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Sterols in Laodelphax striatellus with special reference to the intracellular yeastlike symbiotes as a sterol source
0022-1910/79/0501+43
J. Insect Ph_vsiol., Vol. 25, pp. 443 to 447. c’ Pergamon Press Ltd. 1979. Printed in Great Britain.
SO2.00/0
STEROLS IN LAODELPHAX STRIATELLUS WITH SPECIAL REFERENCE TO THE INTRACELLULAR YEASTLIKE SYMBIOTES AS A STEROL SOURCE HIROAKI NODA*, KOJIROWADA? and TETSLJO SAITO Laboratory of Applied Entomology and Nematology. Faculty of Agriculture, Nagoya University, Chikusa-ku. Nagoya 464. Japan (Received 28 September
1978: revised 6 Novemher
1978)
Abstract-Sterols wereanalysed to investigate the sterol source in Laodelphax srriatellus and three other rice plant-sucking homopterous insects. In L. striatellus. cholesterol, 24-methylenecholesterol and /?-sitosterol were detected. The host plant (rice) contained campesterol, stigmasterol and B-sitosterol. From the honeydew excreted by L. striafellus, cholesterol, p-sitosterol and negligible amounts of campesterol were recovered. Laodelphax striatellus possesses yeastlike symbiotes which can be destroyed by high temperature. Fifth instar nymphs, which have been exposed to 35°C for 3 days in their 1st instar, showed lower cholesterol concentration and markedly reduced amounts of 24-methylenecholesterol. From the results it is concluded that L. striatellus has two sterol sources: one from the host plant and the other from the yeastlike symbiotes which appear to provide 24-methylenecholesterol. Ke_v Word Index: Yeastlike symbiotes. honeydew, rice plant, cholesterol, 24-methylenecholesterol. /I-sitosterol. campesterol, stigmasterol
INTRODUCTION
insects, i.e. those from which the symbiotes have been eliminated, are required in order to evaluate the role of IT IS generally acknowledged that insects have no the symbiotes. The yeastlike symbiotes in L. striatellus ability to synthesize sterols and that a dietary supply of can be reduced in number by exposure to high sterol is indispensable for normal growth (CLAYTON, temperature (NODA and SAITO, unpublished 1964). ROBBINSet al. (1971) stated that the only observations). When the newly-hatched 1st instar exceptions are those insects in which a sterol source nymphs are reared at 35°C for 3 days (heat treatment) may be attributed to associated symbiotes. Aphids can and reared under a temperature of 25°C the resultant be raised on a chemically defined artificial diet without 5th instar nymphs possess about 5% of the yeastlike sterols (DADD and MITTLER, 1966; DADD and symbiotes per individual in comparison with normal KRIEGER, 1968; EHRHARDT,1968a; SRIVASTAVA and 5th instar nymphs. The bacteria in L. striatellus are AUCLAIR, 1971), and are considered not to require hardly affected by this treatment. Though many heatsterols owing to their provision by symbiotes (DADD treated insects fail to moult to the adult stage, they and MITTLER, 1966; SRIVASTAVA and AUCLAIR, 1971; usually develop to the 5th instar after some delay. The AKEYand BECK,1972). EHRHARDT(1968b) and HOUK heat-treated insects can be employed in comparative et al. (1976) support this hypothesis. GRIFFITHS and sterol analysis to clarify the possible participation of BECK (1977a, b) also suggest cholesterol biosynthesis the yeastlike symbiotes in sterol metabolism. by the symbiotes of Acyrthosiphon pisum using In the present paper, in order to locate the sterol electron microscopical techniques. source in L. striatellus, the sterols were analysed in the The smaller brown planthopper, Laodelphax insects, their host plant and the excreted honeydew. striatellus (Homoptera: Delphacidae), like aphids, Secondly. sterols in the heat-treated insects were feeds on the plant juice from the vascular bundle. This investigated and the sterols of three other rice plantinsect harbours yeastlike symbiotes in the fat body and sucking species were compared with those of L. the female transmits them to the next generation via the striatellus. The role played by the yeastlike symbiores ovary (NASU. 1963; NODA, 1977). It also possesses in the host sterol requirement is discussed. bacteria in the mycetocytes in the fat body or in the intercellular space between mycetocytes (NODA and SAITO. unpublished observations). MITSUHASHI and MATERIALS AND METHODS KOYAMA (1972) raised L. striateflus successfully on a Animals chemically defined artificial diet not containing sterol. Thus there is the possibility that the symbiotes of this The following insects were used in the present species synthesise sterols to provide for the insect. striate/b (Fall&), Laodelphax experiments: For the precise study of nutrition, aposymbiotic Niluparvata iugens (StBl), Sogatella furcifera (HorvBth) (Delphacidae) and Nephotettix cincticeps (Uhler) (Deltocephalidae). The insects were reared on *Present address: Shimane Agricultural Experiment rice seedlings at 25°C and subjected to a 16L:8D Station. 2440 Ashiwata. Izumo, Shimane 693, Japan. photoperiod. Heat-treated L. striatellus was also ‘r Laboratory of Pesticides Chemistry. 443
HIKOAI(I NOIIA, KOJIRO WADA AND TETSU~ SAITO
444
employed for sterol analysis. Newly-hatched 1st instar nymphs were exposed to 35 C for 3 days, then transferred to the normal temperature, 25 C. and allowed to feed on rice seedlings ( NODA and SAITO. unpublished observations). E.vtrac/ion
of .strrol.r
Fresh insects or insects stored at -80’ C were chloroform-methanol (2: 1) homogenized with (FOLCH et rd., 1957). After the extracts were filtered and partitioned with 0.2 their volume of water; the chloroform phase was dried down in a rotary evaporator. The extracted whole lipids were refluxed in lo”,, methanolic KOH at 75 ‘C for I hr. The nonsaponifiable matter, after being partitioned between ether and water, was developed using thin layer chromatography (TLC. Merck Silica Gel 60F-254) with the solvent system. chloroform-ethyl acetate (20: I v/v). The spots corresponding to the authentic cholesterol were scraped together and the sterols were extracted with ethyl acetate. For extracting sterols from rice plants, the buds of rice seedlings or the leaves at the 2-3 leaf stage were cut and dried at room temperature for several days. The dried samples were refluxed in methanol at 70’ C for 90 mm and the solvent was removed in IYIJCUU.The extracted matter was partitioned in ether and water, and the ether phase dried down. Honeydew was obtained by allowing female adults to feed on the rice plant (S~)GAWA, 1970). with a filter paper placed under the feeding site to absorb the honeydew excreted. The filter papers were refluxed in methanol at 70°C for 90 min and the solvent was evaporated. The extracts were also partitioned in ether and water. and the ether phase was retained. The ether-soluble substances from the rice seedling, 2-3 leaf stage, and honeydew were saponified separately and the sterols purified with TLC as mentioned above.
Fig. I Gaschromatogram of sterols in the I st instar nymph (OV-I ). (A) Cholesterol; (B) 24-methylenecholesterol; (C)Bsitosterol.
Sterols were analysed by gas chromatography (GLC. Jeol Ltd., JGC-20K) equipped with a FID detector and I m glass column. Two column packings were employed; 3”,, OV-1 (Gaschrome Q, 100-l 20 mesh) at 240’ C and 1.5”,, OV-17 (Shimalite, W-100 mesh) at 260°C. The identification of sterols was
achieved by comparing the retention times to those of authentic samples in two systems. The authentic samples of campesterol (24a-methylcholest-5-en-3jI01) and 24-methylenecholesterol (24-methylcholesta5. 24 (28)-dien-3&ol) were provided by Dr. Ikekawa (Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology). Cholesterol, stigmasterol (24cr-ethylcholesta-5, 22-dien-3fi-ol) and /I-sitosterol (24a-ethylcholest-5-en-3jl-ol) were obtained from Katayama Chemical Co., Ltd. Quantitative analysis by GLC was carried out based on the standard line of the authentic cholesterol in the same condition. The sterols were also analysed by a gas chromatograph-mass spectrometer system (GC-MS, Jeol Ltd.. Model JMS-DIOO) using 30;) OV-1 (1 m column) at 260°C.
100
RESULTS Sterols
in L. striatellus
Sterols were analysed in the following stages: 1st instar; 5th instar nymphs (with a few 4th instar); adult 314
r
HOJYF-+ ’
271
299 229 213
llilhl
200
I
281 11lu
~hh~L
300
398(M+)
111 IIIk
m/e Fig. 2. Mass spectrum
of 24-methylenecholesterol
in the 1st instar nymph.
II I
I 400
Sterols in L. slriatellus Table
1. Sterol concentration Cholesterol
445
of L. striarellus
(A)
in each stage
24-Methylenecholesterol
(B)
Stages*
(pg/g wt)
(flgig wt)
B/A
1st instar nymph 5th instar nymph Adult male aged under 5 days Adult female aged under 5 days Adult male aged over 7 days
9.8 13.7 32.6 50.7 39.0
6.2 8.7 20.4 33.7 17.9
0.64 0.64 0.63 0:66 0.46
* Each reading
represents
only one batch of animals.
males and females under 5 days old; and adult males over 7 days old. In all samples, two major and one minor sterol were detected by GLC (Fig. 1). The two major sterols were identified as cholesterol and 24methylenecholesterol, and the minor sterol was psitosterol. The sterols were also identified by GC-MS. The mass spectra of cholesterol and /?-sitosterol gave the molecular ion peak at m/e 386 and m/e 414 respectively, and were identical to those of the authentic samples. The mass spectrum of 24methylenecholesterol showed the molecular ion peak at m/e 398 (M+) and the base peak at m/e 314 (M+--C,H,,) (Fig. 2). The concentrations of cholesterol and 24methylenecholesterol in each sample measured by means of GLC are shown in Table 1. The concentrations of cholesterol and 24-methylenecholesterol increased with the growth of the insect. The ratio of 24-methylenecholesterol to cholesterol was generally the same. ranging from 0.63 to 0.66, except in males more than 7 days old. Sterols in rice plant
In the samples of rice seedling and 2-3 leaf rice plants, three sterols were detected: campesterol, stigmasterol and jY-sitosterol (Fig. 3). The mass spectra of these gave molecular ion peaks at m/e 400,412 and 414 respectively and were identical to those of each authentic sterol.
feeding on rice plants. cholesterol and p-sitosterol were found. When a large amount of the sample was analysed, there was a small peak between the peaks for cholesterol and /3-sitosterol (Fig. 4). This was identified as campesterol from the retention time using the column packing OV-17: OV-17 showed a small difference in retention times between campesterol and 24-methylenecholesterol. Therefore, three sterols were detected in honeydew: cholesterol, /%sitosterol and a minute amount of campesterol. L. striatellus Heat-treated 5th instar nymphs (with a small number of 4th instar nymphs, about,13-14 days after hatching) were analysed. The heat-treated nymphs contained cholesterol, /?-sitosterol and a small amount of 24-methylenecholesterol (Fig. 5). The concentrations of cholesterol were 1.2 and 1.3 pg/g in two replicates. The amount of cholesterol in the heattreated insect was greatly reduced by comparison with that of the normal 5th instar nymph (13.7 pg/g). Though the gas chromatograph of the heat-treated insects (Fig. 5) showed that the amount of /?-sitosterol was relatively larger than that in the normal 5th instar nymphs (Fig. 6). there were no marked differences in the concentration of p-sitosterol because in the former Sterols in heat-treated
Sterols in honeydew
In the honeydew excreted by the female adults
:
I 5
IO I-Ill"
Fig. 3. Gas chromatogram of sterols in the rice seedling (OVI ). (C) p-Sitosterol: (D) campesterol: (E) stigmasterol.
I IO
5 m,n
Fig. 4. Gas chromatogrdm of sterols in honeydew (OV(A) Cholesterol; (C) p-sitosterol; (D) campesterol.
17).
HIROAKI NODA.KOJIRO WADAANDTETSUO SAITO
446
I, A
min
6. Gas chromatogram of sterols in the normal 5th instar nymph (OV-I). (A) Cholesterol: (B) 24methylenecholes-
Fig. 5. Gas chromatogram of sterols in the heat-treated 5th
instar
nymph (OV-I). (A) Cholesterol; (B) methylenecholesterol; (C) fi-sitosterol.
Fig.
24-
terol; (C) /Lsitosterol.
an essential nutrient for almost all insects. In Manduca sexta, Tribolium confusum (SVOBODAet al.. 1975) and Bombxy mori (MORISAKIet al., 1972). the conversion
a large amount of material was injected into the GLC owing to the low concentration of cholesterol. Sterols in other rice plant-sucking species
of phytosterols into cholesterol has been demonstrated. Campesterol. stigmasterol and bsitosterol can be detected in the rice plant, but in the honeydew of L. striatellus /I-sitosterol and a negligible amount of campesterol is recovered. As this planthopper imbibes the plant juice from the vascular bundles excessively and excretes a large amount of honeydew, phytosterols in the honeydew almost certainly come from the sap in the vascular bundles. Cholesterol is synthesized within the insect. Stigmasterol, which is found in the whole rice plant, does not seem to be in the vascular bundles. L. striatellus can utilize mainly fi-sitosterol from the rice plant as a cholesterol source. 24-Methylenecholesterol is found in L. striatellus. and is one of the major sterols in some species of algae (PATTERSON,1971). This sterol is found in the honey bee, Apis mellifca (BARBIER et al., 1959) and is considered to be a constituent of pollens of various flowers which the honey bee visits (BARBIERet al., 1960). 24-Methylenecholesterol is also identified as an intermediate in the conversion of campesterol into cholesterol in Manduca scvtu (SWBODA et cd..1972). In L. striatellus 24-methylenecholesterol can be detected in abundance in all stages examined. The heat-treated insects, which have a smaller number of the yeastlike
Sterols in three other rice plant-sucking species, N. lugens, S. furcifera and N. cincticeps, were analysed by GLC. These species drink the sap in the vascular bundle as well as L. striatellus. N. lugens and S. furcifira possess yeastlike symbiotes in their fat body (NASU and SUENAGA, 1958; NODA, unpublished observations), whereas N. cincticeps harbours prokaryotic symbiotes in the mycetomes (NASU, 1965; MITSUHASHIand KONO, 1975). Sterols were extracted from the 4th and 5th instar nymphs in these species. In N. lugens and S. furcifera. three sterols, cholesterol, 24-methylenecholesterol and /I-sitosterol, were detected. On the other hand, 24-methylenecholesterol was not recovered in N. cincticeps. This leafhopper had cholesterol, /I-sitosterol and a small amount of campesterol.
DISCUSSION Insects are considered to lack a sterol biosynthetic mechanism (ROBBINS et al., 1971). The insect. therefore. requires a dietary or additional source of sterol. Phytophagous insects generally ingest phytosterols to convert them into cholesterol which is Rice plant _
Vascular bundle
Laodelphax 7
strlatellus
Alimentary
Honeydew
canal -
+ Sitosterol
* Wosterol
Cholesterol Cholesterol Campesterol
I
Stigmasterol
I-ig. 7. Schematic
sterol
flow
in L. .vt~Yrrre//u.\
Sterols in L. striatellus
symbiotes, have little 24-methylenecholesterol. This strongly suggests that this sterol originates from the symbiotes. The reduction in sterol supply from the yeastlike symbiotes in the heat-treated insects appears to account for the lower concentration of cholesterol. The ratio of the two sterols in the adult male more than 7 days old is lower than that in the other stages. This could be explained by the fact that the number of yeastlike symbiotes in male adults decreases after emergence (NODA, 1974, 1977). Niluparvuta lugens, S. furctfera and N. cincticeps are all vascular feeders. The first two species which possess the yeastlike symbiotes 24-methylenecholesterol. However, 24contain methylenecholesterol is not present in N. cincticeps, which does not harbour the same symbiotes. It is, therefore, concluded that L. striatellus obtains the sterol from its yeastlike symbiotes and has two sterol sources: rice plant sap and the symbiotes. Figure 7 represents diagrammatically the sterol flow between the rice plant. L. striatellus and the honeydew. Acknowledgements-The authors are grateful to Dr. S. National Institute of Agricultural Science. for helpful advice. to Dr. N. IKEKAWA of Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology, for provision of sterol samples, and to Dr. M. A. BRINKS of Department of Entomology, Fisheries and Wildlife, University of Minnesota. for reviewing this manuscript. NASU of
REFERENCES AKEY D. H. and BECK S. D. (1972) Nutrition ofthe pea aphid. Acyrthosiphon pisum: requirements for trace metals. sulphur and cholesterol. J. Insecr Physiof. 18, 1901-1914. BARBIER M.. REICHSTEN T., SCHINDLER 0. and LEDERER E. (1959) Isolation of 24-methylenecholesterol from honey bees (Apis mellifica L). Nature, Lond. 184, 732-733. BARBIERM., H~~GELM. F. and LEDERER E. (1960) lsolement du 24-mtthyltnecholesterol a partir du pollen du differentes plantes. Bull. Sot. Chim. biol. 42, 91-97. CLAYTON R. B. (1964) The utilization of sterols by insects. J. Lipid Res. 5, 3-19. DADD R. H. and KRIEGER D. L. (1968) Dietary amino acid requirements of the aphid, Mv-_us persicae. J. Insect Physiol. 14, 741-764. DADD R. H. and MITTLER T. E. (1966) Permanent culture of an aphid on a totally synthetic diet. Experientia 22, 832-835. EHRHARDT P. (1968a) Die Wirkung verschiedener Spurenelemente auf Wachstum, Reproduktion und Symbionten von Neomyzus circumflexus Buckt. (Aphidae. Homoptera. lnsecta) bei kiinstlicher Ernahrung. Z. vergl. Physiol. 58, 47-75. EHRHARDT P. (1968b) Nachweis einer durch symbiontische Microorganismen bewirkten Sterinsynthese in kiinstlich Aphiden. (Homoptera, Rhynchota, Insecta). Experientia 24, 82-83. FOLCH J. M. S., LEES M. and STANLEY G. H. S. (1957) A
447
simple method for the isolation and purification of total lipides from animal tissues. J. biol. Chem. 226, 497-509. GRIFFITHS G. W. and BECK S. D. (1977a) In viva sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography. CL,// Tiss. Res. 176, 179-190. GRIFFITHS G. W. and BECK S. D. (1977b) Effect of dietary cholesterol on the pattern of osmium deposition in the symbiote-containing cells of the pea aphid. Cell 7%~. Res. 176, 191-203. HOUK E. J., GRIFFITHS G. W. and BECK S. D. (1976) Lipid metabolism in the symbiotes of the pea aphid, Acyrthosiphon pisum. Comp. Biochem. Physiol. 54B, 427-43 1. MITSUHASHI J. and KONO Y. (1975) Intracellular microorganisms in the green rice leafhopper, Nephotettix cincficeps Uhler (Hemiptera: Deltocephalidae). Appl. En/. Zool. 10, l-9. MITSUHASHIJ. and KOYAMAK. (I 972) Artificial rearing of the smaller brown planthopper. Laodelphax striatellus Fallen. with special reference to rearing conditions for the first instar nymphs. Jap. J. appt. Em. Zool. 16. 8-17. (In Japanese with English summary). MORISAKI M.. OHTAKA H., OKABAYASHIM. and IKEKAWA N. (1972) Fucosterol-24,2&epoxide. as a probable intermediate in the conversion of/I-sitosterol to cholesterol in the silkworm. J. them. Sot. Chem. Comm. 1275-1276. NASU S. (1963) Studies on some leafhoppers and planthoppers which transmit virus disease of rice plant in Japan. Bull. Kyushu agr. expr. Sm 8, 153-349. (In Japenese with English summary). NASU S. (1965) Electron microscopic studies on transovarial passage of rice dwarf virus. Jup. J. appl. Em. Zoo/. 9, 225-237. NASU S. and SUENAGA H. (1958) On the embryonic development of planthoppers. Bull. K,vushu agr. e.xpt. S/n 5, 71-84. (In Japanese with English summary). NODA H. (1974) Preliminary histological observation and population dynamics of intracellular yeast-like symbiotes in the smaller brown planthopper. Laodelphax striate//as (Homoptera: Delphacidae). Appl. Em. Zool. 9. 275-277. NODA H. (1977) Histological and histochemical observations of intracellular yeastlike symbiotes in the fat body of the smaller brown planthopper, Laode1pha.x striatellus (Homoptera: Delphacidae). Appl. Em. Zoo/. 12, 134-141. PATTERSONG. W. (1971) The distribution of sterols in algae. Lipids 6, 120-127. ROBBINS W. E.. KAPLANIS J. N.. SVOBODA J. A. and THOMPSONM. J. (1971) Steroid metabolism in insects. ~lnn. Rev. Em. 16, 53-72. SAGAWA K. (1970) Studies on the feeding habits of the brown planthopper~~ll. Honeydew excretion. Jap. J. appl. Ent. Zoo/. 14, 134-139. (In Japenese with English summary). SRIVASTAVAP. N. and AUCLAIR J. L. (1971) An improved chemically defined diet for the pea aphid, Acvrrhosiphon . pisum. Ann. enl. Sot. Am. 64, 4?4-4?8. SVOBO~A J. A.. KAPLANIS J. N.. ROBBINS W. E. and THOMPSON M. J. (1975) Recent developments in insect steroid metabolism. Ann. Rev. Em. 20, 205-220. SVOBODAJ. A.. THOMPSON M. J. and ROBBINS W. E. (1972) 24-Methylenecholesterol: isolation and identification as an intermediate in the conversion of campesterol to cholesterol in the tobacco hornworm. Lipids 7, 156-158.
J. Insect Ph_vsiol., Vol. 25, pp. 443 to 447. c’ Pergamon Press Ltd. 1979. Printed in Great Britain.
SO2.00/0
STEROLS IN LAODELPHAX STRIATELLUS WITH SPECIAL REFERENCE TO THE INTRACELLULAR YEASTLIKE SYMBIOTES AS A STEROL SOURCE HIROAKI NODA*, KOJIROWADA? and TETSLJO SAITO Laboratory of Applied Entomology and Nematology. Faculty of Agriculture, Nagoya University, Chikusa-ku. Nagoya 464. Japan (Received 28 September
1978: revised 6 Novemher
1978)
Abstract-Sterols wereanalysed to investigate the sterol source in Laodelphax srriatellus and three other rice plant-sucking homopterous insects. In L. striatellus. cholesterol, 24-methylenecholesterol and /?-sitosterol were detected. The host plant (rice) contained campesterol, stigmasterol and B-sitosterol. From the honeydew excreted by L. striafellus, cholesterol, p-sitosterol and negligible amounts of campesterol were recovered. Laodelphax striatellus possesses yeastlike symbiotes which can be destroyed by high temperature. Fifth instar nymphs, which have been exposed to 35°C for 3 days in their 1st instar, showed lower cholesterol concentration and markedly reduced amounts of 24-methylenecholesterol. From the results it is concluded that L. striatellus has two sterol sources: one from the host plant and the other from the yeastlike symbiotes which appear to provide 24-methylenecholesterol. Ke_v Word Index: Yeastlike symbiotes. honeydew, rice plant, cholesterol, 24-methylenecholesterol. /I-sitosterol. campesterol, stigmasterol
INTRODUCTION
insects, i.e. those from which the symbiotes have been eliminated, are required in order to evaluate the role of IT IS generally acknowledged that insects have no the symbiotes. The yeastlike symbiotes in L. striatellus ability to synthesize sterols and that a dietary supply of can be reduced in number by exposure to high sterol is indispensable for normal growth (CLAYTON, temperature (NODA and SAITO, unpublished 1964). ROBBINSet al. (1971) stated that the only observations). When the newly-hatched 1st instar exceptions are those insects in which a sterol source nymphs are reared at 35°C for 3 days (heat treatment) may be attributed to associated symbiotes. Aphids can and reared under a temperature of 25°C the resultant be raised on a chemically defined artificial diet without 5th instar nymphs possess about 5% of the yeastlike sterols (DADD and MITTLER, 1966; DADD and symbiotes per individual in comparison with normal KRIEGER, 1968; EHRHARDT,1968a; SRIVASTAVA and 5th instar nymphs. The bacteria in L. striatellus are AUCLAIR, 1971), and are considered not to require hardly affected by this treatment. Though many heatsterols owing to their provision by symbiotes (DADD treated insects fail to moult to the adult stage, they and MITTLER, 1966; SRIVASTAVA and AUCLAIR, 1971; usually develop to the 5th instar after some delay. The AKEYand BECK,1972). EHRHARDT(1968b) and HOUK heat-treated insects can be employed in comparative et al. (1976) support this hypothesis. GRIFFITHS and sterol analysis to clarify the possible participation of BECK (1977a, b) also suggest cholesterol biosynthesis the yeastlike symbiotes in sterol metabolism. by the symbiotes of Acyrthosiphon pisum using In the present paper, in order to locate the sterol electron microscopical techniques. source in L. striatellus, the sterols were analysed in the The smaller brown planthopper, Laodelphax insects, their host plant and the excreted honeydew. striatellus (Homoptera: Delphacidae), like aphids, Secondly. sterols in the heat-treated insects were feeds on the plant juice from the vascular bundle. This investigated and the sterols of three other rice plantinsect harbours yeastlike symbiotes in the fat body and sucking species were compared with those of L. the female transmits them to the next generation via the striatellus. The role played by the yeastlike symbiores ovary (NASU. 1963; NODA, 1977). It also possesses in the host sterol requirement is discussed. bacteria in the mycetocytes in the fat body or in the intercellular space between mycetocytes (NODA and SAITO. unpublished observations). MITSUHASHI and MATERIALS AND METHODS KOYAMA (1972) raised L. striateflus successfully on a Animals chemically defined artificial diet not containing sterol. Thus there is the possibility that the symbiotes of this The following insects were used in the present species synthesise sterols to provide for the insect. striate/b (Fall&), Laodelphax experiments: For the precise study of nutrition, aposymbiotic Niluparvata iugens (StBl), Sogatella furcifera (HorvBth) (Delphacidae) and Nephotettix cincticeps (Uhler) (Deltocephalidae). The insects were reared on *Present address: Shimane Agricultural Experiment rice seedlings at 25°C and subjected to a 16L:8D Station. 2440 Ashiwata. Izumo, Shimane 693, Japan. photoperiod. Heat-treated L. striatellus was also ‘r Laboratory of Pesticides Chemistry. 443
HIKOAI(I NOIIA, KOJIRO WADA AND TETSU~ SAITO
444
employed for sterol analysis. Newly-hatched 1st instar nymphs were exposed to 35 C for 3 days, then transferred to the normal temperature, 25 C. and allowed to feed on rice seedlings ( NODA and SAITO. unpublished observations). E.vtrac/ion
of .strrol.r
Fresh insects or insects stored at -80’ C were chloroform-methanol (2: 1) homogenized with (FOLCH et rd., 1957). After the extracts were filtered and partitioned with 0.2 their volume of water; the chloroform phase was dried down in a rotary evaporator. The extracted whole lipids were refluxed in lo”,, methanolic KOH at 75 ‘C for I hr. The nonsaponifiable matter, after being partitioned between ether and water, was developed using thin layer chromatography (TLC. Merck Silica Gel 60F-254) with the solvent system. chloroform-ethyl acetate (20: I v/v). The spots corresponding to the authentic cholesterol were scraped together and the sterols were extracted with ethyl acetate. For extracting sterols from rice plants, the buds of rice seedlings or the leaves at the 2-3 leaf stage were cut and dried at room temperature for several days. The dried samples were refluxed in methanol at 70’ C for 90 mm and the solvent was removed in IYIJCUU.The extracted matter was partitioned in ether and water, and the ether phase dried down. Honeydew was obtained by allowing female adults to feed on the rice plant (S~)GAWA, 1970). with a filter paper placed under the feeding site to absorb the honeydew excreted. The filter papers were refluxed in methanol at 70°C for 90 min and the solvent was evaporated. The extracts were also partitioned in ether and water. and the ether phase was retained. The ether-soluble substances from the rice seedling, 2-3 leaf stage, and honeydew were saponified separately and the sterols purified with TLC as mentioned above.
Fig. I Gaschromatogram of sterols in the I st instar nymph (OV-I ). (A) Cholesterol; (B) 24-methylenecholesterol; (C)Bsitosterol.
Sterols were analysed by gas chromatography (GLC. Jeol Ltd., JGC-20K) equipped with a FID detector and I m glass column. Two column packings were employed; 3”,, OV-1 (Gaschrome Q, 100-l 20 mesh) at 240’ C and 1.5”,, OV-17 (Shimalite, W-100 mesh) at 260°C. The identification of sterols was
achieved by comparing the retention times to those of authentic samples in two systems. The authentic samples of campesterol (24a-methylcholest-5-en-3jI01) and 24-methylenecholesterol (24-methylcholesta5. 24 (28)-dien-3&ol) were provided by Dr. Ikekawa (Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology). Cholesterol, stigmasterol (24cr-ethylcholesta-5, 22-dien-3fi-ol) and /I-sitosterol (24a-ethylcholest-5-en-3jl-ol) were obtained from Katayama Chemical Co., Ltd. Quantitative analysis by GLC was carried out based on the standard line of the authentic cholesterol in the same condition. The sterols were also analysed by a gas chromatograph-mass spectrometer system (GC-MS, Jeol Ltd.. Model JMS-DIOO) using 30;) OV-1 (1 m column) at 260°C.
100
RESULTS Sterols
in L. striatellus
Sterols were analysed in the following stages: 1st instar; 5th instar nymphs (with a few 4th instar); adult 314
r
HOJYF-+ ’
271
299 229 213
llilhl
200
I
281 11lu
~hh~L
300
398(M+)
111 IIIk
m/e Fig. 2. Mass spectrum
of 24-methylenecholesterol
in the 1st instar nymph.
II I
I 400
Sterols in L. slriatellus Table
1. Sterol concentration Cholesterol
445
of L. striarellus
(A)
in each stage
24-Methylenecholesterol
(B)
Stages*
(pg/g wt)
(flgig wt)
B/A
1st instar nymph 5th instar nymph Adult male aged under 5 days Adult female aged under 5 days Adult male aged over 7 days
9.8 13.7 32.6 50.7 39.0
6.2 8.7 20.4 33.7 17.9
0.64 0.64 0.63 0:66 0.46
* Each reading
represents
only one batch of animals.
males and females under 5 days old; and adult males over 7 days old. In all samples, two major and one minor sterol were detected by GLC (Fig. 1). The two major sterols were identified as cholesterol and 24methylenecholesterol, and the minor sterol was psitosterol. The sterols were also identified by GC-MS. The mass spectra of cholesterol and /?-sitosterol gave the molecular ion peak at m/e 386 and m/e 414 respectively, and were identical to those of the authentic samples. The mass spectrum of 24methylenecholesterol showed the molecular ion peak at m/e 398 (M+) and the base peak at m/e 314 (M+--C,H,,) (Fig. 2). The concentrations of cholesterol and 24methylenecholesterol in each sample measured by means of GLC are shown in Table 1. The concentrations of cholesterol and 24-methylenecholesterol increased with the growth of the insect. The ratio of 24-methylenecholesterol to cholesterol was generally the same. ranging from 0.63 to 0.66, except in males more than 7 days old. Sterols in rice plant
In the samples of rice seedling and 2-3 leaf rice plants, three sterols were detected: campesterol, stigmasterol and jY-sitosterol (Fig. 3). The mass spectra of these gave molecular ion peaks at m/e 400,412 and 414 respectively and were identical to those of each authentic sterol.
feeding on rice plants. cholesterol and p-sitosterol were found. When a large amount of the sample was analysed, there was a small peak between the peaks for cholesterol and /3-sitosterol (Fig. 4). This was identified as campesterol from the retention time using the column packing OV-17: OV-17 showed a small difference in retention times between campesterol and 24-methylenecholesterol. Therefore, three sterols were detected in honeydew: cholesterol, /%sitosterol and a minute amount of campesterol. L. striatellus Heat-treated 5th instar nymphs (with a small number of 4th instar nymphs, about,13-14 days after hatching) were analysed. The heat-treated nymphs contained cholesterol, /?-sitosterol and a small amount of 24-methylenecholesterol (Fig. 5). The concentrations of cholesterol were 1.2 and 1.3 pg/g in two replicates. The amount of cholesterol in the heattreated insect was greatly reduced by comparison with that of the normal 5th instar nymph (13.7 pg/g). Though the gas chromatograph of the heat-treated insects (Fig. 5) showed that the amount of /?-sitosterol was relatively larger than that in the normal 5th instar nymphs (Fig. 6). there were no marked differences in the concentration of p-sitosterol because in the former Sterols in heat-treated
Sterols in honeydew
In the honeydew excreted by the female adults
:
I 5
IO I-Ill"
Fig. 3. Gas chromatogram of sterols in the rice seedling (OVI ). (C) p-Sitosterol: (D) campesterol: (E) stigmasterol.
I IO
5 m,n
Fig. 4. Gas chromatogrdm of sterols in honeydew (OV(A) Cholesterol; (C) p-sitosterol; (D) campesterol.
17).
HIROAKI NODA.KOJIRO WADAANDTETSUO SAITO
446
I, A
min
6. Gas chromatogram of sterols in the normal 5th instar nymph (OV-I). (A) Cholesterol: (B) 24methylenecholes-
Fig. 5. Gas chromatogram of sterols in the heat-treated 5th
instar
nymph (OV-I). (A) Cholesterol; (B) methylenecholesterol; (C) fi-sitosterol.
Fig.
24-
terol; (C) /Lsitosterol.
an essential nutrient for almost all insects. In Manduca sexta, Tribolium confusum (SVOBODAet al.. 1975) and Bombxy mori (MORISAKIet al., 1972). the conversion
a large amount of material was injected into the GLC owing to the low concentration of cholesterol. Sterols in other rice plant-sucking species
of phytosterols into cholesterol has been demonstrated. Campesterol. stigmasterol and bsitosterol can be detected in the rice plant, but in the honeydew of L. striatellus /I-sitosterol and a negligible amount of campesterol is recovered. As this planthopper imbibes the plant juice from the vascular bundles excessively and excretes a large amount of honeydew, phytosterols in the honeydew almost certainly come from the sap in the vascular bundles. Cholesterol is synthesized within the insect. Stigmasterol, which is found in the whole rice plant, does not seem to be in the vascular bundles. L. striatellus can utilize mainly fi-sitosterol from the rice plant as a cholesterol source. 24-Methylenecholesterol is found in L. striatellus. and is one of the major sterols in some species of algae (PATTERSON,1971). This sterol is found in the honey bee, Apis mellifca (BARBIER et al., 1959) and is considered to be a constituent of pollens of various flowers which the honey bee visits (BARBIERet al., 1960). 24-Methylenecholesterol is also identified as an intermediate in the conversion of campesterol into cholesterol in Manduca scvtu (SWBODA et cd..1972). In L. striatellus 24-methylenecholesterol can be detected in abundance in all stages examined. The heat-treated insects, which have a smaller number of the yeastlike
Sterols in three other rice plant-sucking species, N. lugens, S. furcifera and N. cincticeps, were analysed by GLC. These species drink the sap in the vascular bundle as well as L. striatellus. N. lugens and S. furcifira possess yeastlike symbiotes in their fat body (NASU and SUENAGA, 1958; NODA, unpublished observations), whereas N. cincticeps harbours prokaryotic symbiotes in the mycetomes (NASU, 1965; MITSUHASHIand KONO, 1975). Sterols were extracted from the 4th and 5th instar nymphs in these species. In N. lugens and S. furcifera. three sterols, cholesterol, 24-methylenecholesterol and /I-sitosterol, were detected. On the other hand, 24-methylenecholesterol was not recovered in N. cincticeps. This leafhopper had cholesterol, /I-sitosterol and a small amount of campesterol.
DISCUSSION Insects are considered to lack a sterol biosynthetic mechanism (ROBBINS et al., 1971). The insect. therefore. requires a dietary or additional source of sterol. Phytophagous insects generally ingest phytosterols to convert them into cholesterol which is Rice plant _
Vascular bundle
Laodelphax 7
strlatellus
Alimentary
Honeydew
canal -
+ Sitosterol
* Wosterol
Cholesterol Cholesterol Campesterol
I
Stigmasterol
I-ig. 7. Schematic
sterol
flow
in L. .vt~Yrrre//u.\
Sterols in L. striatellus
symbiotes, have little 24-methylenecholesterol. This strongly suggests that this sterol originates from the symbiotes. The reduction in sterol supply from the yeastlike symbiotes in the heat-treated insects appears to account for the lower concentration of cholesterol. The ratio of the two sterols in the adult male more than 7 days old is lower than that in the other stages. This could be explained by the fact that the number of yeastlike symbiotes in male adults decreases after emergence (NODA, 1974, 1977). Niluparvuta lugens, S. furctfera and N. cincticeps are all vascular feeders. The first two species which possess the yeastlike symbiotes 24-methylenecholesterol. However, 24contain methylenecholesterol is not present in N. cincticeps, which does not harbour the same symbiotes. It is, therefore, concluded that L. striatellus obtains the sterol from its yeastlike symbiotes and has two sterol sources: rice plant sap and the symbiotes. Figure 7 represents diagrammatically the sterol flow between the rice plant. L. striatellus and the honeydew. Acknowledgements-The authors are grateful to Dr. S. National Institute of Agricultural Science. for helpful advice. to Dr. N. IKEKAWA of Laboratory of Chemistry for Natural Products, Tokyo Institute of Technology, for provision of sterol samples, and to Dr. M. A. BRINKS of Department of Entomology, Fisheries and Wildlife, University of Minnesota. for reviewing this manuscript. NASU of
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