Journal of Chemical Ecology, Vol. 15, No. 1, 1989

CHEMICAL ASSOCIATION IN SYMBIOSIS Sterol Donors in Planthoppers

B R Y A N K. E Y A , 1 P E T E R T . M . K E N N Y , I S U S A N Y. T A M U R A , I M A Y U M I O H N I S H I , I Y O K O N A Y A , I K O J I N A K A N I S H I , I and MIYOJI SUGIURA 2 Suntory Institute for Bioorganic Research Shimamoto, Mishima, Osaka 618 Japan 'National Institute of Agrobiological Resources Yatabe, Tsukuba, Ibaraki 305 Japan (Received September 14, 1987; accepted December 14, 1987) Abstract--The role of intracetlular symbionts contributing to their host has been investigated in the planthoppers, Nilaparvata lugens Stal and Laodelphax striatellus Fallen. We have found that the isolated yeastlike symbionts, identified as a member of the genus Candida, from the host's egg produce ergosterol when cultured. A comparative study of sterols in the cultured symbionts, the host insects, aposymbiotic host insects, and dietary plants demonstrated that ergosterol produced in the symbiotes is provided to the host insects and possibly transformed in the host insects into cholesterol via 24methylenecholesterol. The conversion of injected 24-methylenecholesterold3 into cholesterol has been shown in the brown planthopper (N. lugens). Key Words--Biotransformation, cholesterol, 24-methylenecholesterol, ergosterol, brown planthopper, Nilaparvata lugens Stal, smaller brown planthopper, Laodelphax striatellus Fallen, Homoptera, Fulgoridae, symbiosis, intracellular symbiont.

INTRODUCTION Most arthropoda, such as insects and crustaceans, require cholesterol, since they are incapable o f steroid synthesis de n o v o (Clayton, 1964) and utilize dietary steroids as the source o f m e m b r a n e constituent as well as the p r e c u r s o r o f 2 0 - h y d r o x y e c d y s o n e ( S v o b o d a et al., 1975), the c o m m o n m o l t i n g h o r m o n e . In contrast with m o s t o t h e r insects, plant-sucking h o m o p t e r a n s , such as planthoppers, aphids, and leafhoppers, can be reared on a c h e m i c a l l y defined artificial 373 0098-033118910100-0373506.0010 © 1989 Plenum Publishing Corporation

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diet free of sterols (Noda and Mittler, 1983), an observation suggesting the nutritional role of symbionts harbored (Nasu, 1965) in these insects. However, this is disputed in a recent study on the aphid Schizaphis graminum (Campbell and Nes, 1983). The intracellular symbiotic microorganisms contained within the mycetocytes or fat bodies of aphids, leafhoppers, and rice planthoppers are transmitted into the eggs to the next generation via transovarial infection (Noda and Mittler, 1983). The success in isolating yeastlike symbionts ofL. striatellus and N. lugens and maintaining them in permanent culture (Kusumi et al., 1979, 1980; Nasu et al., 1981) has made it feasible to study the biosynthetic capabilities of these symbionts in the culture medium. Preliminary studies with several symbiotic cultures have demonstrated that symbionts play a dual role (Fredenhagen et al., 1987a), i.e., provision of sterols to the host insect and production of antibiotics for the self-defense of hosts (Fredenhagen et al., 1987a,b). We describe here qualitative and quantitative analysis of the steroids in the cultured symbionts, the host insects, the aposymbiotic host insects, and the dietary plants. The results indicate that the yeastlike symbionts produce ergosterol and provide it to the host as the possible source of 24-methylenecholesterol and cholesterol. This hypothesis was supported by the tranformation (35%) of injected 24-methylenecholesterol-d3 into cholesterol in the brown planthopper (N. lugens).

METHODS AND MATERIALS

bisects and Host Plants. The brown planthopper (rice BPH), N. lugens, and the smaller brown planthopper, L. striatellus, were obtained from the International Rice Research Institute in the Philippines, while the leersia BPH was collected in Indonesia. The insects were reared in plastic cages with their host plants, Oriza sativa L. Koshihikari for rice BPH and Leersia hexandra for leersia BPH, the plants being grown in potted soil, 5-10 cm high. The cages containing insects and host plants were kept in a greenhouse at 25-28°C, with a 16-hr light and 8-hr dark photoperiod. For a comparative study, both sexes of third- to fourth-instar nymphs, adults, and aposymbiotic insects were used; the third-instar nymphs and adults were maintained at 35°C for three days and then transferred to room temperature, 25°C, and allowed to feed on rice plant for seven days. These aposymbiotic insects have greatly reduced numbers of symbionts and fail in ecdysis. However, dietary administration of cholesterol rather than sitosterol partly overcomes this problem (Noda and Saito, 1979). The leaves and stems of the dietary plants, Koshihikari, and L. hexandra were extracted and analyzed, respectively, as described below. Symbionts. The morphologically and physiologically different yeastlike symbionts were isolated from the eggs (Kusumi et al., 1979); i.e., Ls-1 and

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Ls-2 were from L. striatellus and NI-1 and N1-2 were from N. lugens. After pasteurization of the surface, the eggs were homogenized in saline and cultured on Grace's agar plate. The symbionts thus isolated were cultured in 1 liter of sterol-free Burkholder medium for four weeks at 30°C. The work-up of the culture broths was as described for the isolation of the sterols from insects (see below). These yeastlike symbionts were identified as a member of the genus Candida (Amano, unpublishedl). Biotransformation. Brown planthoppers N. lugens, of third- and fourthinstar nymphs were injected (between the midcoxa and metasternum) with a glass microsyringe with deuterium-labeled 24-methylenecholesterol (see below) emulsified in 20% glycerol and 0.2 % Emulgen 913 in water, at a concentration of 2mg/100 ~1 (about 0.001 /~l/insect). The injected insects were transferred to cages with rice plants and sacrificed on day 3 or 7 by chilling in a - 2 0 ° C freezer to see the relative rate of biotransformation. Isolation of Steroids. Using a Tenbrock grinder 130-200 insects were homogenized in 10 ml chloroform-methanol (2: 1) and sonicated for 1 hr. The suspension was centrifuged at 3000 rpm for 5 min, and the organic layer was evaporated to dryness. The residue was then extracted with 40 ml hexane-methanol (1:1) and divided into two fractions. The upper layer, containing free sterols and their esters, was hydrolyzed (90°C, 3 hr) with 0.5 ml 10% methanolic potassium hydroxide in 5 ml water. The lower layer, containing steroidal glycosides, was treated with 5% methanolic hydrogen chloride at 90°C for 3 hr. After work-up of each hydrolysate, the combined free sterol fraction was purified on a silica gel TLC (Kieselgel 60 F254 DC-Fertigplatten Art. 5744 and HPTLC-Fertigplatten Art. 5628, Merck) using ether-hexane (1 : 1) and the Rf region corresponding to sterols was removed and eluted with chloroform-methanol (2 : 1). After evaporation of the solvent, the residue was silylated in the usual manner using pyridine, hexamethyldisilazane, and trimethylchlorosilane (2:1 : 1 v/v). The TMS-derivatized sterols were dissolved in 20 tzl chloroform and 5-/zl aliquots of this solution were analyzed using GC-MS. The TMS-derivatized sterols from the control insects of the same colony reared under identical conditions were used for comparative purposes in the insect biotransformation. Analysis. Sterols were analyzed with a Hitachi M-80 GC-MS spectrometer equipped with a OV-1 packed column (3 mm x 1 m). GC conditions were as follows: oven temperature 175-250°C (2°C/min), He gas flow rate 4.7 ml/sec. The Hitachi mass spectrometer was linked to a model M-003 data processing system. The transformation of [23,25-d3]-24-methylenecholesterol to cholesterol in the insects was measured by intensity enhancement of the M + 3 isotope t Referring to Kreger-vanRij (1984), we identified both yeast strains, Ls-I and NL-2 as a member of the genus Candida; Kreger-van Rij, N.J.W. (ed.), 1984. The Yeasts, A Taxonomic Study. XVI + 1082 pp., Elsevier, Amsterdam.

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peak due to deuterium incorporation. The dispersion of the M + 3 (m/z 461) isotope peak intensity of the standard TMS cholesterol (or recovered TMS cholesterol from insects) at 1 /zg/5 ~1 chloroform was maximally 3.5% in 12 repeated runs of 10-20 scans, the m/z 458 M + peak being adjusted to 100%. Chemicals. Sterol standards and reagents were purchased from Nakarai Chemicals, Ltd. and Tokyo Kasei Kogyo Co,, Ltd. Emulgen 913 (biological detergent) was a gift from Kao Co., Ltd. 0

x

X Y

X

V.

°

2

X.Y =OH

5

X=OH. Y = H

7

X=O

3

X, Y = OTHP

6

X=Y=O

8

X=CHz

4

X = O'[HP. Y = H

°

1

SCHEME 1. Synthetic scheme of 24-methylenecholesterol-d3.

Synthesis. The deuterium-labeled [23,25-da]-24-methylenecholesterol 1 was synthesized in seven steps from 3fl-hydroxy-AS-cholenic acid 2 (Scheme 1). The THP-ester 3 (2 and 3 equiv, of dihydropyran in benzene with pTSOH at room temperature under N2 for 18 hr, 76%) was transformed into the corresponding aldehyde 4 : 3 equiv, of diisobutylaluminium hydride in 1 M hexane solution in toluene at - 7 8 ° C for 15 min under N2 (Zakharkin and Khorlina, 1962), quenched with 50% aq. MeOH at - 7 8 ° C for 30 min and chromatography on alumina, hexane-ether, 85:15, 73%. Grignard coupling of 4 with isopropyl magnesium iodide gave 5 (3 equiv, in diethylether at 25°C for 1 hr followed by usual treatment), which was oxidized to ketosteroid 6 (pyridinium chlorochromate in dichloromethane under Nz at 25°C for 30 min). The ketosteroid 6 was transformed into the desired product 1, overall 80%, by the following sequence: (1) Deuteration of ketosteroid (41.5 mg of 6 in 4 ml methanol-d4, 0.5 mg of deuterium oxide and 3 mg of anhydrous sodium carbonate at 70°C for 20 min, evaporated to dryness and the cycle repeated four times; Nolin and Jones, 1952); (2) Wittig coupling to give the 24-methylene steroid 8 (deuterated ketosteroid 7 with 2 equiv, of triphenylmethylphosphorane in ether under N2 at 25°C for 15 min; Newman, 1960); (3) hydrolysis (1 N HC1 in methanol for 1 hr at 25°C, neutralization with sat. aq. sodium bicarbonate, extraction and flash chromatography on Kieselgel 60 Art 9385, hexaneethyl acetate). The synthetic product 1 was identified by the NMR spectrum recorded on a Nicolet NT-360 spectrometer, 6(CDC13): 5.35 (1H, m, 6-H), 4.71 and 4.66 (2H, dd, J = 2, 28 Hz, 24-H), 3.48-3.58 (1H, m, 3-H); m/z

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401, C28H43D30: (M+), (26%); 386(M-CH3) +, (39%); 314 (M-C6H9D3) +, (100%). The percentages of d3 (m/z 401), d2 (m/z 400), and dj (m/z 399) of the deuterated 24-methylenecholesterol after subtraction of natural abundance 13C isotope were 54.0, 42.6, and 5.0% respectively.

RESULTS AND DISCUSSION

Sterols from Cultured Intracellular Symbionts. Ergosterol was the only sterol found in the culture broth (I liter) of Ls-1 and Ls-2. Mixed cultures of Ls-1 and Ls-2 or addition of mevalonic acid (5 mg/1) did not change the products. No sterols could be detected, however, from the cultured medium of symbionts NI-1 and N1-2. Instead, a series of compounds i.e., lanosterol, 24methylenelanosterol, dihydroergosterol, and ergosterol (major), suggestive of an intracellular biosynthetic buildup of ergosterol from mevalonic acid was detected in the cells of NI-1 and N1-2. The cells of Ls-1 and Ls-2 also contained the same set of compounds, although in much smaller quantities. The above result indicates that ergosterol was found in intact N. lugens but only a trace amount in L. striatellus (see below). Detection of 24-methylenecholesterol in L. striatellus and N. lugens may demonstrate differences in the mode in which the symbiont-synthesized sterol was made available to the host, an observation in agreement with the hypothesis that different symbionts fulfill different physiological roles (Griffiths and Beck, 1975; Hinde, 1971). Sterols in Host Insects. In both rice brown planthopper (BPH) (N. lugens) and leersia BPH, the sterols consisted of cholesterol, 24-methylenecholesterol, and ergosterol (Figure 1). It is noted that the relative content of 24-methylenecholesterol is greater in adults than in nymphs. In both adult and nymph of aposymbiotic insects seven days after heat treatment, the amounts of ergosterol and 24-methylenecholesterol relative to cholesterol, as well as the total amount of sterols, were reduced drastically. On the other hand, in the smaller brown planthopper, L. striatellus, a small amount of sitosterol was found. This result is in agreement with a previous report (Noda and Saito, 1979). The ratio of nonconverted dietary steroids to insect steroids was rather small. Approximately 4-8 mg of each sterol was detectable in each of the isolated samples from 130 insects by the mass chromatographic GC-MS of the TMS derivatives. The body cholesterol content of the apoinsects was reduced to ~o of that of the control, in agreement with the previous studies (Noda and Saito, 1979). Sterols in Host Plants vs. Rice BPH and Leersia BPH. Sterols of the rice plant Koshihikari and L. hexandra showed that the former contained sitosterol, stigmasterol, campesterol and their respective esters, while the latter contained sitosterol, stigmasterol, campesterol, cholesterol, and their repective esters and

378

EYA ET AL. N./ugerts ratio

( rice BPH )

1:2:1

7I

~

1 : 0.25

-- n o r m a l -

-- apo--

1:1.2:1.1 1 : 0.03

-- normal--

ADULT

N. lugens

--apo--

NYMPH

(Leersia BPH)

7 o.o~

ratio

1:0.4 B

- - normal - -

[] [] []

cholesterol 24-methylenechl. ergosterol

-- apo--

ADULT

F~G. 1. Sterol constituents in combined sterol fraction from normal and aposymbiotic N. lugens (rice BPH and leersia BPH); relative contents of sterols are normalized to cholesterol. glycosides. The presence of cholesterol in L. hexandra may account for the higher ratio of cholesterol in leersia BPH (Figure 1). Thus, the possibility that ergosterol originates from the food chain can be ruled out. Biotransformation of 24-Methylenecholesterol-d3 to Cholesterol in BPH. In vivo conversion was demonstrated upon adminstration of labeled compound emulsified in glycerin and Emulgen 913 ® to BPH. The cholesterol-d 3 was found in insects harvested on both days 3 or 7 after injection. On the basis of the injected 24-methylenecholesterol-d3, 54%, the cholesterol-d3 recovered from insects was found to be 13 and 19% upon GC-MS (after subtraction of the +3.5% natural abundance isotope effect, see above), corresponding to conversions of 24 and 35 %, respectively. Dealkylation of the 24-methylenecholesterol to cholesterol in BPH was thus proven, the conversion of which was shown previously in Manduca sexta (Thompson et al., 1973). The high level of this conversion in the insects demonstrates the utilization of 24-methylenecholesterol as the main source of cholesterol. Although the transformation of ergos-

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379

terol to 24-methylenecholesteroI and sitosterol to cholesterol has as yet not been proven quantitatively, comparative study among insects may indicate 24-methylenecholesterol is accumulated after transformation. The conspicuous decrease of 24-methylenecholesterol in the apoinsects may suggest the fast turnover of the ergosterol supplied by the symbionts and not of campesterol from the host plants. Ergosterol is the obligatory nutrient in the ambrosia beetle (Xyleborus ferrugineus), which carries endosymbiotic fungi (Rao et al., 1983). The hypothesis that the sterols are synthesized and supplied, at least partly, by their associated symbionts has been elucidated in the present studies. It has previously been reported that the prokaryotic symbionts in the pea aphids (Neomyzus circumflexlus and Acrythosyphon pisum) synthesize sterols (Ehrhardt, 1968; Houk et al., 1976) in agreement with their possible role as a steroid supplier. However, Campbell et al. (1983) reported that dietary [2~4C]mevalonic acid was not converted to steroids in the aphid but dietary [4HC]sitosterol was converted to cholesterol. Therefore, reinvestigation using isolated symbiont cultures is desirable, because bacteria and mycoplasmal prokaryotes generally lack the ability of steroid synthesis (Nes and Nes, 1980). Acknowledgments--This research was supported in part by Special CoordinationFunds from the Science and Technology Agency of Japanese Government.We thank Dr. Hiroshi Kita, latron Laboratories Inc., for technical information of the symbiote culture; in addition we thank Dr. Norihide Amano, Suntory Ltd., for classificationof the yeastlike symbionts.

REFERENCES CAMPBELL, B.C., and NES, W.D. 1983. A reappraisal of sterol biosynthesisand metabolism in

aphids. J. Insect Physiol. 29:149-156. CLAYTON,R.B. 1964. The utilizationof sterols by insects. J. Lipid Res. 5:3-19. EHRHARDT, P. 1968. Nachweis einer durch symbiontische Microorganismen bewirkten Sterinsynthese in kunstlichern~ihrtenAphiden(Homoptera, Phynchota, Insecta). E.rperientia 24:8283. FREDENHAGEN,A., KENNY,P.T.M., KITA,H., KOMURA,H., NAYA,Y., NAKANISHI,K. NISHIYAMA, K., SUOIURA,M., and TAMURA,S.Y. 1987a. Role of intmcetlular symbiotes in planthoppers, pp. 101-108, in R. Greenhalghand T.R. Roberts (eds.). Pesticide Scienceand Biotechnology;6th IUPAC Congress of Pesticide Chemistry. BlackwellScientificPubl., London. FREDENHAGEN, A., TAMURA, S.Y., KENNY, P.T.M., KOMURA,H., NAYA,Y., NAKANISHI,K., NISHWAMA,K., SUGXURA,M., and KITA,H. 1987b. Andrimid, a new peptide antibioticproduced by an intracellularbacterial symbiontisolated from a brown planthopper.J. Am. Chem. Soc. 109:4409-4411. GRIEFITrtS,G.W., and BECK,S.D. 1975. Ultrastructureof pea aphids mycetocytes: Evidence for symbiote secretion. Cell Tissue Res. 159:351-367. HJNDE, R. 1971. The control of the mycetome symbiotes of the aphids Brevicoo,ne brassicae, Myzus persicae, and Macrosiphum rosae. J. hasect Physiol. 17: 1791-1800.

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HOUK, E.J., GRIFFITHS, G.W., and BECK, S.D. 1976. Lipid metabolism in the symhiotes of the pea aphid, Acyrthosiphon pisum. Comp. Biochem. Physiol. 54B:427-431. KUSOMI,T., SUWA,T., KITA, H., and NASU,S. 1979. Symbiotes of planthoppers: I. The isolation of intracellular symbiotes of the smaller brown planthopper Laodelpha.r striatellus Fallen (Hemiptera: Delphacidae). Appl. Entomol. Zool. 14:459-463. KUSUMI,T., SUWA,T., KITA, H., and NASU, S. 1980. Properties of intracellular symbiotes of the smaller brown planthopper Laodelphax striateUus Fallen (Hemiptera: Delphacidae). Appl. Entomol. Zool. 15:129-134. NASU, S. 1965. Electron microscopic studies on transovarial passage of rice dwarf virus. Jpn. J. Appl. Entomol. Zool. 9:225-237. NAStJ, S., KOSUMI,T., SUwA, T., and KtTA, H. 1981. Symbiotes of planthopper: II. Isolation of intracellular symbiotic microorganism from the brown planthopper, Nilaparvata lugens Stal, and immunological comparison of the symbiotes associated with rice planthopper (Hemiptera: Delphacidae). Appl. Entomol. Zool. 16:88-93. NES, W.R., and NES, W.D. 1980. Lipids in Evolution. Monographs in Lipid Research. Plenum Press, New York. 244 pp. NEWMAN, M.S. (ed.). 1960. Org. Synth. 40:66-68. NODA, H., and MITTLER, T.E. 1983. Sterol biosynthesis by symbiotes of aphids and leafhoppers, pp. 41-55, in T.E. Mittler and R.H. Dadd (eds.). Metabolic Aspects of Lipid Nutrition in Insects. Westview Press, Boulder, Colorado. NODA, H., and SAITO, T. 1979. The role of intracellular yeastlike symbiotes in the development of Laodelphax striatellus (Homoptera: Delphacidae). Appl. Ent. Zool. 14:453-458. NOOA, H., WAOA,K., and SArro, T. 1979. Sterols in Laodelphax striatellus with special reference to the intracellular yeastlike symbiotes as a sterol source. J. Insect Physiol. 25:443-447. NOUN, B., and JONES, R.N. 1952. The preparation of some steroids containing deuterium. Can. J. Chem. 30(10):727. RAO, K.D.P., NORRIS, D.M., and CHu, H.M. 1983. Lipid interdependencies between Xyleborus ambrosia beetles and their ectosymbiotic microbes, pp. 27-40, in T.E. Mittler and R.H. Dadd (eds.). Metabolic Aspects of Lipid Nutrition in Insects. Westview Press, Boulder, Colorado. SVOBODA,J.A., KAPLANIS,J.N., ROBBINS,W.E., and THOMPSON,M.J. 1975. Recent developments in insect steroid metabolism. Annu. Rev. Entomol. 20:205-220. THOMPSON. M.J., KAPLANIS,J.N., ROBBINS,W.E., and SVOBODA,J.A. 1973. Metabolism of steroids in insects. Adv. Lipid Res. 11:219-265. ZAKHARKIN,L.I., and KHORLINA,I.M. 1962. Reduction of esters of carboxylic acids into aldehydes with diisobutylaluminum hydride. Tetrahedron Lett. 14:619.