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Sulphate utilization in an aphid symbiosis
Insect Biochem. Vol. 18, No. 6, pp. 599-605, 1988 Printed in Great Britain. All rights reserved
0020-1790/88 $3.00+ 0.00 Copyright © 1988Pergamon Press plc
SULPHATE UTILIZATION IN AN APHID SYMBIOSIS A. E. DOUGLAS School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ and AFRC Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich NR4 7UH, England
(Received 26 October 1987; revised and accepted 6 April 1988) Abstract--When two clones of Myzus persicae were maintained on a defined diet with inorganic sulphate as sole sulphur source, their growth and survival were inferior to that on diets containing the sulphur amino acid, methionine. This discrepancy is due, at least in part, to the phagostimulatory properties of methionine, which stimulated aphid feeding rate by 50-150%. Myzus persicae incorporated radioactivity from dietary [35S]sulphate into protein and low molecular weight compounds, including cysteine and methionine. Two lines of evidence indicate that the mycetocyte-symbionts are responsible for the reductive assimilation of sulphate. (1) [35S]sulphate incorporation is abolished by treatment of the aphids with the antibiotic chlortetracycline, which disrupts the symbionts; and (2) [35S]sulphate is utilized by isolated embryos (which contain mycetocyte--symbionts but no gut flora) but not by isolated guts. Tracer studies suggest that 20% of dietary radiosulphur is translocated to the aphid tissues, and it is hypothesized that methionine may be the principal product released by the symbionts.
Key Word Index: sulphate, aphid, Myzus persicae, symbiosis, mycetocyte, amino acids
INTRODUCTION The symbiosis between aphids and prokaryotic microorganisms located in mycetocyte cells in the insect haemocoel is generally believed to have a nutritional basis. The phloem sap on which aphids feed is nutritionally-poor, with an exceptionally high C/N ratio, unbalanced amino acid composition and low levels of lipids and vitamins (Dixon, 1975; Raven, 1983). The mycetocyte-symbionts of aphids have been proposed to complement this diet by the synthesis of essential amio acids, B-vitamins, unsaturated fatty acids and sterols, but the evidence is fragmentary and often contradictory (see reviews of Ehrhardt, 1968; Houk and Griffiths, 1980; Dadd, 1985; de Renobles et al., 1987). The aphid symbionts have also been shown to synthesize a single major protein of molecular weight 63,000 (Ishikawa, 1982a), and it has been argued that this protein is essential for the aphid, although its function is unknown (Ishikawa, 1984). The most clear-cut evidence for the synthesis of amino acids by the mycetocyt¢--symbionts has been obtained for the green peach aphid, Myzus persicae. This species can be maintained on holidic (i.e. chemically defined) diets in which the total concentration and composition of amino acids can be varied over a wide range (Dadd and Krieger, 1968). Furthermore, from analyses of the performance of aphids over two generations on diets from which individual amino acids are omitted, Dadd and Krieger (1968) concluded that M. persicae requires only three of the 10 essential amino acids, namely methionine, isoleucine and histidine. In contrast, when the comPresent address: Department of Zoology, University of Oxford, South Parks Road, Oxford, England. 599
plement of mycetocyte-symbionts in M. persicae was depleted by treatment with the antibiotic chlortetracycline (aureomycin), growth of the aphid was substantially reduced by omission of any of the essential amino acids from the diet (Mittler, 1971). Although the implication of these results is that the symbionts synthesize at least seven of the essential amino acids and make them available to the insect, this has not been examined directly. A particularly useful approach to the study of nutritional interactions in an intact symbiosis is to examine incorporation of a precursor that can be utilized only by the microbial partner. This obviates the need for "selective" metabolic inhibitors, which may have deleterious side-effects. For the study of amino acid synthesis in the aphid symbiosis, incorporation of sulphur from inorganic sulphate into sulphur amino acids, cysteine and methionine, is suitable because animals, including insects, are unable to reduce sulphate to the - 2 oxidation state (Schiff, 1980). Both Neomyzus circumflexus and Acyrthosiphon pisum can be maintained on holidic diets in which sulphur amino acids are replaced by sulphate (Ehrhardt, 1968; Retnakaran and Beck, 1967) and incorporation of [35S]sulphate into methionine and cysteine has been demonstrated in N. circumflexus (Ehrhardt, 1968). However, inorganic sulphate could replace cysteine, but not methionine for M. persicae (Dadd and Krieger, 1968; Massonie, 1974) and both sulphur amino acids are dietary essentials for Aphis gossypii (Turner, 1971). In this study, the capacity of inorganic sulphate to support the growth and reproduction of M. persicae is assessed. Evidence is presented that sulphate is assimilated by the mycetocyte-symbionts and sulphur compounds are translocated to the aphid tissues.
600
A . E . DOUGLAS MATERIALS AND METHODS
Maintenance of M. persicae on plants and holidic diets Two clonal cultures of virginoparae of M. persicae Sulzer were used: clone UEA-3, derived from an isolate from sugar beet at Brooms Barn in 1985 (D. Akers, personal communication) and clone JlI-2, from a long-standing culture of M. persicae at John Innes Institute, Norwich, originally obtained from A. Cockbain, Rothamsted Experimental Station, Harpenden, Herts. (P. G. Markham, personal communication). Both clones were maintained on 7-to-14-day-old radish seedlings var. French Breakfast, clone UEA-3 at 20°C with 16 h light/8 h dark light regime at 150#Einstein m -2 s -~ P.A.R. and clone JII-2 18°C with 20 h light/4 h dark regime at 300 #Einstein m -2 s ~ P.A.R. The aphids were maintained on holidic diets under the same conditions as on plants, but with light intensity 40-60 #einstein m -2 s -1 P.A.R. The diet cages used in most experiments are described in Douglas (1988) and the diets were prepared by the method of Kunkel (I 976), as modified by Douglas (1988). Two diet formulations were regularly used: the "standard diet" (diet a of Kunkel, 1976), which contains the following sulphur sources: methionine (5.4mM), cysteine (2.6mM) and magnesium sulphate (5.0 mM), and the "sulphate diet", which contains 10.0 mM magnesium sulphate as sole sulphur source. For the sulphate diet and other diet formulations in which methionine and/or cysteine were omitted, those amino acids were replaced by an equimolar concentration of serine (as recommended by Dadd and Krieger, 1968). In sulphate-free diets, magnesium sulphate was replaced by an equimolar concentration of magnesium chloride. For growth experiments and determination of feeding rates, adult apterae were transferred from radish seedlings to the standard diet and offspring born on the fourth day were transferred to the experimental diet in three replicate groups of 10 individuals/treatment. To disrupt the mycetocyte-symbionts, these "fourth-day larvae" were raised for 2 days on the standard diet and then maintained on diet containing 0.00 l q). 1% (w/v) chlortetracycline for 7 days.
Table 1. Composition of GRS Medium (mgl i). The medium was sterilized by filtration through 0.22 gm pore Millipore filter before use
Inorganic salts MgCI2'6H20 MgSO4' 7H20 NaHCO3 NaH2PO4-H20 KCI CaCI2' 2H20 Organic' acids Fumaric acid ~-ketoglutaric acid Malic acid Succinic acid Sugars Glucose Trehalose
pH 7.5
2280 2780 350 1013 4100 993 100 200 100 100 39,400 27,600
Amino acid (L-form) Alanine 425 Arginine 700 Asparagine 350 Asparticacid 350 Cysteine 22 Glutamicacid 600 Glutamine 600 Glycine 650 Histidine 250 Isoleucine 50 Leucine 75 Lysine 625 Methionine 50 Phenylalanine 150 Proline 350 Serine 1100 Threonine 175 Tryptophan 100 Tyrosine 50 Valine 100
sulphur-free GRS, in which the magnesium sulphate of sulphate--GRS was replaced by an equimolar concentration of magnesium chloride.
Incorporation of radioisotopes by intact M. persicae and isolated embryos The radioisotopes L-[U-t4C]leucine and inorganic [3SS]sulphate (Amersham plc) were used at a final sp. act. of 1 MBq ml ~of diet or GRS medium. Aphids at a density of 30M0 individuals/diet cage were allowed to feed on holidic diets containing the radioisotope. Incorporation was assayed over 72h, with two to four replicates of 150-200 aphids/treatment and time of assay. Three replicate groups of freshly-isolated embryos (of total biomass approx. 1 mg protein ml- ~) were incubated for up to 24 h either in standard GRS medium containing [U-14C]leucine or sulphate-GRS medium containing [35S]sulphate. At the end of the experiment, the embryos were washed in excess Isolation and short-term culture o f embryos and guts non-radioactive medium. Adult apterae were surface-sterilized by immersion in The aphids or washed embryos were homogenized in 70% (v/v) ethanol for 30 s, and washed in five changes of 0.2 ml ice-cold buffer, comprising 35 mM Tris-HCl, 25 mM sterile distilled water. Either embryos (within the ovariole KC1, 10mM MgCI2'6H20, l mM dithiothreitol, 0.25M sheath) or the gut distal to the pharyngeal duct were sucrose, pH 7.0 (pH determined at 20°C). Inorganic suldissected out with sterile forceps and pins and washed free phate was quantitatively precipitated by addition of 0.2 ml of contaminating insect tissue with ice-cold phosphate- 0.4M barium chloride to the homogenate, which was buffered saline (0.14 M NaC1, 1.8 mM NaH2PO 4, 8.5 mM centrifuged at 10,000g for 5 min. In test studies, all radioNa2HPO4). Tissues were incubated with shaking at 20°C in sulphur recovered from the pellet was inorganic sulphate filter-sterilized medium for up to 24h. Routine sterility and more than 99% of the radioactivity in the supernatant testing (see Douglas, 1988) confirmed the asepsis of the was in the low molecular weight and protein fractions. For experiments with [~4C]leucine, radioactivity was recovered preparations. Several media were tested for short-term culture of em- from only free and protein-bound leucine. Therefore, a bryos and guts. The tissues were viable over 24 h in two simplified protocol for the quantification of radioactivity in published media, Grace's Insect Tissue Culture Medium different tissue fractions was developed. The crude homoge(Grace, 1962) and JRS410 (Hardie, 1987), as determined by nate (for [~4C]leucine incorporation) or supernatant after the Neutral Red/Trypan Blue method of De Reizis and barium chloride treatment ([35S]sulphate incorporation) was Schechtman (1973), but they incorporated L-[U-14C]leucine incubated with 5% (w/v) trichloroacetic acid (TCA) for 30 into protein at very low rates [< 1 nmol exogenous leucine min at room temperature and then centrifuged at 10,000g (mg total protein)- i h - i]. The incorporation of [14C]leucine for 15min. The pellet was washed once in 0.4ml 50mM was higher [3-14 nmol (mg protein) -I h -1 in embryos and Tris-HCl, pH 7.0 and radioactivity in the pooled super1 4 nmol (mg protein)-I h - t in guts] when the tissues were natants (low molecular weight fraction) and washed pellet incubated in an alternative formulation, based on these (protein fraction) was assayed. For determination of 35S-labelled amino acids in the low media and modified Grace's medium (Gibco Life Technologies Inc., Product No. 041-1490). The composition of molecular weight fraction, advantage was taken of the insolubility of inorganic sulphate in ethanol. The aphids the alternative medium, which I have called standard GRS medium, is shown in Table 1. Several modified GRS media were homogenized in 0.2 ml ice-cold 80% ethanol and the were also used: leucine-free GRS, in which leucine was homogenate was centrifuged at 10,000 g and washed 3 times replaced by an equimolar concentration of serine; with centrifugation in 80% ethanol. (In test studies, sulphate-GRS, in which methionine and cysteine were [35S]sulphate was added to the aphid sample immediately replaced by an equimolar concentration of serine; and before homogenization; all ninhydrin-positive material was
Sulphate and aphids
601
recovered from the supernatant after three washes and Table 2. Growth of larvae of M. persicae clone UEA-3 on diets neither [35S]sulphate nor protein were detected in the pooled containing sulphur sources for 7 days. The aphids were transferred supernatant.) The ethanolic extracts were diluted 4-fold to the experimental diets within 24 h of birth, when they weighed 0.024 + 0.001 mg (mean _+SE) with 0.5 M HCI and were passed through 0.75 ml Dowex-50 Final weight (mg) of aphids (H ÷) ion exchange column, previously equilibrated with mean + SE (No. aphids) 0.5 M HCI. The eluate and a wash of 5 ml distilled water Sulphur source in diet" were combined to yield the "soluble non-amino acid Met + cys + SO4 0.370 _+0.021 (23) 0.374 + 0.019 (22) fraction". The amino acids were eluted with 6 ml 1.6 M Met + cys 0.321 + 0.024 (19) ammonium hydroxide, which was subsequently removed by Met + SO4 0.072 + 0.005 (19) vortex evaporation. Test studies with amino acid standard Cys + SO4 Met 0.146 + 0.009 (21) solutions (Sigma) and solutions of methionine and cysteine Cys 0.050 + 0.003 (8) confirmed that the amino acids were quantitatively recov- SO4 0.057 + 0.003 (15) ered (yield was >95%). Radiolabelled amino acids in the None 0.019 + 0.001 (13) soluble amino acid fraction were identified by I-D thin F(132, 140) = 53.41, P < 0.01. layer chromatography on silica gel plates (Merck 60, F254) concentration of each sulphur amino acid [methionine (met) run in n-butanol/acetic acid/water (3 : 1 : 1 v/v) under satur- ITheand cysteine (cys)] was 3 mM and that of magnesium sulphate ation against standards (Randerath, 1963). The amino acids (SO4) was 10mM. were visualized by spraying with ninhydrin [0.3% (w/v) in n-butanol/acetic acid (50: 3, v/v)] and heating to 110-130°C. Radiochromatograms were prepared using Fuji ted. At the end of the experiment, the weight of the X-ray film. larvae varied significantly and, using Scheffe's multiFeeding rates of M. persicae ple range test, the treatments could be divided into Food uptake was assayed by a modification of the four groups, significantly different from each other method of Wright et al. (1985), using [t4C]methylated inulin (P < 0.05): diets containing methionine plus other (Sigma). Each group of I0 aphids was allowed to feed on sulphur source(s) (mean relative growth rate the non-radioactive test diet in cages of the design of 0.37-0.39mg mg -l day-l); methionine as sole sulMarkham (1982) for 2 h, after which the diet droplet was phur source (0.26 mg m g - l day-l); sulphur source(s) replaced by 0.1 ml diet containing 0.1 MBq [t4C]methylated other than methionine (0.10-0.16 mg m g - l d a y - l); inulin. At the end of the experiment, the diet and aphids and sulphur-free diet, on which the aphids did not were removed and the t4C in the scintillation vial was quantified (see below). grow. The capacity of inorganic sulphate to sustain culAssay of 14C- and 35S-labelled samples tures of M. persicae suggests that this sulphur source All samples were brought to 0.9 ml with distilled water, is assimilated, with the net synthesis of cysteine and shaken with 10 ml Picofluor 15 scintillation fluid (Packard methionine. The superior performance of the aphids Instrument Co. Inc.) and counted in a Philips counter PW4700 or LKB 1219 Rackbeta counter, with pre-set on diets containing methionine may arise from two factors: growth of the insects on methionine-free diets 14C-window. may be limited by the rate of endogenous synthesis Protein content of aphids of methionine; and methionine is a phagostimulant The protein content of a portion of the homogenate of for M. persicae (Mittler, 1967), in the absence of aphids was assayed by the method of Bradford (1976), with which the aphids may feed at a reduced rate. These bovine serum albumin as standard. possibilities are investigated below in experiments on the phagostimulatory properties of methionine The weight of aphids and the incorporation of inorganic sulphate by M. The aphids were weighed individually on a Cahn 21 persicae. microbalance. RESULTS
Performance o f M. persicae on diets containing different sulphur sources M. persicae clones U E A - 3 and JII-2 were successfully maintained on the standard diet (Douglas, 1988). Both clones also grew on the sulphate diet: clone U E A - 3 survived for two to three generations and the cultures of clone JII-2 were vigorous when the experiment was terminated after four generations. It was routinely observed that aphids on the sulphate diet were smaller than insects on the standard diet. To investigate the capacity of inorganic sulphate to support the growth and development of M. persicae in more detail, larvae were fed on diets containing methionine, cysteine and sulphate, in combination and as sole sulphur sources. The results for the two clones did not differ substantially and data for clone U E A - 3 are shown in Table 2. Over the experimental period of 7 days, the aphids developed from the first to the third or fourth larval instar on all diets except the sulphur-free diet, on which no individual moulI.B. 1 8 / 6 ~ E s
Feeding rates o f M. persicae on diets containing different sulphur sources Larvae (4-day-old) of clone JII-2, reared from birth on the standard diet, fed at a linear rate over 1-2 days. On diets containing methionine (e.g. 3 m M methionine as sole sulphur source, standard diet), each aphid fed at a rate of 20-30 ni day -l, but their feeding rate on sulphate diet or diet with 3 m M cysteine as sole sulphur source was 10-15 nl day -l. These data confirm the conclusion of Mittler (1967) that methionine is a phagostimulant. To examine the effect of dietary pretreatment on the feeding response of larval M. persicae to methionine, aphids were reared on either standard diet or sulphate diet and each class of aphid was tested on both diets over 4 h (Table 3). Feeding rate was significantly increased by methionine both in the test diet [F(1, 8 ) = 16.71, P <0.01] and in the diet on which the larvae had been raised [F(I, 8 ) = 22.18, P < 0.01]. It is concluded that methionine is a phagostimulant, whether or not the aphids had previously been fed on diet containing methionine. The lower
602
A.E. DOUGLAS Table 3. Feeding rate by 4-day-old larvae of M. persicae clone Jll-2 on diets containing different sulphur sources, as determined from the [14C]methylated inulin content of honeydew Volume of diet (nl) ingested/aphid over 4 h (mean _+ SE) Diet on which aphids were reared Standard diet Sulphate diet
Standard diet
Sulphate diet
5.12 _+0.21 2.58 _+0.80
2.87 _+0.40 1.05 _+0.12
feeding rate of aphids raised on sulphate diet is probably a consequence of their small size (see Table 2).
Utilization of inorganic sulphate by intact M. persicae Initial experiments demonstrated that M. persicae maintained on the sulphate diet incorporated radioactivity from dietary [35S]sulphate at a linear rate over 1-3 days. The rate of incorporation by aphids transferred directly from plants to the diet was 18-25 nmol dietary sulphur (mg aphid protein)-l day-'. Maintenance on the sulphate diet, but not on diets containing sulphur amino acids, for 2-4 days prior to the experiment enhanced uptake to 30-50 nmol sulphur (mg protein)-~ day-~. Between 70-75% of the dietary radiosulphur was recovered from the protein fraction, with the remainder in the low molecular weight (TCA-soluble) fraction (Fig. 1). The amount of 35S in ethanolic extracts was closely similar to that in the TCA-soluble fraction of parallel samples and, within the ethanolic extracts, amino acids accounted for 11-13% of the total 35S. Approximately half of the radioactivity in this fraction co-chromatographed with methionine standard (R~ = 0.4-0.43). The remainder of the radioactivity was of RF 0.08-0.3; this is the location of the cysteine standard and its autoxidation products. These results suggest that sulphur from exogenous sulphate is incorporated into methionine and cysteine.
Utilization of dietary sulphate by M. persicae treated wtih the antibiotic chlortetracycline No insects are known to be capable of reductive assimilation of inorganic sulphate. It is therefore very probable that microorganisms in the aphid are responsible for the incorporation of dietary sulphate. To check this interpretation, the capacity of M. persicae to incorporate 35S after treatment with the antibiotic chlortetracycline was examined. Of the
100
-
"gT
ao
B 2
6o-
~~
40
•
/
-
o
.e Q vE
20
0
24
48
72
T i m e (h)
Fig. 1. Incorporation of radiosulphur from 35S-labelled sulphate diet by adult apterae of M. persicae clone UEA-3; O, protein fraction; ll, low molecular weight fraction.
three concentrations of dietary chlortetracycline tested, 0.01 and 0.005%, but not 0.001% cause the disruption of symbionts over 7 days (Douglas, 1988). The incorporation of dietary leucine into protein by parallel samples of aphids was determined, as an approach to establish the degree to which aphid metabolism was directly affected by chlortetracycline. Aphids transferred directly from diet containing 0.001% chlortetracycline to [35S]sulphate diet incorporated radiosulphur to half the extent of the controls and those treated with 0.005 and 0.01% chlortetracycline incorporated no detectable 35S. The capacity for [35S]suiphate incorporation was recovered over 10 days in individuals treated with 0.001%, but not 0.005 or 0.01% antibiotic (Table 4) and the latter aphids also could not be maintained on the sulphate diet. The effect of the antibiotic treatment on [14C]leucine incorporation was less marked and for every concentration of chlortetracycline used, the level of leucine incorporation increased over 10 days after the treatment, e.g. from 30 to 64% of the
Table 4. Incorporation of inorganic sulphate and leucine by larvae of M. persicae clone UEA-3 previously fed for 7 days on diet containing the antibiotic chlortetracycline. Data for aphids assayed immediately after antibiotic t r e a t m e n t and maintained on the standard diet for 10 days prior to assay are shown Incorporation of radioisotope aver 2 days nmol (rag protein) -I (mean _+SE, N = 4) 0-2 Days after 10-12 Days after antibiotic treatment antibiotic treatment
Dietary concentration of chlortetracycline (%, w/v)
[U)4C]leucine=
[3SS]sulphateb
[U)4C]leucine=
135Slsulphateb
0 0.001 0.005 0.01
169_+ 12 148 __ 10 51 _+6 25_+9
62-I-5 32_+ 3 <2 <2
132_+9 148 _+ 15 84_+ 10 81 _+9
58_+4 47 _+4 <2 <2
=Incorporation from standard diet (containing 6 mM leucine) into aphid protein. bIncorporation from sulphate diet into the combined low molecular weight and protein fractions.
Sulphate and aphids 30
100
E7 ='d
~
10
-/ /
W 0
603
I
I
I
12
18
80--
o
~2
6o-
~ .O
40-
O
6
--
J
°
Time (h)
Fig. 2. Incorporation of radiosulphur from 35S-labelled sulphate43RS medium by embryos isolated from M. persicae clone JII-2. control values for aphids fed on diet containing 0.005% chlortetracycline. The sustained abolition of [35S]sulphate but not [~4C]leucine incorporation into aphids treated with chlortetracycline at concentrations which disrupt the symbionts, supports the view that sulphate is utilized by microorganisms in the aphids.
0
24
I
I
48
72
Timo (h) Fig. 3. Incorporation of radiosulphur from 3~S-labelled
sulphate diet into protein fraction of the anterior (1, symbiont-free) and posterior (0, symbiont-containing) portions of M. persicae JII-2.
aphid's abdominal haemocoel (Ponsen, 1976). Apterous adult aphids fed on [35S]sulphate diet were bisected at the thorax and the incorporation of 35S into organic sulphur compounds in the anterior (symbiont-free) and posterior (symbiont-containing) portions of the aphid was determined (Fig. 3). Radioactivity was not detected in the anterior tissues of aphids that had fed on the diet for 8 h [i.e. < 0.6 nmol (mg protein) -l or < 4 % of total 35S incorporated], but these tissues contained appreciable 35S after 24 h. Both the amount and proportion of the total incorporated 35S that was recovered from the anterior tissues increased with time, to a value equivalent to 20-25 nmol dietary sulphur (mg protein)-~, or 20% of the total, at 72 h. In experiments with clone JII-2, all 35S in the anterior tissue was recovered from the protein fraction, but in clone UEA-3 35S-labelled low molecular weight compounds represented 30-50% of the total radiosulphur in the anterior portion.
Utilization of inorganic sulphate by isolated embryos and guts of M. persicae In principle, the utilization of sulphate in M. persicae may be mediated by the mycetocytesymbionts or microorganisms in the gut. The mycetocyte-symbionts are transmitted to embryos at the blastoderm stage in aphid virginoparae, but insects acquire their gut flora after birth (Buchner, 1965). Therefore, to examine the contribution of these two classes of microorganisms, the capacity of isolated guts and isolated embryos to incorporate [35S]sulphate from sulphate-GRS medium was investigated. No incorporation of [35S]sulphate into isolated guts was detected, whether the gut preparations were incubated in sulphate-GRS for 24 h as whole pieces, fragments or macerates. Isolated embryos did utilize exogenous [35S]sulphate. Incorporation was linear, at a rate of 2-8 nmol (mg protein) -~ h -1 over 4-6 h and continued, at a lower rate, over the subsequent 12 h of the experiDISCUSSION ment (Fig. 2). Radiosulphur was recovered from the Studies on the incorporation of [35S]sulphate by low molecular weight and protein fractions. Over 24h, the incorporation into these fractions was Neomyzus circumflexus (Ehrhardt, 1968) and M. (this study) indicate that the 17.6+ 1.6 and 12.3 + 0 . 4 n m o l (mg protein) -l, re- persieae mycetocyte--symbionts are capable of reductive asspectively. These results are consistent with the hypothesis similation of inorganic sulphate, with the net synthat the assimilation of inorganic sulphate by M. thesis of cysteine and methionine, and that products persieae is mediated exclusively by the of sulphate assimilation are made available to the mycetocyte-symbionts. The absence of sulphate re- aphid tissues. The mycetocyte-symbionts are production by isolated guts supports the results of posed to be exclusively responsible for sulphate asstructural studies (Douglas, 1988), in which no micro- similation by M. persicae because no microorganisms have been identified in the gut, fat body or other organisms were observed in the gut of M. persicae. organs of the clones used in this study (Douglas, Transport of 3SSfrom the mycetocyte-symbionts to the 1988) and isolated guts of M. persicae incorporate no aphid detectable [35S]sulphate. However, studies of isolated To investigate whether the 35S-products of dietary mycetocytes and symbionts are required to demonsulphate incorporation are transported from the strate unequivocally that the mycetocyte-symbionts mycetocyte-symbionts to the aphid tissues, advan- synthesize methionine and cysteine from inorganic tage was taken of the location of mycetocytes in the sulphate.
604
A.E. DOUGLAS
At first sight, these metabolic data are difficult to reconcile with the phagostimulatory properties of methionine for M. persicae and various published studies which indicate that methionine is a dietary essential. For example, in the classic study of Dadd and Krieger (1968), few individuals reached adulthood and no second-generation larvae were produced on diet with sulphate as sole sulphur source. However, some variation in the performance of different clones on sulphate diets is apparent; in this study, clone UEA-3 persisted for two to three generations and JII-2 for at least four generations on sulphate diet. Differences in formulation of diets and culture conditions and variation between clones in the rate of endogenous synthesis of sulphur amino acids and gustatory response to dietary methionine may all contribute to these discrepancies. In more general terms, one may argue that, although the mycetocyte-symbionts can utilize inorganic sulphate, the reduced sulphur products made available to the aphid are often inadequate to support the insect's requirements over long periods. For this reason, it is of selective advantage for M. persicae to respond to dietary methionine by feeding at an increased rate. Virtually nothing is known of the mechanism by which nutrients synthesized by mycetocytesymbionts are made available to the aphid partner, although the finding that up to 20% of incorporated radiosulphur is recovered from the anterior tissues of the aphids over 3 days suggests that the release of sulphate reduction products is substantial. Unfortunately, the absolute amount of sulphur transported cannot be calculated from these data because the specific activity of 35S in the mobile products is unknown. The location of [35S]sulphate in the anterior tissues of the aphid remains to be determined, but one may speculate that it is preferentially incorporated into the salivary glands. Microbial symbionts of other associations, especially the rhizobium-legume symbioses and alga-invertebrate symbioses, display substantial release of nutrients, the characteristics of which have several common features (Smith and Douglas, 1987). Firstly, the nutrients are released from living cells of the microorganisms; and secondly, release is selective, involving one or a few low molecular weight products (e.g. glucose, ammonia) that are not abundant intracellular products of the microorganism. If these generalities apply to the aphid symbiosis, then one may hypothesize that the symhionts selectively release methionine, the sulphur of which may be derived from the assimilation of inorganic sulphate. It is proposed that methionine is not metabolized in the mycetocyte cytoplasm, beyond that required for the mycetocyte's own requirements, e.g. for protein, and that no other sulphur compound, including cysteine, is released from the symbionts. Dietary studies suggest that M. persieae possesses the trans-sulphuration pathway of cysteine synthesis from methionine. [Both untreated and chlortetracycline-treated M. persieae thrive on holidic diets with methionine as the sole sulphur amino acid (Mittler, 1971; Douglas, this study)]. However, both Aphis fabae and Aphis gossypii have been reported to lack the capacity to synthesize cysteine from methionine (Leckstein and Llewellyn, 1973; Turner, 1971) and therefore the
interactions between these species and their mycetocyte-symbionts may be different from those proposed for M. persieae. The validity of the proposed scheme can be explored by studies on preparations of freshly isolated symbionts. These preparations are viable and capable of DNA, RNA and protein synthesis (Ishikawa, 1982b). By analogy with other symbiotic microorganisms, one would expect the fundamental metabolic capabilities of these preparations to closely mirror those of symbionts in the intact association (Smith and Douglas, 1987), even though gene expression in the symbionts is substantially altered on isolation (Ishikawa, 1982b). Two specific predictions of this scheme are that symbionts isolated from aphids reared on sulphate diet synthesize methionine from exogenously-applied [35S]sulphate and possess high activities of the key enzymes of sulphate reduction, cysteine synthesis and methionine synthesis. The significance o f sulphate utilization to aphid nutrition Virtually all the data on the utilization of inorganic sulphate by the aphid symbiosis come from studies of aphids on defined diets. It is therefore pertinent to consider the relevance of these studies to aphids on their natural food source, phloem sap. Unfortunately, only fragmentary information is available on phloem transport of sulphur compounds. Chemical analyses indicate that phosphate, and not sulphate, is the major inorganic anion (Ziegler, 1975); the concentration of inorganic sulphate is 0.5-1 meq 1-1. This had led to the suggestion that sulphate plays only a minor role in phloem transport of sulphur (Ziegler, 1975; Raven, 1983). In contrast, studies of phloem transport of sulphur from leaves treated with [3SS]sulphate or [35S]sulphite indicate that at least half of the radiosuiphur is translocated as sulphate. The remainder of 35S is in the form of reduced organic sulphur compounds, predominantly glutathionine (glutamylcysteinylglycine), with free cysteine and methionine as minor components (Garsed and Read, 1977; Rennenberg et al., 1979; Bonas et al., 1982). In these radiotracer experiments, the specific activity, and hence absolute quantity, of radiolabelled sulphur compounds was not determined, but the implication, namely that the levels of free cysteine and methionine in phloem are low, is supported by direct analyses of phloem sap (Ziegler, 1975). These considerations indicate that the capacity of the mycetocyte-symbionts to assimilate inorganic sulphate into sulphur amino acids and to synthesize methionine de novo may make an important contribution to the nutrition of aphids feeding on plants. •4cknowledgements--I thank Mr P. Kenworthy and Dr N. Shaw for their advice and practical suggestions on the study of 35S utilization, Mrs J. Geeson who helped with the preparation of diets, and Mr G. Cleveland who cheerfully resolved many technical problems. I am grateful to Professor A. F. G. Dixon, Dr P. G. Markham and Dr J. B. Searle for helpful discussions and to Dr J. Hardie, who persuaded me of the potential of isolated embryos for metabolic studies. This project was funded by grants from the Science and Engineering Research Council and the Royal Society of London.
Sulphate and aphids REFERENCES
Bonas U., Schmitz K., Rennenberg H. and Bergmann L. (1982) Phloem transport of sulfur in Ricinus. Planta 155, 82-88. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye study. Analyt. Biochem. 72, 248-254. Buchner P. (1965) Endosymbiosis of Animals with Plant Microorganisms. Interscience, New York. Dadd R. H. (1985) Nutrition: organisms. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 4 (Edited by Kerkut G. A. and Gilbert L. I.), pp. 313-391. Pergamon Press, Oxford. Dadd R. H. and Krieger D. L. (1968) Dietary amino acid requirements of the aphid Myzus persicae. J. Insect Physiol. 14, 741-764. De Reizis F. A. and Schechtman A. (1973) Staining by neutral red and trypan blue in sequence for assaying vital and non-vital cultured cells. Stain Technol. 48, 135-136. Dixon A. F. G. (1975) Aphids and translocation. Encyclopedia of Plant Physiology New Series, Vol. I. (Edited by Zimmerman M. H. and Milburn J. A.), pp. 154-170. Springer, Berlin. Douglas A. E. (1988) On the source of sterols for the green peach aphid, Myzus persicae, on bolidic diets. J. Insect Physiol. 34, 403-408. Ehrhardt P. (1968) Einfluss yon Ernahrungsfaktoren auf die Entwicklung yon Safte saugenden Insekten unter besonderer Beruchsichtung yon Symbionten. Z. Parasitenkd. 31, 38-66. Garsed S. G. and Read D. J. (1977) Sulphur dioxide metabolism in soy-bean Glycine max vat. Biloxi. II. Biochemical distribution of 35SO2 products. New Phytol. 74, 583-592. Grace T. C. C. (1962) Establishment of four strains of cells from insect tissues grown in vitro. Nature 195, 788-789. Hardie J. (1987) Juvenile hormone stimulation of ooeyte development and embryogenesis in the parthenogenetic ovaries of an aphid, Aphis fabae. Int. J. Invert. Reprod. Dev. 11, 189-203. Houk E. J. and Gritfiths G. W. (1980) Intracellular symbiotes of the Homoptera. A. Rev. Entl. 25, 161-187. Ishikawa H. (1982a) Host-symbiont interactions in the protein synthesis in the pea aphid Acyrthosiphon pisum. Insect Biochem. 12, 613-622. Ishikawa H. (1982b) DNA, RNA and protein synthesis in the isolated symbionts from the pea aphid, Acyrthosiphon pisum. Insect Biochem. 12, 605-612. lshikawa H. (1984) Molecular aspects of intracellular symbiosis in the aphid mycetocyte. Zool. Sci. 1, 509-522. Kunkel H. (1976) Membrane feeding systems in aphid research. In Aphids as Virus Vectors (Edited by Harris
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K. F. and Maramorosch K.), pp. 311-337. Academic Press, New York. Leckstein P. M. and Llewellyn M. (1973) Effect of dietary amino acids on size and alary polymorphism of Aphis fabae. J. Insect Physiol. 19, 973-980. Markham P. G. (1982) Insect vectors. In Plant and Insect Mycoplasma Techniques (Edited by Daniels M. J. and Markham P. G.), pp. 307-326. Croom Helm, London. Massonie G. (1974) Elevage d'un biotype de Myzus persicae Sulzer sur milieu synthetique. III. Valeur alimentaire respective de la l-methionine, de la 1-cysteine et des sulfates. Annls Nutr. Aliment. 29, 45-50. Mittler T. E. (1967) Effect of aphid feeding of dietary methionine. Nature 214, 386. Mittler T. E. (1971) Dietary amino acid requirements of the aphid Myzus persicae affected by antibiotic uptake. J. Nutr. 101, 1023-1038. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Edited by Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press, New York. Randerath K. (1963) Thin-Layer Chromatography. Academic Press, New York. Raven J. A. (1983) Phytophages of xylem and phloem: a comparison of animal and plant sap-feeders. Adv. ecol. Res. 13, 136-234. Rennenberg H., Schmitz K. and Bergmann L. (1979) Longdistance transport of sulfur in Nicotinia tabacum. Planta 147, 57-62. Renobales M. de, Cripps C., Stanley-Samuelson D. W., Jurenka R. A. and Blomquist G. J. (1987) Biosynthesis of linoleic acid in insects. Trends Biochem. Sci. 12, 364-366. Retnakaran A. and Beck S. D. (1967) Amino acid requirements and sulphur amino acid metabolism in the pea aphid, Acyrthosiphon pisum (Harris). Comp. Biochem. Physiol. 24, 611-619. Schiff J. A. (1980) Pathways of assimilatory sulphate reduction in plants and microorganisms. In Sulphur in Biology, Ciba Foundation Vol. 72, pp. 49-64. Excerpta Medica, Amsterdam. Smith D. C. and Douglas A. E. (1987) The Biology of Symbiosis. Arnold, London, Turner R. B. (1971) Dietary amino acid requirements of the cotton aphid, Aphis gossypii: the sulphur-containing amino acids. J. Insect Physiol. 17, 2451 2456. Wright J. P., Fisher D. B. and Mittler T. E. (1985) Measurement of aphid feeding rates on artificial diets using 3H-inulin. Ent. exp. Appl. 37, 9-11. Ziegler H. (1975) Nature of transported substances. In Encyclopedia of Plant Physiology New Series, Vol. I. (Edited by Zimmerman M. H. and Milburn J. A.), pp. 59-99. Springer, Berlin.
0020-1790/88 $3.00+ 0.00 Copyright © 1988Pergamon Press plc
SULPHATE UTILIZATION IN AN APHID SYMBIOSIS A. E. DOUGLAS School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ and AFRC Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich NR4 7UH, England
(Received 26 October 1987; revised and accepted 6 April 1988) Abstract--When two clones of Myzus persicae were maintained on a defined diet with inorganic sulphate as sole sulphur source, their growth and survival were inferior to that on diets containing the sulphur amino acid, methionine. This discrepancy is due, at least in part, to the phagostimulatory properties of methionine, which stimulated aphid feeding rate by 50-150%. Myzus persicae incorporated radioactivity from dietary [35S]sulphate into protein and low molecular weight compounds, including cysteine and methionine. Two lines of evidence indicate that the mycetocyte-symbionts are responsible for the reductive assimilation of sulphate. (1) [35S]sulphate incorporation is abolished by treatment of the aphids with the antibiotic chlortetracycline, which disrupts the symbionts; and (2) [35S]sulphate is utilized by isolated embryos (which contain mycetocyte--symbionts but no gut flora) but not by isolated guts. Tracer studies suggest that 20% of dietary radiosulphur is translocated to the aphid tissues, and it is hypothesized that methionine may be the principal product released by the symbionts.
Key Word Index: sulphate, aphid, Myzus persicae, symbiosis, mycetocyte, amino acids
INTRODUCTION The symbiosis between aphids and prokaryotic microorganisms located in mycetocyte cells in the insect haemocoel is generally believed to have a nutritional basis. The phloem sap on which aphids feed is nutritionally-poor, with an exceptionally high C/N ratio, unbalanced amino acid composition and low levels of lipids and vitamins (Dixon, 1975; Raven, 1983). The mycetocyte-symbionts of aphids have been proposed to complement this diet by the synthesis of essential amio acids, B-vitamins, unsaturated fatty acids and sterols, but the evidence is fragmentary and often contradictory (see reviews of Ehrhardt, 1968; Houk and Griffiths, 1980; Dadd, 1985; de Renobles et al., 1987). The aphid symbionts have also been shown to synthesize a single major protein of molecular weight 63,000 (Ishikawa, 1982a), and it has been argued that this protein is essential for the aphid, although its function is unknown (Ishikawa, 1984). The most clear-cut evidence for the synthesis of amino acids by the mycetocyt¢--symbionts has been obtained for the green peach aphid, Myzus persicae. This species can be maintained on holidic (i.e. chemically defined) diets in which the total concentration and composition of amino acids can be varied over a wide range (Dadd and Krieger, 1968). Furthermore, from analyses of the performance of aphids over two generations on diets from which individual amino acids are omitted, Dadd and Krieger (1968) concluded that M. persicae requires only three of the 10 essential amino acids, namely methionine, isoleucine and histidine. In contrast, when the comPresent address: Department of Zoology, University of Oxford, South Parks Road, Oxford, England. 599
plement of mycetocyte-symbionts in M. persicae was depleted by treatment with the antibiotic chlortetracycline (aureomycin), growth of the aphid was substantially reduced by omission of any of the essential amino acids from the diet (Mittler, 1971). Although the implication of these results is that the symbionts synthesize at least seven of the essential amino acids and make them available to the insect, this has not been examined directly. A particularly useful approach to the study of nutritional interactions in an intact symbiosis is to examine incorporation of a precursor that can be utilized only by the microbial partner. This obviates the need for "selective" metabolic inhibitors, which may have deleterious side-effects. For the study of amino acid synthesis in the aphid symbiosis, incorporation of sulphur from inorganic sulphate into sulphur amino acids, cysteine and methionine, is suitable because animals, including insects, are unable to reduce sulphate to the - 2 oxidation state (Schiff, 1980). Both Neomyzus circumflexus and Acyrthosiphon pisum can be maintained on holidic diets in which sulphur amino acids are replaced by sulphate (Ehrhardt, 1968; Retnakaran and Beck, 1967) and incorporation of [35S]sulphate into methionine and cysteine has been demonstrated in N. circumflexus (Ehrhardt, 1968). However, inorganic sulphate could replace cysteine, but not methionine for M. persicae (Dadd and Krieger, 1968; Massonie, 1974) and both sulphur amino acids are dietary essentials for Aphis gossypii (Turner, 1971). In this study, the capacity of inorganic sulphate to support the growth and reproduction of M. persicae is assessed. Evidence is presented that sulphate is assimilated by the mycetocyte-symbionts and sulphur compounds are translocated to the aphid tissues.
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A . E . DOUGLAS MATERIALS AND METHODS
Maintenance of M. persicae on plants and holidic diets Two clonal cultures of virginoparae of M. persicae Sulzer were used: clone UEA-3, derived from an isolate from sugar beet at Brooms Barn in 1985 (D. Akers, personal communication) and clone JlI-2, from a long-standing culture of M. persicae at John Innes Institute, Norwich, originally obtained from A. Cockbain, Rothamsted Experimental Station, Harpenden, Herts. (P. G. Markham, personal communication). Both clones were maintained on 7-to-14-day-old radish seedlings var. French Breakfast, clone UEA-3 at 20°C with 16 h light/8 h dark light regime at 150#Einstein m -2 s -~ P.A.R. and clone JII-2 18°C with 20 h light/4 h dark regime at 300 #Einstein m -2 s ~ P.A.R. The aphids were maintained on holidic diets under the same conditions as on plants, but with light intensity 40-60 #einstein m -2 s -1 P.A.R. The diet cages used in most experiments are described in Douglas (1988) and the diets were prepared by the method of Kunkel (I 976), as modified by Douglas (1988). Two diet formulations were regularly used: the "standard diet" (diet a of Kunkel, 1976), which contains the following sulphur sources: methionine (5.4mM), cysteine (2.6mM) and magnesium sulphate (5.0 mM), and the "sulphate diet", which contains 10.0 mM magnesium sulphate as sole sulphur source. For the sulphate diet and other diet formulations in which methionine and/or cysteine were omitted, those amino acids were replaced by an equimolar concentration of serine (as recommended by Dadd and Krieger, 1968). In sulphate-free diets, magnesium sulphate was replaced by an equimolar concentration of magnesium chloride. For growth experiments and determination of feeding rates, adult apterae were transferred from radish seedlings to the standard diet and offspring born on the fourth day were transferred to the experimental diet in three replicate groups of 10 individuals/treatment. To disrupt the mycetocyte-symbionts, these "fourth-day larvae" were raised for 2 days on the standard diet and then maintained on diet containing 0.00 l q). 1% (w/v) chlortetracycline for 7 days.
Table 1. Composition of GRS Medium (mgl i). The medium was sterilized by filtration through 0.22 gm pore Millipore filter before use
Inorganic salts MgCI2'6H20 MgSO4' 7H20 NaHCO3 NaH2PO4-H20 KCI CaCI2' 2H20 Organic' acids Fumaric acid ~-ketoglutaric acid Malic acid Succinic acid Sugars Glucose Trehalose
pH 7.5
2280 2780 350 1013 4100 993 100 200 100 100 39,400 27,600
Amino acid (L-form) Alanine 425 Arginine 700 Asparagine 350 Asparticacid 350 Cysteine 22 Glutamicacid 600 Glutamine 600 Glycine 650 Histidine 250 Isoleucine 50 Leucine 75 Lysine 625 Methionine 50 Phenylalanine 150 Proline 350 Serine 1100 Threonine 175 Tryptophan 100 Tyrosine 50 Valine 100
sulphur-free GRS, in which the magnesium sulphate of sulphate--GRS was replaced by an equimolar concentration of magnesium chloride.
Incorporation of radioisotopes by intact M. persicae and isolated embryos The radioisotopes L-[U-t4C]leucine and inorganic [3SS]sulphate (Amersham plc) were used at a final sp. act. of 1 MBq ml ~of diet or GRS medium. Aphids at a density of 30M0 individuals/diet cage were allowed to feed on holidic diets containing the radioisotope. Incorporation was assayed over 72h, with two to four replicates of 150-200 aphids/treatment and time of assay. Three replicate groups of freshly-isolated embryos (of total biomass approx. 1 mg protein ml- ~) were incubated for up to 24 h either in standard GRS medium containing [U-14C]leucine or sulphate-GRS medium containing [35S]sulphate. At the end of the experiment, the embryos were washed in excess Isolation and short-term culture o f embryos and guts non-radioactive medium. Adult apterae were surface-sterilized by immersion in The aphids or washed embryos were homogenized in 70% (v/v) ethanol for 30 s, and washed in five changes of 0.2 ml ice-cold buffer, comprising 35 mM Tris-HCl, 25 mM sterile distilled water. Either embryos (within the ovariole KC1, 10mM MgCI2'6H20, l mM dithiothreitol, 0.25M sheath) or the gut distal to the pharyngeal duct were sucrose, pH 7.0 (pH determined at 20°C). Inorganic suldissected out with sterile forceps and pins and washed free phate was quantitatively precipitated by addition of 0.2 ml of contaminating insect tissue with ice-cold phosphate- 0.4M barium chloride to the homogenate, which was buffered saline (0.14 M NaC1, 1.8 mM NaH2PO 4, 8.5 mM centrifuged at 10,000g for 5 min. In test studies, all radioNa2HPO4). Tissues were incubated with shaking at 20°C in sulphur recovered from the pellet was inorganic sulphate filter-sterilized medium for up to 24h. Routine sterility and more than 99% of the radioactivity in the supernatant testing (see Douglas, 1988) confirmed the asepsis of the was in the low molecular weight and protein fractions. For experiments with [~4C]leucine, radioactivity was recovered preparations. Several media were tested for short-term culture of em- from only free and protein-bound leucine. Therefore, a bryos and guts. The tissues were viable over 24 h in two simplified protocol for the quantification of radioactivity in published media, Grace's Insect Tissue Culture Medium different tissue fractions was developed. The crude homoge(Grace, 1962) and JRS410 (Hardie, 1987), as determined by nate (for [~4C]leucine incorporation) or supernatant after the Neutral Red/Trypan Blue method of De Reizis and barium chloride treatment ([35S]sulphate incorporation) was Schechtman (1973), but they incorporated L-[U-14C]leucine incubated with 5% (w/v) trichloroacetic acid (TCA) for 30 into protein at very low rates [< 1 nmol exogenous leucine min at room temperature and then centrifuged at 10,000g (mg total protein)- i h - i]. The incorporation of [14C]leucine for 15min. The pellet was washed once in 0.4ml 50mM was higher [3-14 nmol (mg protein) -I h -1 in embryos and Tris-HCl, pH 7.0 and radioactivity in the pooled super1 4 nmol (mg protein)-I h - t in guts] when the tissues were natants (low molecular weight fraction) and washed pellet incubated in an alternative formulation, based on these (protein fraction) was assayed. For determination of 35S-labelled amino acids in the low media and modified Grace's medium (Gibco Life Technologies Inc., Product No. 041-1490). The composition of molecular weight fraction, advantage was taken of the insolubility of inorganic sulphate in ethanol. The aphids the alternative medium, which I have called standard GRS medium, is shown in Table 1. Several modified GRS media were homogenized in 0.2 ml ice-cold 80% ethanol and the were also used: leucine-free GRS, in which leucine was homogenate was centrifuged at 10,000 g and washed 3 times replaced by an equimolar concentration of serine; with centrifugation in 80% ethanol. (In test studies, sulphate-GRS, in which methionine and cysteine were [35S]sulphate was added to the aphid sample immediately replaced by an equimolar concentration of serine; and before homogenization; all ninhydrin-positive material was
Sulphate and aphids
601
recovered from the supernatant after three washes and Table 2. Growth of larvae of M. persicae clone UEA-3 on diets neither [35S]sulphate nor protein were detected in the pooled containing sulphur sources for 7 days. The aphids were transferred supernatant.) The ethanolic extracts were diluted 4-fold to the experimental diets within 24 h of birth, when they weighed 0.024 + 0.001 mg (mean _+SE) with 0.5 M HCI and were passed through 0.75 ml Dowex-50 Final weight (mg) of aphids (H ÷) ion exchange column, previously equilibrated with mean + SE (No. aphids) 0.5 M HCI. The eluate and a wash of 5 ml distilled water Sulphur source in diet" were combined to yield the "soluble non-amino acid Met + cys + SO4 0.370 _+0.021 (23) 0.374 + 0.019 (22) fraction". The amino acids were eluted with 6 ml 1.6 M Met + cys 0.321 + 0.024 (19) ammonium hydroxide, which was subsequently removed by Met + SO4 0.072 + 0.005 (19) vortex evaporation. Test studies with amino acid standard Cys + SO4 Met 0.146 + 0.009 (21) solutions (Sigma) and solutions of methionine and cysteine Cys 0.050 + 0.003 (8) confirmed that the amino acids were quantitatively recov- SO4 0.057 + 0.003 (15) ered (yield was >95%). Radiolabelled amino acids in the None 0.019 + 0.001 (13) soluble amino acid fraction were identified by I-D thin F(132, 140) = 53.41, P < 0.01. layer chromatography on silica gel plates (Merck 60, F254) concentration of each sulphur amino acid [methionine (met) run in n-butanol/acetic acid/water (3 : 1 : 1 v/v) under satur- ITheand cysteine (cys)] was 3 mM and that of magnesium sulphate ation against standards (Randerath, 1963). The amino acids (SO4) was 10mM. were visualized by spraying with ninhydrin [0.3% (w/v) in n-butanol/acetic acid (50: 3, v/v)] and heating to 110-130°C. Radiochromatograms were prepared using Fuji ted. At the end of the experiment, the weight of the X-ray film. larvae varied significantly and, using Scheffe's multiFeeding rates of M. persicae ple range test, the treatments could be divided into Food uptake was assayed by a modification of the four groups, significantly different from each other method of Wright et al. (1985), using [t4C]methylated inulin (P < 0.05): diets containing methionine plus other (Sigma). Each group of I0 aphids was allowed to feed on sulphur source(s) (mean relative growth rate the non-radioactive test diet in cages of the design of 0.37-0.39mg mg -l day-l); methionine as sole sulMarkham (1982) for 2 h, after which the diet droplet was phur source (0.26 mg m g - l day-l); sulphur source(s) replaced by 0.1 ml diet containing 0.1 MBq [t4C]methylated other than methionine (0.10-0.16 mg m g - l d a y - l); inulin. At the end of the experiment, the diet and aphids and sulphur-free diet, on which the aphids did not were removed and the t4C in the scintillation vial was quantified (see below). grow. The capacity of inorganic sulphate to sustain culAssay of 14C- and 35S-labelled samples tures of M. persicae suggests that this sulphur source All samples were brought to 0.9 ml with distilled water, is assimilated, with the net synthesis of cysteine and shaken with 10 ml Picofluor 15 scintillation fluid (Packard methionine. The superior performance of the aphids Instrument Co. Inc.) and counted in a Philips counter PW4700 or LKB 1219 Rackbeta counter, with pre-set on diets containing methionine may arise from two factors: growth of the insects on methionine-free diets 14C-window. may be limited by the rate of endogenous synthesis Protein content of aphids of methionine; and methionine is a phagostimulant The protein content of a portion of the homogenate of for M. persicae (Mittler, 1967), in the absence of aphids was assayed by the method of Bradford (1976), with which the aphids may feed at a reduced rate. These bovine serum albumin as standard. possibilities are investigated below in experiments on the phagostimulatory properties of methionine The weight of aphids and the incorporation of inorganic sulphate by M. The aphids were weighed individually on a Cahn 21 persicae. microbalance. RESULTS
Performance o f M. persicae on diets containing different sulphur sources M. persicae clones U E A - 3 and JII-2 were successfully maintained on the standard diet (Douglas, 1988). Both clones also grew on the sulphate diet: clone U E A - 3 survived for two to three generations and the cultures of clone JII-2 were vigorous when the experiment was terminated after four generations. It was routinely observed that aphids on the sulphate diet were smaller than insects on the standard diet. To investigate the capacity of inorganic sulphate to support the growth and development of M. persicae in more detail, larvae were fed on diets containing methionine, cysteine and sulphate, in combination and as sole sulphur sources. The results for the two clones did not differ substantially and data for clone U E A - 3 are shown in Table 2. Over the experimental period of 7 days, the aphids developed from the first to the third or fourth larval instar on all diets except the sulphur-free diet, on which no individual moulI.B. 1 8 / 6 ~ E s
Feeding rates o f M. persicae on diets containing different sulphur sources Larvae (4-day-old) of clone JII-2, reared from birth on the standard diet, fed at a linear rate over 1-2 days. On diets containing methionine (e.g. 3 m M methionine as sole sulphur source, standard diet), each aphid fed at a rate of 20-30 ni day -l, but their feeding rate on sulphate diet or diet with 3 m M cysteine as sole sulphur source was 10-15 nl day -l. These data confirm the conclusion of Mittler (1967) that methionine is a phagostimulant. To examine the effect of dietary pretreatment on the feeding response of larval M. persicae to methionine, aphids were reared on either standard diet or sulphate diet and each class of aphid was tested on both diets over 4 h (Table 3). Feeding rate was significantly increased by methionine both in the test diet [F(1, 8 ) = 16.71, P <0.01] and in the diet on which the larvae had been raised [F(I, 8 ) = 22.18, P < 0.01]. It is concluded that methionine is a phagostimulant, whether or not the aphids had previously been fed on diet containing methionine. The lower
602
A.E. DOUGLAS Table 3. Feeding rate by 4-day-old larvae of M. persicae clone Jll-2 on diets containing different sulphur sources, as determined from the [14C]methylated inulin content of honeydew Volume of diet (nl) ingested/aphid over 4 h (mean _+ SE) Diet on which aphids were reared Standard diet Sulphate diet
Standard diet
Sulphate diet
5.12 _+0.21 2.58 _+0.80
2.87 _+0.40 1.05 _+0.12
feeding rate of aphids raised on sulphate diet is probably a consequence of their small size (see Table 2).
Utilization of inorganic sulphate by intact M. persicae Initial experiments demonstrated that M. persicae maintained on the sulphate diet incorporated radioactivity from dietary [35S]sulphate at a linear rate over 1-3 days. The rate of incorporation by aphids transferred directly from plants to the diet was 18-25 nmol dietary sulphur (mg aphid protein)-l day-'. Maintenance on the sulphate diet, but not on diets containing sulphur amino acids, for 2-4 days prior to the experiment enhanced uptake to 30-50 nmol sulphur (mg protein)-~ day-~. Between 70-75% of the dietary radiosulphur was recovered from the protein fraction, with the remainder in the low molecular weight (TCA-soluble) fraction (Fig. 1). The amount of 35S in ethanolic extracts was closely similar to that in the TCA-soluble fraction of parallel samples and, within the ethanolic extracts, amino acids accounted for 11-13% of the total 35S. Approximately half of the radioactivity in this fraction co-chromatographed with methionine standard (R~ = 0.4-0.43). The remainder of the radioactivity was of RF 0.08-0.3; this is the location of the cysteine standard and its autoxidation products. These results suggest that sulphur from exogenous sulphate is incorporated into methionine and cysteine.
Utilization of dietary sulphate by M. persicae treated wtih the antibiotic chlortetracycline No insects are known to be capable of reductive assimilation of inorganic sulphate. It is therefore very probable that microorganisms in the aphid are responsible for the incorporation of dietary sulphate. To check this interpretation, the capacity of M. persicae to incorporate 35S after treatment with the antibiotic chlortetracycline was examined. Of the
100
-
"gT
ao
B 2
6o-
~~
40
•
/
-
o
.e Q vE
20
0
24
48
72
T i m e (h)
Fig. 1. Incorporation of radiosulphur from 35S-labelled sulphate diet by adult apterae of M. persicae clone UEA-3; O, protein fraction; ll, low molecular weight fraction.
three concentrations of dietary chlortetracycline tested, 0.01 and 0.005%, but not 0.001% cause the disruption of symbionts over 7 days (Douglas, 1988). The incorporation of dietary leucine into protein by parallel samples of aphids was determined, as an approach to establish the degree to which aphid metabolism was directly affected by chlortetracycline. Aphids transferred directly from diet containing 0.001% chlortetracycline to [35S]sulphate diet incorporated radiosulphur to half the extent of the controls and those treated with 0.005 and 0.01% chlortetracycline incorporated no detectable 35S. The capacity for [35S]suiphate incorporation was recovered over 10 days in individuals treated with 0.001%, but not 0.005 or 0.01% antibiotic (Table 4) and the latter aphids also could not be maintained on the sulphate diet. The effect of the antibiotic treatment on [14C]leucine incorporation was less marked and for every concentration of chlortetracycline used, the level of leucine incorporation increased over 10 days after the treatment, e.g. from 30 to 64% of the
Table 4. Incorporation of inorganic sulphate and leucine by larvae of M. persicae clone UEA-3 previously fed for 7 days on diet containing the antibiotic chlortetracycline. Data for aphids assayed immediately after antibiotic t r e a t m e n t and maintained on the standard diet for 10 days prior to assay are shown Incorporation of radioisotope aver 2 days nmol (rag protein) -I (mean _+SE, N = 4) 0-2 Days after 10-12 Days after antibiotic treatment antibiotic treatment
Dietary concentration of chlortetracycline (%, w/v)
[U)4C]leucine=
[3SS]sulphateb
[U)4C]leucine=
135Slsulphateb
0 0.001 0.005 0.01
169_+ 12 148 __ 10 51 _+6 25_+9
62-I-5 32_+ 3 <2 <2
132_+9 148 _+ 15 84_+ 10 81 _+9
58_+4 47 _+4 <2 <2
=Incorporation from standard diet (containing 6 mM leucine) into aphid protein. bIncorporation from sulphate diet into the combined low molecular weight and protein fractions.
Sulphate and aphids 30
100
E7 ='d
~
10
-/ /
W 0
603
I
I
I
12
18
80--
o
~2
6o-
~ .O
40-
O
6
--
J
°
Time (h)
Fig. 2. Incorporation of radiosulphur from 35S-labelled sulphate43RS medium by embryos isolated from M. persicae clone JII-2. control values for aphids fed on diet containing 0.005% chlortetracycline. The sustained abolition of [35S]sulphate but not [~4C]leucine incorporation into aphids treated with chlortetracycline at concentrations which disrupt the symbionts, supports the view that sulphate is utilized by microorganisms in the aphids.
0
24
I
I
48
72
Timo (h) Fig. 3. Incorporation of radiosulphur from 3~S-labelled
sulphate diet into protein fraction of the anterior (1, symbiont-free) and posterior (0, symbiont-containing) portions of M. persicae JII-2.
aphid's abdominal haemocoel (Ponsen, 1976). Apterous adult aphids fed on [35S]sulphate diet were bisected at the thorax and the incorporation of 35S into organic sulphur compounds in the anterior (symbiont-free) and posterior (symbiont-containing) portions of the aphid was determined (Fig. 3). Radioactivity was not detected in the anterior tissues of aphids that had fed on the diet for 8 h [i.e. < 0.6 nmol (mg protein) -l or < 4 % of total 35S incorporated], but these tissues contained appreciable 35S after 24 h. Both the amount and proportion of the total incorporated 35S that was recovered from the anterior tissues increased with time, to a value equivalent to 20-25 nmol dietary sulphur (mg protein)-~, or 20% of the total, at 72 h. In experiments with clone JII-2, all 35S in the anterior tissue was recovered from the protein fraction, but in clone UEA-3 35S-labelled low molecular weight compounds represented 30-50% of the total radiosulphur in the anterior portion.
Utilization of inorganic sulphate by isolated embryos and guts of M. persicae In principle, the utilization of sulphate in M. persicae may be mediated by the mycetocytesymbionts or microorganisms in the gut. The mycetocyte-symbionts are transmitted to embryos at the blastoderm stage in aphid virginoparae, but insects acquire their gut flora after birth (Buchner, 1965). Therefore, to examine the contribution of these two classes of microorganisms, the capacity of isolated guts and isolated embryos to incorporate [35S]sulphate from sulphate-GRS medium was investigated. No incorporation of [35S]sulphate into isolated guts was detected, whether the gut preparations were incubated in sulphate-GRS for 24 h as whole pieces, fragments or macerates. Isolated embryos did utilize exogenous [35S]sulphate. Incorporation was linear, at a rate of 2-8 nmol (mg protein) -~ h -1 over 4-6 h and continued, at a lower rate, over the subsequent 12 h of the experiDISCUSSION ment (Fig. 2). Radiosulphur was recovered from the Studies on the incorporation of [35S]sulphate by low molecular weight and protein fractions. Over 24h, the incorporation into these fractions was Neomyzus circumflexus (Ehrhardt, 1968) and M. (this study) indicate that the 17.6+ 1.6 and 12.3 + 0 . 4 n m o l (mg protein) -l, re- persieae mycetocyte--symbionts are capable of reductive asspectively. These results are consistent with the hypothesis similation of inorganic sulphate, with the net synthat the assimilation of inorganic sulphate by M. thesis of cysteine and methionine, and that products persieae is mediated exclusively by the of sulphate assimilation are made available to the mycetocyte-symbionts. The absence of sulphate re- aphid tissues. The mycetocyte-symbionts are production by isolated guts supports the results of posed to be exclusively responsible for sulphate asstructural studies (Douglas, 1988), in which no micro- similation by M. persicae because no microorganisms have been identified in the gut, fat body or other organisms were observed in the gut of M. persicae. organs of the clones used in this study (Douglas, Transport of 3SSfrom the mycetocyte-symbionts to the 1988) and isolated guts of M. persicae incorporate no aphid detectable [35S]sulphate. However, studies of isolated To investigate whether the 35S-products of dietary mycetocytes and symbionts are required to demonsulphate incorporation are transported from the strate unequivocally that the mycetocyte-symbionts mycetocyte-symbionts to the aphid tissues, advan- synthesize methionine and cysteine from inorganic tage was taken of the location of mycetocytes in the sulphate.
604
A.E. DOUGLAS
At first sight, these metabolic data are difficult to reconcile with the phagostimulatory properties of methionine for M. persicae and various published studies which indicate that methionine is a dietary essential. For example, in the classic study of Dadd and Krieger (1968), few individuals reached adulthood and no second-generation larvae were produced on diet with sulphate as sole sulphur source. However, some variation in the performance of different clones on sulphate diets is apparent; in this study, clone UEA-3 persisted for two to three generations and JII-2 for at least four generations on sulphate diet. Differences in formulation of diets and culture conditions and variation between clones in the rate of endogenous synthesis of sulphur amino acids and gustatory response to dietary methionine may all contribute to these discrepancies. In more general terms, one may argue that, although the mycetocyte-symbionts can utilize inorganic sulphate, the reduced sulphur products made available to the aphid are often inadequate to support the insect's requirements over long periods. For this reason, it is of selective advantage for M. persicae to respond to dietary methionine by feeding at an increased rate. Virtually nothing is known of the mechanism by which nutrients synthesized by mycetocytesymbionts are made available to the aphid partner, although the finding that up to 20% of incorporated radiosulphur is recovered from the anterior tissues of the aphids over 3 days suggests that the release of sulphate reduction products is substantial. Unfortunately, the absolute amount of sulphur transported cannot be calculated from these data because the specific activity of 35S in the mobile products is unknown. The location of [35S]sulphate in the anterior tissues of the aphid remains to be determined, but one may speculate that it is preferentially incorporated into the salivary glands. Microbial symbionts of other associations, especially the rhizobium-legume symbioses and alga-invertebrate symbioses, display substantial release of nutrients, the characteristics of which have several common features (Smith and Douglas, 1987). Firstly, the nutrients are released from living cells of the microorganisms; and secondly, release is selective, involving one or a few low molecular weight products (e.g. glucose, ammonia) that are not abundant intracellular products of the microorganism. If these generalities apply to the aphid symbiosis, then one may hypothesize that the symhionts selectively release methionine, the sulphur of which may be derived from the assimilation of inorganic sulphate. It is proposed that methionine is not metabolized in the mycetocyte cytoplasm, beyond that required for the mycetocyte's own requirements, e.g. for protein, and that no other sulphur compound, including cysteine, is released from the symbionts. Dietary studies suggest that M. persieae possesses the trans-sulphuration pathway of cysteine synthesis from methionine. [Both untreated and chlortetracycline-treated M. persieae thrive on holidic diets with methionine as the sole sulphur amino acid (Mittler, 1971; Douglas, this study)]. However, both Aphis fabae and Aphis gossypii have been reported to lack the capacity to synthesize cysteine from methionine (Leckstein and Llewellyn, 1973; Turner, 1971) and therefore the
interactions between these species and their mycetocyte-symbionts may be different from those proposed for M. persieae. The validity of the proposed scheme can be explored by studies on preparations of freshly isolated symbionts. These preparations are viable and capable of DNA, RNA and protein synthesis (Ishikawa, 1982b). By analogy with other symbiotic microorganisms, one would expect the fundamental metabolic capabilities of these preparations to closely mirror those of symbionts in the intact association (Smith and Douglas, 1987), even though gene expression in the symbionts is substantially altered on isolation (Ishikawa, 1982b). Two specific predictions of this scheme are that symbionts isolated from aphids reared on sulphate diet synthesize methionine from exogenously-applied [35S]sulphate and possess high activities of the key enzymes of sulphate reduction, cysteine synthesis and methionine synthesis. The significance o f sulphate utilization to aphid nutrition Virtually all the data on the utilization of inorganic sulphate by the aphid symbiosis come from studies of aphids on defined diets. It is therefore pertinent to consider the relevance of these studies to aphids on their natural food source, phloem sap. Unfortunately, only fragmentary information is available on phloem transport of sulphur compounds. Chemical analyses indicate that phosphate, and not sulphate, is the major inorganic anion (Ziegler, 1975); the concentration of inorganic sulphate is 0.5-1 meq 1-1. This had led to the suggestion that sulphate plays only a minor role in phloem transport of sulphur (Ziegler, 1975; Raven, 1983). In contrast, studies of phloem transport of sulphur from leaves treated with [3SS]sulphate or [35S]sulphite indicate that at least half of the radiosuiphur is translocated as sulphate. The remainder of 35S is in the form of reduced organic sulphur compounds, predominantly glutathionine (glutamylcysteinylglycine), with free cysteine and methionine as minor components (Garsed and Read, 1977; Rennenberg et al., 1979; Bonas et al., 1982). In these radiotracer experiments, the specific activity, and hence absolute quantity, of radiolabelled sulphur compounds was not determined, but the implication, namely that the levels of free cysteine and methionine in phloem are low, is supported by direct analyses of phloem sap (Ziegler, 1975). These considerations indicate that the capacity of the mycetocyte-symbionts to assimilate inorganic sulphate into sulphur amino acids and to synthesize methionine de novo may make an important contribution to the nutrition of aphids feeding on plants. •4cknowledgements--I thank Mr P. Kenworthy and Dr N. Shaw for their advice and practical suggestions on the study of 35S utilization, Mrs J. Geeson who helped with the preparation of diets, and Mr G. Cleveland who cheerfully resolved many technical problems. I am grateful to Professor A. F. G. Dixon, Dr P. G. Markham and Dr J. B. Searle for helpful discussions and to Dr J. Hardie, who persuaded me of the potential of isolated embryos for metabolic studies. This project was funded by grants from the Science and Engineering Research Council and the Royal Society of London.
Sulphate and aphids REFERENCES
Bonas U., Schmitz K., Rennenberg H. and Bergmann L. (1982) Phloem transport of sulfur in Ricinus. Planta 155, 82-88. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye study. Analyt. Biochem. 72, 248-254. Buchner P. (1965) Endosymbiosis of Animals with Plant Microorganisms. Interscience, New York. Dadd R. H. (1985) Nutrition: organisms. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 4 (Edited by Kerkut G. A. and Gilbert L. I.), pp. 313-391. Pergamon Press, Oxford. Dadd R. H. and Krieger D. L. (1968) Dietary amino acid requirements of the aphid Myzus persicae. J. Insect Physiol. 14, 741-764. De Reizis F. A. and Schechtman A. (1973) Staining by neutral red and trypan blue in sequence for assaying vital and non-vital cultured cells. Stain Technol. 48, 135-136. Dixon A. F. G. (1975) Aphids and translocation. Encyclopedia of Plant Physiology New Series, Vol. I. (Edited by Zimmerman M. H. and Milburn J. A.), pp. 154-170. Springer, Berlin. Douglas A. E. (1988) On the source of sterols for the green peach aphid, Myzus persicae, on bolidic diets. J. Insect Physiol. 34, 403-408. Ehrhardt P. (1968) Einfluss yon Ernahrungsfaktoren auf die Entwicklung yon Safte saugenden Insekten unter besonderer Beruchsichtung yon Symbionten. Z. Parasitenkd. 31, 38-66. Garsed S. G. and Read D. J. (1977) Sulphur dioxide metabolism in soy-bean Glycine max vat. Biloxi. II. Biochemical distribution of 35SO2 products. New Phytol. 74, 583-592. Grace T. C. C. (1962) Establishment of four strains of cells from insect tissues grown in vitro. Nature 195, 788-789. Hardie J. (1987) Juvenile hormone stimulation of ooeyte development and embryogenesis in the parthenogenetic ovaries of an aphid, Aphis fabae. Int. J. Invert. Reprod. Dev. 11, 189-203. Houk E. J. and Gritfiths G. W. (1980) Intracellular symbiotes of the Homoptera. A. Rev. Entl. 25, 161-187. Ishikawa H. (1982a) Host-symbiont interactions in the protein synthesis in the pea aphid Acyrthosiphon pisum. Insect Biochem. 12, 613-622. Ishikawa H. (1982b) DNA, RNA and protein synthesis in the isolated symbionts from the pea aphid, Acyrthosiphon pisum. Insect Biochem. 12, 605-612. lshikawa H. (1984) Molecular aspects of intracellular symbiosis in the aphid mycetocyte. Zool. Sci. 1, 509-522. Kunkel H. (1976) Membrane feeding systems in aphid research. In Aphids as Virus Vectors (Edited by Harris
605
K. F. and Maramorosch K.), pp. 311-337. Academic Press, New York. Leckstein P. M. and Llewellyn M. (1973) Effect of dietary amino acids on size and alary polymorphism of Aphis fabae. J. Insect Physiol. 19, 973-980. Markham P. G. (1982) Insect vectors. In Plant and Insect Mycoplasma Techniques (Edited by Daniels M. J. and Markham P. G.), pp. 307-326. Croom Helm, London. Massonie G. (1974) Elevage d'un biotype de Myzus persicae Sulzer sur milieu synthetique. III. Valeur alimentaire respective de la l-methionine, de la 1-cysteine et des sulfates. Annls Nutr. Aliment. 29, 45-50. Mittler T. E. (1967) Effect of aphid feeding of dietary methionine. Nature 214, 386. Mittler T. E. (1971) Dietary amino acid requirements of the aphid Myzus persicae affected by antibiotic uptake. J. Nutr. 101, 1023-1038. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Edited by Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press, New York. Randerath K. (1963) Thin-Layer Chromatography. Academic Press, New York. Raven J. A. (1983) Phytophages of xylem and phloem: a comparison of animal and plant sap-feeders. Adv. ecol. Res. 13, 136-234. Rennenberg H., Schmitz K. and Bergmann L. (1979) Longdistance transport of sulfur in Nicotinia tabacum. Planta 147, 57-62. Renobales M. de, Cripps C., Stanley-Samuelson D. W., Jurenka R. A. and Blomquist G. J. (1987) Biosynthesis of linoleic acid in insects. Trends Biochem. Sci. 12, 364-366. Retnakaran A. and Beck S. D. (1967) Amino acid requirements and sulphur amino acid metabolism in the pea aphid, Acyrthosiphon pisum (Harris). Comp. Biochem. Physiol. 24, 611-619. Schiff J. A. (1980) Pathways of assimilatory sulphate reduction in plants and microorganisms. In Sulphur in Biology, Ciba Foundation Vol. 72, pp. 49-64. Excerpta Medica, Amsterdam. Smith D. C. and Douglas A. E. (1987) The Biology of Symbiosis. Arnold, London, Turner R. B. (1971) Dietary amino acid requirements of the cotton aphid, Aphis gossypii: the sulphur-containing amino acids. J. Insect Physiol. 17, 2451 2456. Wright J. P., Fisher D. B. and Mittler T. E. (1985) Measurement of aphid feeding rates on artificial diets using 3H-inulin. Ent. exp. Appl. 37, 9-11. Ziegler H. (1975) Nature of transported substances. In Encyclopedia of Plant Physiology New Series, Vol. I. (Edited by Zimmerman M. H. and Milburn J. A.), pp. 59-99. Springer, Berlin.