Lipid metabolism in the symbiotes of the pea aphid, Acyrthosiphon pisum

Comp. Biocllem. Physiol., 1976. Vol. 5411, pp. 427 to 431. Pergamon Press. Printed in Great BritaiJl

LIPID METABOLISM IN THE SYMBIOTES OF THE PEA APHID, A C Y R T H O S I P H O N PISUM* EDWARD J. HOUKt, GARETHW. GRIFFITHS~AND STANLEYD. BECK Department of Entomology, University of Wisconsin, Madison, WI 53706, U.S.A. (Received 6 June 1975) Abstract--1. The synthesis of lipids in the primary symbiote of the pea aphid was examined. The

amount of radioactive acetate incorporated into the symbiote lipid fraction in vitro was found to increase as the concentration of symbiotes in culture decreased. 2. The symbiotes synthesized free fatty acids, mono- and diglycerides, phosphatidyl choline, phosphatidyl ethanolamine, and cholesterol. 3. The level of cholesterol in the symbiote fraction appeared to be more than an order of magnitude higher than sterol levels previously reported for any bacteria-like microorganism.

INTRODUCTION Sterols play essential structural and physiological roles in insect development (Thompson et al., 1973). The latter authors point out that no insect has been shown to possess the biochemical machinery for synthesizing its own cholesterol and that insects, in general, obtain cholesterol (or other related sterols) from their diet. The few insects known that can develop without an exogenous sterol source also contain symbiotic microorganisms that are believed to synthesize sterols. The pea aphid, Acyrthosiphon pisum Harris possesses two kinds of intracellular symbiotes (Griffiths & Beck, 1973). This insect can be reared indefinitely on chemically-defined diets completely lacking sterol (Akey & Beck, 1971). Similar results have been seen with three other aphid species (Dadd & Krieger, 1967; Erhardt, 1968a). This is strong indirect evidence suggesting a role for the symbiotes in supplying sterol to the aphids. This hypothesis is supported by the fact that the aphid Neomyzus circumflexus could incorporate [~4C]acetate into sterols in vivo but lacked this capacity when the symbiotes were removed by the administration of chlortetracycline (Erhardt, 1968b). This result does not, however, preclude the possibility that the sterol is synthesized by the gut flora or that the antibiotic is directly inhibiting an aphid sterol synthetic pathway. The primary intracellular symbiotes of the pea aphid, Acyrthosiphon pisum, have been isolated en masse from whole aphid homogenates (Houk & McLean, 1974). Houk (1974a) examined the lipid chemistry of the isolated symbiotes and found significant amounts of cholesterol and other lipids that are not commonly seen in Eubacteriales. This author con* Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a grant (GB-31840X2) from the National Science Foundation. t Present address: University of California, Naval Biomedical Research Laboratory, Oakland CA 94625, U.S.A. :~Present address: Max Planck Institut fur Biologische Kybernetik, Tubingen, W. Germany.

cluded that the ancestral affinities of the symbiote was still an open question; a number of authors had previously concluded, primarily on morphological grounds, that the symbiotes were Rickettsia (Lamb & Hinde, 1967), L- or secondarily similar forms of bacteria (Vago & Laporte, 1965; Hinde, 1971), or Mycoplasma (McLean & Houk, 1973). In an attempt to reconcile the large sterol component with the possibility that the symbiotes were Mycoplasma, Houk (1974a) argued that the symbiotes may be sequestering sterol from the aphid host rather than synthesizing it de novo. This hypothesis was based on the fact that, aside from the large cholesterol fraction, the lipid chemistry of the symbiotes showed similarities to the lipid chemistry of the Mycoplasmatales and the latter are known to sequester sterols from their surrounding media (Smith, 1971). This paper presents the results of an investigation into the lipid biosynthetic capacity of the pea aphid primary symbiotes. The purpose of this work was to show whether the symbiotes could synthesize cholesterol and other lipids in vitro.

MATERIALS AND METHODS Symbiote isolation

Pea aphids were cultured on Vicia fava (Broadbeans) for approx l0 days; the aphids were then shaken from the plants and collected. The aphids were rinsed for about 1 rain in 70% ethanol and twice with sterile water. They were then macerated and the primary symbiotes isolated under aseptic conditions by the method of Houk & McLean (1974}. Culture Experimental culture media, utilizing a modification of Grace's Insect Tissue Culture Medium (Houk, 1974b), were prepared in a sterile chamber and antibiotics (penicillin, 500/l/ml; streptomycin, 500 #g/ml; and neomycin, 5/~g/ml) and isotope (5 izC/ml of [14C]acetate, [3H]mevalonate or [aH](Methyl)-methionine; 2.51tC/ml of [14C]serine or [l'~C]choline) added. The media were then filtered through a sterile 0.22/~ millipore® filter, fitted to a syringe, directly onto the symbiote pellet obtained in the above described

427

EDWARD J. HOUK et al.

428

isolation. The symbiotes were resuspended in the culture media by gentle stirring with a sterile glass rod. The symbiotes were maintained in a shaking water bath (160 oscillations/min; 27°C) for 8hr. At the end of the culture period, a small aliquot of the material was examined by light microscopy for contamination. The remainder was centrifuged at 2000 g for 15 min. The supernatant was discarded and the pellet extracted with acidified chloroform-methanol (2:1) as described by Houk (1974a). The total lipid in the extract was determined gravimetrically for specific activity calculations.

Thin-layer chromatography ( TLC) The classes of lipids present in the lipid extract were separated as described by Houk (1974a). The symbiote lipids were chromatographed simultaneously with a series of standards (cholesterol acetate, triolein, cholesterol, myristic acid phosphatidylcholine and phosphatidylethanolamine). The standards were visualized with 70~o sulfuric acid saturated with dichromate; phospholipids by molybdenum blue; or the entire plate was visualized by spraying with deionized, distilled water (Randerath, 1966). The symbiote lipid classes were scraped from the plate and the material, including silica gel, was added directly to a simplified scintillation fluid (5 g PPO/1 toluene). The radioactivity of the fractions was determined in a Packard Tri-Carb scintillation counter, utilizing an external standard for conversion of counts/min into disintegrations per min.

75

50

25

I

,5 [] © I i.o

I 20

r 3.0

Symbiote concentrotion,

I 4.0

rng/mt

I 50

protein

Fig. 2. Changes in radiolabel [~4C]acetate distribution within the various lipid classes examined ( I - t , triglyceride; A - - i , , diglyceride; t~---O, phospholipids; E ] ~ , monoglycerides; A A free fatty acids; O--O, cholesterol) with a change in symbiote concentration.

Gas-liquid chromatography (GLC) GLC of the sterol fraction was necessary in order to identify, specifically, the symbiote synthetic product. The sterol fraction was applied to a 1~o OV-I column (29°C; N2 20 ml/min) and the retention time of the peaks compared to a series of standards (cholesterol, stigmasterol, sitosterol, and dihydrocholesterol). RESULTS When the symbiote fraction was incubated with ['4C]acetate and the amounts of label incorporated into total lipid examined, the specific activity of the lipid fraction was found to increase as the concentration of symbiotes decreased (Fig. 1). This response resembled the classic bacterial growth curve--the lowest symbiote concentration (2mg symbiote protein/ml culture medium) being analogous to an exponential growth phase and the highest concen-

%

4C

3C

2"C

tration (4-5 mg/ml) analogous to a stationary phase. Examination of the distribution of radioactivity in the various lipid classes also revealed a typical bacterial pattern (Fig. 2). At the highest concentration the symbiotes exhibited a stationary phase metabolism in which approx 83~o of the total radioactivity appeared in the storage form; triglyceride. With lower concentrations, the symbiotes showed an increasing tendency toward the synthesis of more polar lipids; i.e. phospholipids (Table 1). The effects of adding exogenous sources of ATP, N A D P H , and a combination of both were tested. The results indicated that as the symbiote concentration was lowered, the effects of N A D P H or ATP and the combination became more marked (Table 2). Thus, at the 2 mg/ml symbiote concentration, the levels of radioactivity in total lipid was increased approx 72~o over the control with A T P ; 120~o with N A D P H ; and about 300~o when both ATP and N A D P H were added. Experiments subsequent to these were routinely carried out using a combination of ATP and N A D P H (at a concn of 1 mg/ml) and a symbiote concentration of 2 mg symbiote protein/ml. The levels of radioactivity in the monoglyceride and free fatty acid fractions remained at levels considered transitory regardless of the symbiote concentration (Fig. 2). The radioactivity levels associated with the diglyceride fractions were, however, signifiTable 1. Concentration dependence of polar:nonpolar lipid classes

u3 I 1.0

Symbiote

I 2"0

I 3"0

concentrotion,

I 4"0

I 5'0

Concentration

(~/ml)

~/~NF

4.5

0.066

3.0

0.159

2.0

0.198

mg/mL protein

Fig. 1. Relationship of [ 14C]acetate incorporation between specific activity and concentration of symbiotes in culture.

Lipid metabolism in the symbiotes of the pea aphid Table 2. Effects of cofactor addition on [14C]acetate incorporation

429

cantly higher and paralleled the levels associated with the phospholipid fraction. This was surprising, since the synthesis of triglycerides and phospholipids from diglyceride precursors via established bacterial pathways does not involve levels of diglyceride above what could be considered low, transitory levels. Houk (1974a), in his comparison of the phospholipid chemistry of isolated symbiotes to that of existing groups of microorganisms, suggested that the symbiote might obtain some of its phospholipid amine bases via a salvage mechanism from the aphid host like the established pathway in animals (Fig. 3). The high levels of diglyceride found here tend to support this hypothesis.

reaction required for the synthesis of phosphatidylethanolamine from a phosphatidylserine precursor. It was thought that this inhibition would allow detection of a previously transitory concentration of phosphatidylserine. This effect was observed as the phosphatidylserine fraction was much more heavily labelled in the hydroxylamine-inhibited system (Table 3). In the presence of hydroxylamine, two additional components appeared in the phospholipid fraction. These two components were chromatographically distinct from the standards (phosphatidylethanolamine, phosphatidylcholine, and lyso-phosphatidylcholine). The unknown components were tentatively identified as mono- and dimethylethanolamine derivatives reported as intermediates in the synthesis of phosphatidylcholine from phosphatidylethanolamine (Kaneshiro & Law, 1964). This evidence appears to negate any sort of salvage pathway in the synthesis of symbiote phospholipid. Attempts to demonstrate the methylation of phosphatidylethanolamine by use of [3H]methionine, to yield labelled phosphatidylcholine were of limited success. Levels of activity were low for all fractions, but a definite increase in the activity of the phosphatidylcholine and lyso-phosphatidylcholine fractions, in excess of phosphatidylethanolamine, was observed (Table 3).

Phospholipid synthesis

Sterol synthesis

The symbiotes synthesized labelled phosphatidyl choline and phosphatidylethanolamine from labelled acetate precursor. The synthetic pathway of the phospholipids could not be determined, however, using [ 14C]acetate. Different labelled compounds were used to determine whether the symbiotes synthesized phospholipids via typical bacterial pathways or via the animal salvage pathway (Fig. 3). Separate cultures containing [14C]serine, [~4C]serine with added hydroxylamine and [14C]choline were examined. The results indicated that the symbiotes synthesized phospholipids by established bacterial pathways (Table 4). [14C]Choline was not incorporated into the phosphatidylcholine fraction, in excess of the activity of the other phospholipid fractions, as would be expected for a salvage pathway. Hydroxylamine was added to a [~4C]serine culture to inhibit the decarboxylation

There were good indications of sterol biosynthesis using [14C]acetate precursor. The maximum activity associated with the sterol band on the TLC plates was 2.6~o of the total activity. Since these levels of sterol were relatively low it was felt that further verification was required. It is now established that sterol biosynthesis in plants and animals proceeds via a mevalonic acid intermediate (Mahler & Cordes, 1971). For this reason, the symbiote fraction was incubated with [3H]mevalonic acid. As shown in Table 4, significant label was associated with the sterol fraction. Oxygen is necessary for sterol synthesis since it is necessary for the epoxidation of squalene to form squalene 2,3-epoxide (Mahler & Cordes, 1971). An attempt was made to inhibit sterol biosynthesis by bubbling nitrogen through the culture medium to reduce available oxygen. Under these conditions, the

Concentrat £on

Control

4.5

3.0

2,0

ATe

1.12

1.03

1.72

~,D?I1

1.02

1.15

2.21

AI~ & blkI)I~

1,02

1.60

2.99

Control

Bacteria

Animals

CO2

~ J Phosphotidylserine

PhosphotidyleCha nola rn ine .~ 3 ~r

Ethonolornine

CH3 (Methionlne)

/

Serine CDP-Diglyceride Phosphotidy/choline

=

Oiglyceride

COP - Choline

Fig. 3. Schematic representation of major pathways of phospholipid synthesis.

EDWARD J. HOUK et al.

430

Table 3. Synthesis of phospholipids from various radiolabelled precursors pho0photipid

14C-Serlne ContYol

14C-Choltne

3H-Hethion£ne

Hydroxylam~ne

Phosphat £dyl serine

626.0

1124.0

56.0

50.5

11.4

125.0

25.0

25.0

10.0

150.0

53.8

240.4

102.0

165.0

Phosphat ldy 1 ent hanolmaine

phosphacidy1 choline

Lyso-Ftact~on

45.4

Orlsln

level of radioactivity in the sterol fraction was reduced to approx 25~o of the control value (Table 4). Simultaneously, an increase in the radioactivity associated with the (less-polar) solvent front was noted. It was thought that this increase might be attributed to increases in the levels of squalene, a highly nonpolar precursor of cholesterol. Squalene is soluble in hexane, whereas all the other components expected to be present at the solvent front (i.e. di- and triglycerides and sterol esters) are insoluble (Randerath, 1966). Based on this assumption the solvent fronts were extracted with hexane and the radioactivity counted. An almost equivalent reversal of the sterol situation was observed (Table 4). That is, the hexane extract of aerobic control cultures contained only about 25~o of the radioactivity found in anaerobic (N2) culture hexane extract. Further substantiation of the identity of the hexane-soluble components as squalene or its epoxide was not attempted. The specific identity of the sterol synthesized by the symbiote fraction was determined by GLC. A single peak was seen for the sterol spot; its retention time coincided with that of cholesterol. DISCUSSION

Our experimental results clearly show that the pea aphid symbiotes have the capacity to synthesize cholesterol in vitro from either acetate or mevalonate precursors. A reasonable assumption would be that this symbiote-synthesized cholesterol is available to the aphid in vivo, as other work has suggested (see introduction). Accepting this assumption, it will be of interest to know the mechanism by which the choles-

terol is made available to the aphid. For example, simple secretion must clearly be ruled out on the grounds that cholesterol is a large molecule, and almost completely insoluble in aqueous solutions. The controlled lysosomal breakdown of the symbiotes by the aphid, offers one distinct possibility, as Hinde (1971) suggested for the aphids Brevicoryne brassicae, Myzus persicae, and Macrosiphum rosae. In the pea aphid, however, while the breakdown of secondary symbiotes in extensive cytolysosomes appears significant, the lysosomal breakdown of primary symbiotes occurs at very low rates (Griffiths & Beck, 1973). Two observations concerning sterol synthesis and symbiote chemistry suggest that an alternate mechanism would be required. First, the lipid chemistry of the isolated symbiote fraction (Houk, 1974a) reflects closely the in vitro situation seen here with a stationary phase symbiote culture (4.5mg/ml concn). At this level, the symbiote sterol contribution to the total lipid complement in vitro was reduced to approx 1.1~o. Second, sterol is known to be a feedback inhibitor of its own synthesis (Mahler & Cordes, 1971). Hence, a stationary phase symbiote might be expected to have its sterol synthetic capacity reduced to relatively low levels. This suggests that some kind of mobilization mechanism would be required to shuttle the sterol from the symbiote into the mycetocyte cytoplasm; this would prevent feedback inhibition. Recent observations indicate the formation of membranous vesicles at the outer cell wall layer of the primary symbiote (Griffiths & Beck, 1975). These vesicles collect in the space between the symbiote cell wall and the outer membrane envelope and may subsequently pass out into the cytoplasm by exocytosis.

Table 4. 3H-mevalonate metabolism and sterol biosynthesis Lipid Class

Control

N2

Phospholipid

800

570

HouosIycer£de

850

640

Sterol Solvent Front Bexane Soluble

31,000

8,100

230,000

320p000

9,680

38,200

Lipid metabolism in the symbiotes of the pea aphid It is tempting to suggest that the vesicles contain cholesterol but, as yet, this has not been investigated.

REFERENCES

AKEY D. H. & BECK S. D. (197l) Continuous rearing of the pea aphid, Acyrthosiphon pisum on a holidic diet. Ann. ent. Soc. Am. 64. 353 356. DADD R. H. & KRIEGER D. L. (1967) Continuous rearing of aphids of the Aphisfabae complex on sterile synthetic diet. J. econ. Ent. 60, 1512-1514. ERHARDT P. (1968a) Einfluss yon Frnahrungfaktoren auf die Entwicklung von Safte saugenden Insekten unter besonderer Berucksightigung von Symbionten. Z. ParasitKde. 31, 38-66. ERHARDT P. (1968b) Nachweis einer durch symbiontische mikroorganism bewirken sterinsynthese in kunstlich ernahrten aphiden (Homoptera, Rhynchota, Insecta). Experientia (Basel). 24, 82-83. GRIEFITHS G. W. & BECK S. D. (1973) Intracellular symbiotes of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 17, 1791-1800. (;RIE]-II~tS G. W. & BECK S. D. (1975) Ultrastructure of pea aphid mycetocytes: Evidence for symbiote secretion. Cell Tiss. Res. 159, 351-367. HINDE R. (1971) The fine structure of the mycetome symbiotes of the aphids Brevicoryne brassicae, Myzus persicae and Macrosiphum rosae. J. Insect Physiol. 17, 2035 2O5O

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HOOK E. J. & MCLEAN D. L. (19741 Isolation of the primary intracellular symbiote of the pea aphid, Acyrthosiphon pisum. J. Invert. Pathol. 23, 237-241. HouK E. J. (1974a) Lipids of the primary intracellular sym-' biote of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 20, 471~,78. HOOK E. J. (1974b) Maintenance of the primary symbiote of the pea aphid, Acyrthosiphon pisum in liquid media. J. Invert. Pathol. 24. 24-28. KANESHIRO T. & LAW J. H. (1964) Phosphatidylcholine synthesis in Agrobacterium tumefaciens I. Purification and properties of phosphatidylethanolamine N-methyl transferase. J. biol. Chem. 239, 1705-1713. LAMBK. P. & HINDER. (1967) Structure and development of the mycetome in the cabbage aphid, Brevicoryne brassicae. J. Invert. Pathol. 9, 3 11. MAHLER H. R. & CORDES E. H. (1971) Biological Chemistry. Harper and Row, New York. MCLEAN D. L. & HOUK E. J. (1973) Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum J. Insect Physiol. 19, 625 633. RANDERATH K. (1966) Thin-layer Chromatography. Academic Press, New York. SMITH P. F. (1971) The Biology of the Mycoplasmas. Academic Press, New York. THOMPSON M. J., KAPLANISJ. N., ROBBINS W. E. & SvoBODA J. A. (1973) Metabolism of steroids in insects. Adv. Lipid. Res. l l , 219-265. VAGO C. & LAPORTE M. (1965) Microscope electronique des symbiontes globuleux des aphides (Horn. Aphidoidea). Ann. ent. Soc. Ft. l, 181-196.