefore that the decrease in the sarcosine levels observed in the aposymbiotic pupae was probably the consequence of its elimination with the exuvial fluid during pupal eedysis. The facts that sarcosine and methionine had opposite variations during S. oryzae development and
249
that sarcosine biosynthesis was proportional to methionine content of the diet and also that sarcosine accumulated in larval tissues without being used suggest that sarcosine could have a role in the elimination of an excess of methionine coming from a diet too rich in this amino acid to allow a good growth of the larvae. The wheat which was used in our experiments contained 0.45% methionine, which was higher than the required concentration for an optimal growth, ranged from 0.10 to 0.30% in the absence of L-cystine, these values being lower in the presence of L-cystine (Baker, 1978). It appears therefore that the presence of sarcosine in aposymbiotic S. oryzae larvae could be an adaptative response to diets high in methionine, as it is the case in mammals (Mitchell and Benevenga, 1976) and that symbiotic larvae probably get rid of this amino acid by another metabolic pathway. In conclusion, the presence of sarcosine in a p t symbiotic larvae of S. oryzae seems to result from a glycine N-methyltransferase-like activity, though such an enzyme is at the present time unknown in insects. This work provides a starting point for further /n vitro enzymatic studies aiming at purification and characterization of this transferase.
Acknowledgements--The authors
would like to t h a n k J.
Guilland for excellent assistance throughout this study. This work was supported by grants from CNRS and INSERM. REFERENCES
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