Production of essential amino acids from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum

Pergamon

J. Insect Physiol. Vol. 41, No. I, pp. 4146, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

0022-1910(94)00080-8

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Production of Essential Amino Acids from Glutamate by Mycetocyte Symbionts of the Pea Aphid, Acyrthosiphon pisum TETSUHIKO Received

10 May

SASAKI,*

HAJIME

ISHIKAWA*

1994; revised 24 June 1994

Glutamine was the most abundant amino acid constituent in the hemolymph of pea aphids, Acyrtlrosiphon pisum. While mycetocytes isolated from the aphids took up glutamine actively, the intracellular symbionts isolated from the mycetocytes scarcely took up the amino acid, and instead took up glutamic acid actively. [U-‘4C]Glutamine incorporated by mycetocytes was converted into glutamate. When [E-15N]glutamine was introduced into mycetocytes, [“Nlglutamate was found in the cytosol. These results suggested that glutamine taken up by mycetocytes was hydrolyzed into glutamic acid and ammonia, and at least a portion of ammonia was assimilated into glutamate in the cytosol, probably through reaction with a-ketoglutarate. When isolated symbionts were incubated with [r5N]glutamic acid, the following amino acids were found highly labeled: alanine, aspartic acid, glutamine, isoleucine, leucine, phenylalanine, proline and valine. Pea aphid

Mycetocyte

Endosymbiont

Glutamine

Glutamic acid

INTRODUCTION

munication). In this work, in an attempt to clarify the nitrogen flow in the mycetocyte symbiosis of aphids, we investigated uptake and metabolism of glutamine by isolated mycetocytes and amino acid synthesis by symbionts.

Almost all aphids have an association with obligatory bacterial endosymbionts (Buchner, 1965). These symbionts are harbored by the mycetocyte, a specialized cell in the body cavity of the aphid. When the symbionts are removed by experimental methods such as treatment with antibiotics, growth and reproduction of the aphid are depressed (Mittler, 1971; Ishikawa and Yamaji, 1985). It has often been suggested that symbionts provide the host with several essential amino acids (e.g. Dadd and Krieger, 1968; Srivastava et al., 1985; Sasaki et al., 1991; Prosser et al., 1992). This biosynthetic capability of symbionts is thought to be of great importance for aphids since nitrogen in the phloem sap on which aphids feeds comprises almost exclusively amino acids with grossly unbalanced composition (Barlow and Randolph, 1978). In the meanwhile, it was found that deprivation of symbionts results in striking elevation of glutamine in tissues and honeydew of the aphid, suggesting that glutamine is utilized by the symbionts as a nitrogen source for synthesis of other amino acids (Sasaki et al., 1990; Prosser and Douglas, 1991). In contradiction with this suggestion, the symbionts isolated from aphids did not take up glutamine (A. E. Douglas, personal com-

*Zoological Institute, Faculty Tokyo 113, Japan.

of Science, University

of Tokyo,

MATERIALS AND METHODS Insect A long-established parthenogenetic clone of pea aphids, Acyrthosiphon pisum (Harris) was maintained on young broad bean plants, Vicia faba (L.) at 15°C under a 16 h light : 8 h dark photoperiodic regime. Collection

of hemolymph

An aphid was anesthetized in a stream of carbon dioxide, and the fore legs were pulled out. Pressing the abdomen gently, hemolymph that oozed out was collected with a capillary pipette. Isolation

of mycetocytes

and symbionts

Approximately 20 non-bearing young adult aphids were dissected in ice-cold buffer A [25 mM KCl, 10 mM MgCl,, 250 mM sucrose and 35 mM Tris-HCl, pH 7.5 (Ishikawa, 1982)] in a plastic dish bedded with 1% agarose gel. The mycetocytes scattered on the agarose bed were collected with a glass pipette connected into a peristaltic pump. The protein content of the collected cells, determined using the BCA protein assay reagent

Hongo,

41

42

TETSUHIKO

SASAKI and HAJIME

(Pierce Co.) with bovine serum albumin as a standard, varied between 20 and 30 pg. To collect symbionts from isolated mycetocytes, the mycetocytes were broken by pipetting in buffer A. The symbionts were collected by centrifugation at 3000 g for 1 min and washed once with buffer A. The protein content of the isolated symbionts, thus prepared from approx. 20 aphids, was between 10 and 20 ,ug. Isolated symbionts were also obtained from the homogenate of aphid whole tissues. Insect material was sterilized with 70% ethanol for 30 s and lightly crushed in 50 vol of buffer A containing 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride using a looselyfitting homogenizer. The homogenate was passed through nylon mesh with pore size of 90 pm, centrifuged at 1500 g for 5 min and the pellet was suspended in buffer A. The suspension was filtered through nylon mesh with pore size of 20 pm, nylon mesh with pore size of 10 pm and Isopore-membrane (pore size 5 pm, Millipore Co.) successively. The filtrate was centrifuged at 1500 g for 5 min and the pellet was washed once with buffer A. The final symbiont preparation was free from the contamination by other cell components except a small portion of nuclei when examined under a light microscope. Uptake of radiochemicals symbion ts

by isolated

mycetocytes

and

A small piece of Parafilm membrane was set on a bed of 1% agarose gel in a plastic dish. On this Parafilm membrane, isolated mycetocytes were incubated in 50 p 1 of buffer A containing either [U-‘4C]glutamine or [U-‘4C]glutamic acid at a concentration of 185 kBq/ml. The reaction was stopped by adding 3 ml of ice-cold buffer A, and the mycetocytes, washed once in the same buffer, were dissolved in 50 ~1 of 5% SDS and assayed for radioactivity in a liquid scintillation system. The uptake experiment of isolated symbionts was performed according to the methods described by Whitehead and Douglas (1993) with minor modifications. Symbionts were collected from isolated mycetocytes and suspended in 50 ~1 of incubation medium comprising 0.5 mM MgSO,, 250mM sucrose and 50mM MOPSNaOH, pH 7.0. The reaction was started by adding either [U-‘4C]glutamine or [U-‘4C]glutamic acid to give a final concentration of 0.019 mM and 185 kBq/ml. After incubation at room temperature for 60 s, the suspension was transferred onto GF/F filter (Whatman) on a vacuum filter manifold. The filter was washed with 2 ml of the incubation medium and assayed for radioactivity. Zero-time values were obtained and subtracted from the values.

ISHIKAWA

mycetocytes were extracted with 80% ethanol, separated through reverse phase HPLC, and the eluted fractions were assayed for radioactivity. To trace the amide nitrogen of glutamine, mycetocytes were incubated in 50 ~1 of buffer A containing 5 mM [c-“Nlglutamine (95% “N atom%) in a 0.5 ml Eppendorf tube. After incubation at room temperature for 10 min, 150 ~1 of buffer A was added and the cells were broken by pipetting. The symbionts and the cytosol of the mycetocytes were separated by centrifugation. Free amino acids of the symbionts were extracted with 80% ethanol. Samples of amino acids thus prepared were applied to an anionic exchange column of Dowex 1, and acidic amino acids retained in the column were eluted with 2 M acetic acid. Aspartic acid and glutamic acid were separated through reverse phase HPLC and the fraction of glutamic acid was assayed for “N. Amino

acid synthesis

Symbionts were prepared from the homogenate of approx. 10 g of insect tissues, and suspended (5 mg protein/ml) in the incubation medium containing 10 mM [‘Nlglutamic acid (95% 15N atom%). The suspension was incubated at room temperature for 30 min with gentle shaking and then centrifuged at 3000g for 1 min. The supernatant which contained [‘5N]glutamic acid and the amino acids released from the symbionts during the incubation period was applied to an anionic exchange column of Dowex 1 and a cationic exchange column of Dowex 50 W successively. Acidic amino acids retained in the first column and the other amino acids retained in the second column were eluted with 2 M acetic acid and 3 M ammonia, respectively. The “N content of each amino acid was determined after separation through a reverse phase HPLC. Reverse phase HPLC

of [U-‘4C]glutamine

and [t -'*N]glutamine

in

Mycetocytes were incubated at room temperature for 10 min in buffer A containing [U-‘4C]glutamine at a concentration of 1 mM and 185 kBq/ml, and collected into a tube as described above. Free amino acids in the

of amino acids

Amino acids were derivatized with phenyl isothiocyanate as described previously (Sasaki et al., 1991), and analyzed by reverse phase HPLC, using a solvent system as follows: Solvent A, 0.05% triethylamine in 0.14 M sodium acetate (pH 6.35)-acetonitrile (47: 3, v/v); Solvent B, 60% acetonitrile. The column elutes were monitored at 254 nm. The reference amino acid mixture was Type-H of Wako Co. supplemented with asparagine, glutamine and tryptophan. For assay for 15N in the eluted fraction, the fraction was subjected to re-chromatography to remove sodium acetate, using 10 and 60% acetonitrile containing 0.01% trifluoroacetic acid. Determination

Metabolism mycetocytes

of isolated symbionts

of 15N

The 15N content was measured by the emission spectrometric analysis (Kumazawa, 1986). A sample of amino acid (50-200 nmol) was sealed into a Pyrex glass tube with CuO and CaO under low pressure (below 10e4 torr). The tube was then heated at 560°C for 3 h. During this period, the sample was decomposed taking

AMINO

ACID

PRODUCTION

1.

2 c-0

E = 0.01 3, Asp Asn Gin His Thr Pro Val Ile Phe Lys Glu Ser Gly Arg Ala Tyr Met Leu Trp 1. Amino acids expressed

in hemolymph of A. pisum. as means & SD (n = 3).

Values

FIGURE are

the oxygen atom from CuO and the resulting water and carbon dioxide were absorbed into CaO. After analysis in an emission spectrometer, JASCO-NIA-1, “N abundance was calculated from measurement of intensity ratio of the bandheads of 14N14N, 14N15N and l’N”N molecules in the nitrogen emission spectra.

RESULTS

Amino

I , ,,,,,,’

, ,“_

1 0.1 10 Concentration of glutamine (mM)

Allllrln

FIGURE

43

IN A. PZSUM

acid analysis of the hemolymph

The

hemolymph collected from non-bearing adult aphids contained amino acids at young 60.6 + 9.5 nmol/pl (mean + SD, n = 3). Glutamine, the amino acid focused in the present study, was the most abundant constituent, the second being asparagine (Fig. 1). These two amide amino acids amounted to 56.1 mol% of the total amino acids. The contents of acidic amino acids, aspartic acid and glutamic acid, were very small.

3. Effect of concentration on the initial uptake glutamine. Vertical lines represent SD (n = 3).

Uptake and metabolism

of

of radiochemicals

Isolated mycetocytes actively took up [‘4C]glutamine (Fig. 2). The uptake rate was constant for the first 5 min and decreased with time thereafter. Little uptake of [‘4C]glutamic acid was observed over 10 min. To examine uptake kinetics (Fig. 3), uptake plots of 3 min were used. The uptake was linear at external concentrations between 0.1 M and 2 mM and saturated at higher external concentrations. Uptake of glutamine and glutamic acid by isolated symbionts is shown in Fig. 4. Unlike mycetocytes, symbionts took up glutamic acid much more actively than glutamine, which may suggest that glutamine taken up by mycetocytes is hydrolyzed, and resulting glutamic acid is taken up by the symbionts. To examine conversion of glutamine into glutamic acid in mycetocytes, we analyzed 80% ethanol soluble fraction prepared from the mycetocytes which took up [‘4C]glutamine. The chromatogram of HPLC analysis showed that in the mycetocytes glutamic acid was much more abundant than glutamine, and the radioactivity in the fractions of glutamic acid and glutamine was 1692 and 328 cpm, respectively (Fig. 5).

Time (min) FIGURE 2. Uptake of radiochemicals by isolated mycetocytes. Mycetocytes were incubated in a medium containing either [U-‘4C]glutamine (0) or [U-i4C]glutamic acid (0) at 0.019 mM and 185 kBq/ml. For each experiment, mycetocytes were collected from approx. 20 aphids. Vertical lines represent SD (n = 3).

rates

0

tl5 Gln

FIGURE

Glu

4. Uptake of radiochemicals by isolated symbionts. are expressed as means f SD (n = 5).

Values

TETSUHIKO

SASAKI

and HAJIME

ISHIKAWA

TABLE 1 Asp 2 GI; 3 Asn 4 Ser

5 Gln 6 Gly 7 His

1600~

800-

Retention time (min) FIGURE 5. Metabolism of [U-Ylglutamine in mycetocytes. Mycetocytes were incubated in a buffer containing [‘Qlutamine at I mM and 185 kBq/ml for 10 min, and free amino acids were extracted with 80% ethanol. A sample prepared from approx. 50 aphids was applied to HPLC.

Metabolism

of [E-“N]glutamine

in mycetocytes

Experiments with radiochemicals suggested that glutamine is hydrolyzed into glutamic acid and ammonia in the cytosol of mycetocytes. One of the possible metabolic fates of ammonia thus produced is a formation of glutamic acid with a-ketoglutaric acid. This reaction will occur in the cytosol of the mycetocyte and/or in the symbionts, which was investigated using glutamine whose amide nitrogen was labeled with “N. After [c-“Nlglutamine was introduced into mycetocytes, glutamic acid was purified from the cytosol and the symbionts. As shown in Fig. 6, 15N content of glutamic acid was approx. four times higher in the cytosol than in the symbionts, suggesting that glutamic acid formation occurs mainly in the former.

0

Cytosol Symbiont FIGURE 6. Formation of [“Nlglutamic acid in the mycetocyte. Mycetocytes were incubated in a buffer containing [t-‘5N]glutamine at 5 mM for 10 min. Glutamic acid purified from the cytosol and the symbionts was assayed for “N Values are based on mycetocytes collected from approx. 1500 aphids.

1. Amino

acids released

(nmol/mg

Amount symbiont

from isolated

protein)

symbionts

15N concentration (atom% excess)

Non -essential amino acids Alanine Asparagine Aspartic acid Glutamine Glycine Proline Serine Tyrosine

8.15 4.83 1.49 2.34 1.72 1.24 1.58 3.04

20.8 0.1 5.6 7.1 0.3 12.5 0.4 0.0

Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Tryptophan Valine

1.26 1.02 1.10 1.59 3.68 1.44 1.48 1.02 1.92

0.0 0.1 15.6 20.9 0.0 7.4 0.0 0.0 10.2

Amino

acid synthesis

by isolated symbionts

To examine the utilization of glutamic acids by symbionts, isolated symbionts were incubated in a medium containing 10 mM [“Nlglutamic acid for 30 min. In the medium after incubation, were found various amino acids among which the following amino acids were highly labeled with “N: alanine, aspartic acid, glutamine, proline, isoleucine, leucine, phenylalanine and valine (Table 1).

DISCUSSION

In the present study, it was demonstrated that: (1) glutamine was the most abundant amino acid constituent in the hemolymph of the aphid (Fig. 1); (2) glutamine was taken up by the mycetocytes (Figs 2 and 3) and converted into glutamic acid (Figs 5 and 6); and (3) the symbionts took up glutamic acid (Fig. 4) and utilized it as a nitrogen source for synthesis of other amino acids (Table 1). Glutamine is one of the major amino acids in the phloem sap of the bean plant on which the aphids feed (Sasaki and Ishikawa, 1990). In addition, glutamine is actively synthesized by the aphids (Sasaki and Ishikawa, 1993). Even when the aphids were kept on a synthetic diet (Febvay et al., 1990) from which glutamine was omitted, the most abundant amino acid in the hemolymph was still glutamine (data not shown), implying that glutamine functions as an important nitrogen carrier in the aphid. Investigations on the metabolism of [U-‘4C]-(Fig. 5) and [c-‘ WJglutamine (Fig. 6) suggested that glutamine taken up by mycetocytes is hydrolyzed into glutamic acid and ammonia, and at least a portion of ammonia, thus produced, is assimilated into glutamic acid in the cytosol. It is also possible that glutamic acid is formed

AMINO

ACID

PRODUCTION

by glutamate synthetase which catalyzes the formation of two molecules of glutamic acid from glutamine and a-ketoglutaric acid. Since this enzyme, however, has not been found in animal tissues ever examined, it is more likely that conversion of glutamine into glutamic acid is catalyzed by glutaminase and glutamate dehydrogenase. Capability of amino acid synthesis of symbionts was examined by incubating isolated symbionts with [?Jlglutamic acid (Table 1). In the analyses for “N, we used amino acids found in the medium after incubation rather than free amino acids in symbionts because the HPLC analysis of the free amino acids extracted from the symbionts with 80% ethanol suggested the presence of many unidentified small peaks and it was not feasible to quantify and fractionate these amino acids. According to “N assay, apparent incorporation of 15N was observed in several amino acids, four of which, namely, isoleucine, leucine, phenylalanine and valine were essential amino acids (Table 1). While the symbionts have been often suggested to synthesize essential amino acids for the host, direct evidence has been provided only for methionine (Douglas, 1990) and tryptophan (Douglas and Prosser, 1992; Munson and Baumann, 1993; Lai et al., 1994). The present study indicated that the symbionts synthesize the four essential amino acids using glutamic acid as a nitrogen donor. As for amino acid transfer from symbionts to the host, it was suggested that amino acids are released from intact symbionts rather than by lysis of the symbionts. The amount of amino acids found in the medium increased during the incubation while that of free amino acids in the symbionts were constant for the periods (data not shown). The symbionts of aphids have been assigned to a new genus, Buchnera, in the y-Proteobacteria (Munson et al., 1991). Analysis of the gene for the 16s RNA indicated that the symbiont is closely related to Escherichia coli (Unterman et al., 1989). In E. coli, glutamic acid provides nitrogen for the synthesis of most amino acids whereas glutamine donates its amide nitrogen for the synthesis of purines, pyrimidines, histidine, tryptophan and asparagine (Tyler, 1978). The present study suggested that nitrogen taken up as glutamine by mycetocytes enters the symbionts in the form of glutamic acid, which may function to take full advantage of the metabolic capabilities of the symbionts for amino acid synthesis. The symbionts are surrounded by the symbiosome membrane, a derivative of the host membrane. In other symbioses, it is suggested that the symbiosome membrane controls the transport of nutrients into the symbionts (Udvardi et al., 1988, on leguminous plants-rhizobia symbiosis; Rands et al., 1993, on sea anemone-alga symbiosis). It is possible that aphids control the metabolism of their symbionts by selective transport of nitrogen sources through the symbiosome membrane. Recently, Whitehead and Douglas (1993) conducted a detailed study on uptake of carbon sources by the symbionts of A. pisum. The symbionts took up several

45

IN A. PZSUM

carboxylic acids and amino acids glutamic acid and aspartic acid. Initial uptake rate of glutamic acid was proportional to its external concentrations between 0.015 and lOmM, and 90% of incorporated glutamic acid was metabolized to carbon dioxide over 60min incubation. It is likely that for the symbiont glutamic acid is an important compound as both a nitrogen source and a respiratory substrate. In the hemolymph of aphids, asparagine was the second most abundant amino acid constituent (Fig. 1). Our previous studies suggested that asparagine, as well as glutamine, functions as a nitrogen carrier in aphids (Sasaki and Ishikawa, 1993; Sasaki et al., 1993). In spite of the low level of aspartic acid in the hemolymph, the ratio of aspartic acid to asparagine was high in the mycetocytes (Fig. 5), and the symbionts took up aspartic acid actively (Whitehead and Douglas, 1993). These results may suggest that asparagine is utilized by the mycetocyte in the same manner as glutamine. As for nitrogen flux from aphid to symbionts, another compound of interest is ammonia. Whitehead et al. (1992) reported that isolated symbionts accumulate exogenous methylamine, an analogue of ammonia, through carriermediated transport system. A portion of ammonia produced from glutamine, and possibly from asparagine, may be taken up by the symbionts directly without being assimilated into glutamic acid through reaction with a-ketoglutarate.

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46

TETSUHIKO

SASAKI

Munson M. A. and Baumann P. (1993) Molecular cloning and nucleotide sequence of a putative trpDC(F)BA operon in Euchnera aphidicola (endosymbiont of the aphid Schizaphis graminum). J. Bacterial. 175, 642666432. Munson M. A., Baumann P. and Kinsey M. G. (1991) Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. Int. J. Syst. Bacterial. 41, 566-568. Prosser W. A. and Douglas A. E. (1991) The aposymbiotic aphid: an analysis of chlortetracycline-treated pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 37, 713-119. Prosser W. A., Simpson S. J. and Douglas A. E. (1992) How an aphid (Acyrthosiphon pisum) symbiosis responds to variation in dietary nitrogen. J. Insect Physiol. 38, 301-307. Rands M. L., Loughman B. C. and Douglas A. E. (1993) The symbiotic interface in an alga-invertebrate symbiosis. Proc. R. Sot. Lond. B253, 161-165. Sasaki T. and Ishikawa H. (1993) Nitrogen recycling in the endosymbiotic system of the pea aphid, Acyrthosiphon pisum. Zool. Sci. 10, 7799785. Sasaki T., Aoki T., Hayashi H. and Ishikawa H. (1990) Amino acid composition of the honeydew of symbiotic and aposymbiotic pea aphids Acyrthosiphon pisum. J. Insect Physiol. 36, 35-40. Sasaki T., Hayashi H. and Ishikawa H. (1991) Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets. J. Insect Physiol. 37, 749-756.

and HAJIME

ISHIKAWA

Sasaki T., Fukuchi N. and Ishikawa H. (1993) Amino acid flow through aphid and its symbiont: Study with 15N-labeled glutamine. Zool. Sci. 10, 787-791. Srivastava P. N., Gao Y., Levesque J. and Auclair J. L. (1985) Differences in amino acid requirements between two biotypes of the pea aphid, Acyrthosiphon pisum. Can. J. Zool. 63, 603-606. Tyler B. (1978) Regulation of the assimilation of nitrogen compounds. A. Rev. Biochem. 47, 1127-l 162. Udvardi M. K., Price G. D., Gresshoff P. M. and Day D. A. (1988) A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEMS Lett. 231, 36-40. Unterman B. M., Baumann P. and Mclean D. L. (1989) Pea aphid symbiont relationships established by analysis of 16s rRNAs. J. Bacterial. 171, 2970-2974. Whitehead L. F. and Douglas A. E. (1993) A metabolic study of Buchnera, the intracellular bacterial symbionts of the pea aphid Acyrthosiphon pisum. J. Gen. Microbial. 139, 821-826. Whitehead L. F., Wilkinson T. L. and Douglas A. E. (1992) Nitrogen recycling in the pea aphid (Acyrthosiphon pisum) symbiosis. Proc. R. Sot. Lond. B250, 1155117. Acknowledgements-We thank Dr Y. Arima (Faculty of Agriculture, Tokyo University of Agriculture Technology) for his help with emission spectrometric analysis of 15N. This research was supported by Grants-in-Aid for General Research (No. 03454020) and for scientific Priority Areas, “Symbiotic Biosphere: Ecological Interaction Network Promoting Coexistence of Many Species” (No. 04264103) from the Ministry of Education, Science and Culture of Japan.