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Identification of JH III in haemolymph from adults and larvae of Schistocerca gregaria
Insect Biochem., 1976, Vol. 6, pp. 35 to 38. Pergamon Pre~s. Printed in Great Britain.
IDENTIFICATION OF JH III IN HAEMOLYMPH FROM ADULTS AND LARVAE OF SCHISTOCERCA GREGARDI MAae~a~'r M. BLIGHT and MICHAELJ. Wm'~H~Vt Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, University of Sussex, Falmer, Brighton, BN1 9QJ, England
(Received 19 ffune 1975) Abstract--An analytical method for the separation and identification of JH III and (E,E)-methyl farnesoate from locust haemolymph is described. Using this method, JH III was identified in haemolymph from adults, but no (E,E)-methyl farnesoate was found. JH III was also identified in fourth instar hopper haemolymph fractions, which had previously been shown to be active in the Galleria pupal wax assay. INTRODUCTION RECENTLY (BLIGHT and WENHAM, 1975) we described the separation of three fractions, 'AI', 'A2', and 'BI', with juvenile hormone activity from Schistocerca gregaria. These fractions, which were purified with the aid of the Galleria bioassay, were isolated from whole animal extracts of third instar hoppers, and from haemolymph of fourth instar hoppers and adult females. Fractions ' A I ' and 'A2' did not contain any of the known natural insect juvenile hormones. The chromatographic behaviour of fraction ' B I ' was consistent with its containing JH III. This paper describes an analytical investigation of haemolymph from adults, undertaken when bioassay facilities were no longer available. Its purpose was to discover if (E,E)-methyl famesoate (MF), (1) and JH I I I [(E,E)-methyl-10, l l epoxy-farnesoate], (2), were present, and by analogy,
[
]
H
(3s) R= OH OMe
(31)) R= OCOCF3
R
JH It
Scintillation counting The procedure used was described (BLIGHTand WENHAM,1975).
O
•" ' O ~ ~ ~ O M
" R-...[
MF rE,E)
OMe
• {2)
Preparation of the diol ester of ~ H I I l and its derivatisation JH III was converted to the diol ester (3a) on the nanogram scale, using the method of JODYet aL (1973a). The diol ester was then converted to the bis-trifluoroacetyl derivative (3b) with trifluoroacetic anhydride+ pyridine in dioxan at 25°C for 24 hr. (The reagent and
solvents used were freshly distilled before use). The reaction mixture was taken up in acetone and purified by preparative thin-layer chromatography on Merck silica gel H (0.75 mm layer), 25% ether in benzene being used for development. Marker spots of the Merck lipophilic dye mixture were used to delineate the position of the derivative on the plate. The derivative was eluted from the silica with ether.
o (1]
(P~aaxr¢ et aL, 1966). The preparation of most of the chemicals used has been described previously (BLIGHT and WF2qHAM,1975).
(E,E)
e
to show whether activity ' B I ' was JH I I I . T h e analysis, for JH III, of hopper haemolyrnph ' B I ' fractions, which had previously been shown to be active (BLIGHTand WENHAM, 1975) in the bioassay, was then performed.
previously
Chromatography (1) The Sephadex LH-20 chmmatographie method was the same as that used previously (BLIGHTand W~'qHAM, 1975). (2) Preparative thin-layer chromatographic (TLC) purification of Sephadex column fractions from haemolymph extracts of adults containing putative JH III and EXPERIMENTAL (E,E)-MF were separated on Merck silica gel H, The insect material used and the collection and 750/an layer, pre-run in purified methanol. The extraction of the haemolymph is described by BLIGHT loading was 1 mg material]13 cm sample streak, and the plate was developed with 20% ether in light petroleum and Wm~rH~V[(1975). As before, glassware was heated at 200 to 250°C at 5°C. The fractions which should contain JH III and before use, and all solvents were rigorously purified (E,E).MF were delineated with reference to marker 35
36
MARGARETM. BLIGHTAND MICHAELJ. WENHAM
spots of the 2,4-dinitrophenyl hydrazones of acetone, ethyl methyl ketone and tetradecylaldehyde. They were extracted from the silica with ether. Combined hopper haemolymph 'BI' fractions (see below) were purified by preparative thin layer chromatography in the same manner. (3) High pressure liquid chromatographic (HPLC) separations were obtained using a Perkin-Elmer 1220 instrument equipped with a variable wavelength UV detector (Cecil Instruments CE-212), set at 230 nm. The column used (40 cm×4.6 mm i.d.), was packed using a Waters Model 6000 pump. A slurry of 6# Partisil (Reeve Angel Scientific Ltd.) in a balanced density solvent (tetrabromoethane+tetrachloroethane; 4 5 : 5 5 v/v) was placed in a pre-column, and covered with a layer of water. The slurry was pumped into the column using heptane, at 6000 psi. The column was conditioned by pumping through consecutively methanol, acetone, ethyl acetate, and finally ether, which was 75% saturated with water. HPLC separations were performed using ether-pentane mixtures (75% saturated with water), or pentane containing iso-propanol and acetonitrile. (4) Gas-liquid chromatography (GLC) using wallcoated glass capillary columns and flame ionization detection, was performed in a Pye 104 gas chromatograph, with nitrogen as the carrier gas. The columns used were a 20m × 0.32 mm i.d. OV-101 (Phase Separations Ltd., Flintshire, Wales), a 20m×0"32 mm i.d. Ucon oil 50HB-2000 (H. Jaeggi, CH-9043, Trogen, Switzerland) and a 26m× 0"5 mm i.d. Carbowax 20M (Phase Separations Ltd.). Samples were injected using an aU-glass splitless injector (Phase Separations Ltd.). Electron capture gas chromatography (EC-GC) was performed using a Pye GCV gas chromatograph with nitrogen as the carrier gas. The glass columns (5 ft × 4ram i.d.) were packed with 3% OV-225 and 3% OV-17 on 80[100 mesh Gas Chrom. Q.
Analysis procedurefor the identification offill III and (E,E)-MF in haemolymph from adults Haemolymph was collected from 500 mature adults 14 to 20 days old. The extracts obtained were combined and a one-sixth aliquot was taken. To this aliquot, c. 1.4/tg [3H]-JH III (3.6 × 105 dis/ rain) and c. 1.3/tg [3H]-(E,E)-MF (2-3× 105 dis/rain) were added. The aliquot was then used to design the analytical method to be employed for the detection of any endogenous (E,E)-MF and JH III in the remaining (unlabelled), five-sixths of the extract.
RESULTS
The analysis of haemolymph from adults for ffH I l l and (E,E)-MF Th e method used to separate endogenous (E,E)M F and J H I I I from haemolymph is outlined in Fig. 1. As previously stated (BLIGHT and WENHAM, 1975) J H I I I and (E,E)-MF elute in the same Sephadex fractions, 'B' (Fig. 2). These were combined and separated by T L C to give 'BI ', which should contain any J H I I I and 'B2', which should contain any (E,E)-MF. EXTRACT OF HAEMOLYMPH FROM ADULTS Sephadex LH-20 chromatography
COMBINED FRACTIONS 'B' TLC 202' ether in light petroleum
I FRACTION 'B 1'
FRACTION 'B2'
(i) HPLC
(i) HPLC
(ii) glass capillary
(ii) glass capillary G LC
JH "111"
no MF
Fig. 1. Scheme for the purification of the haemolymph extract from adults.
Before the H P L C separation of ' B I ' and 'B2' was performed, an H P L C study showed that only a partial separation of the (Z,E)- and (E,E)-isomers of J H I I I was obtained on a 40 cm 61~ partisil column, when ether-pentane mixtures, 75% saturated with water, were used as eluants. Deactivation of the stationary phase with water was necessary (SNYD~, 1971) to prevent degradation of J H III. However, complete resolution of the isomers was obtained when pentane containing iso-propanol and acetoAnalysis procedure for the identification of fill 111 in nitrile was used as the eluant. This system also has hopper haemolymph ' B I ' fractions practical advantages because it does not contain Haemolymph had been collected, over a period of water. Gas liquid chromatographic and mass spectrometric analyses of the J H I I I eluted from the 8 months, from fourth instar hoppers 0 to 3 days old. Extracts were prepared immediately and separated by column with the latter eluant mixture confirmed thin-layer chromatography (BLIGHT and WEN'HAM, that no degradation took place. 1975). Seven fractions, which were active in the Fraction ' B I ' (Fig. 1) was therefore separated Galleria assay, and which, according to their thin-layer by H P L C using 1"5% acetonitrile+0"3% isochromatographic behaviour contained 'BI' activity, propanol in pentane at 1 ml/min as the eluant were obtained and stored at --20°C. These fractions were combined and separated by preparative thin-layer mixture. N o peak was seen at the retention time of ] H I I I during replicate H P L C separations, because chromatography to obtain a purified 'BI' fraction.
37
JH III in haemolymph from adults and larvae of Schistocerca gregaria 45--
35--
25--
15--
5--
/ I 5
I 15
Elution volume,
I 25
I 35
rnl
Fig. 2. Sephadex LH-20 chromatography of the haemolymph extract from adults. 'B' denotes the zone where (E,E)-MF and JH III elute. the quantity present was below the detection limit. T h e H P L C fraction which should contain any JH I I I was therefore delineated using other peaks in the chromatogram as internal standards. These fractions from replicate separations were combined. Eight per cent of the combined fraction, when analysed on the 20m OV-101 G L C column at 155°C and 4"5 ml/min flowrate, gave a major peak eluting, and co-chromatographing with J H I I I , at 20.7 min. There was only one other major peak in the chromatogram, which eluted at 25.6 min.
Another 8% aliquot of this fraction, when analysed on the 20 m Ucon oil 50HB-2000 column at 165°C and a 1"5 ml/min flowrate, gave the ehromatogram shown in Fig. 3, the peak at 13"2 rain eluting at the retention time of J H I I I . Confirmation of the identification of J H I I I as one of the two major components of this fraction was obtained by conversion of the remainder to the b i s - T F A derivative (3b) of the diol ester (3a). E C - G C analysis of this derivative on the 3% OV-225 column at 165°C, with a nitrogen carrier flowrate of 30 ml/min gave a peak eluting, and co-chromatographing, with the authentic derivative of J H I I I at 7-5 rain. On the 3% OV-17 column at 170°C and 30 ml/rnin nitrogen flowrate, a peak eluted and co-chromatographed with the authentic JH I I I derivative at 5"8 min. This result was confirmed by G L C analysis (and co-chromatography) on the 20m OV-101 column, using flame ionization detection. F r o m these G L C analyses, and the analysis procedure with labelled JH I I I , which allowed experimental losses during the workup to be estimated, the endogenous JH I I I titre was estimated to be of the order of 500 to 600 pg per adult insect. Fraction 'B2' was separated by H P L C , eluting with 0"75% aeetonitrile+0.15% iso-propanol in pentane at 2 ml/min. No peak was detected by the UV monitor at the retention time of (E,E)-MF b u t the fractions which might contain it were examined by G L C on the 20m OV-101 glass capillary column at 155°C and a nitrogen flowrate of 3 ml/min. N o (E,E)-MF was detected. Allowing for losses during work-up, it would have been possible to detect 20 pg (E,E)-MF per locust, if present.
The analysis of hopper haemolymph fractions which contained 'BI ' activity The purified T L C fraction containing ' B I ' activity was fractionated further by H P L C using the system employed during the analysis of the J H ~a,
I 25
f 20
I 15
I 10
I 5
I 0
Time, minutee
Fig. 3. Gas chromatograrn of HPLC fraction separated from the haemolymph extract from adults. JH I I I eluted at 13.2 min. Column: 20mx0-32 mm i.d. glass capillary, wall-coated with Ucon oil 50HB-2000. Operating conditions: Isothermal at 165°C, with 1.5 ml/min carrier gas, and flame ionization detection.
38
MARGARETM. BLIGHTAND MICHAELJ. WENHAM
haemolymph extract from adults. T h e quantity of J H I I I present was again below the detection limit under the conditions used, and other peaks in the chromatogram had to be used to delineate the fraction containing it. J H I I I was identified in this fraction by G L C analysis on the OV-101 and Carbowax 20M capillary columns. From the G L C results, and making an estimate of the experimental losses, the J H I I I content per fourth instar hopper was of the order of 150 pg.
DISCUSSION This study has confirmed the presence of J H I I I in S. gregaria adults (TRAUTMANe t al., 1974b) and, in addition, has shown that it is present in S. gregaria hoppers. T h e quantity found in adult blood (0"6 ng/insect) is of the same order as that found in older adults (whole bodies) by TRAUTMAN et al. (1974b) (0"5 ng/g body weight, i.e. 1"4 ng/ animal approx.) Th e estimates of the titres may, however, be too low, since the aim of this study was primarily one of identification. At least some of the juvenile hormone activity in fraction ' B I ' (BLIGHT and WENHAM, 1975) from hoppers and adults is due to J H III, but the presence of other juvenilising substances in this fraction cannot be excluded since it was not possible to obtain the desired bioassay data. A search was made for (E,E)-methyl farnesoate in haemolymph from adults because it is probably a biosynthetic precursor, in vivo, of J H III. Failure to detect it in haemolymph may indicate that it is synthesised, hut not released from the corpus aUatum, in vivo. PRATTand TOBE (1974) found that corpora allata from adult female S. gregaria, incubated in vitro with [3H]-(E,E)-farnesenic acid, synthesised (E,E)-MF but it was not released into the medium. T h e presence of J H I I I in many sexually mature adult insects (JUDY et al., 1973a, b; 1975; MULLER et al., 1974; TRAUTMANNet al., 1974a, b; this study) suggests to us that it may contribute primarily to the gonadotropic action of the corpus allatum (WIGGLESWORTH,1970); and that J H I, J H II and other unknown juvenilising substances (e.g. ' A I ' and 'A2' in S. gregaria; BLIGHT and W~HAM, 1975) may be mainly concerned with larval development. On the other hand, lack of evidence so far for the presence of J H I I I in larvae may simply reflect the greater difficulties involved in the isolation of juvenilising substances from larvae.
Note added in proof--JH I, II and III have now been identified in the haemolymph of larvae of the cockroach,
Nauphoeta cinema [LANZREIN B., HASHIMOTO M., PARMAKOVlCH V., NAKANISHI m., WILHELM R., and Li)SCHER M. (1975). Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nauphoeta cinema. Life Sci. 16, 12711284]. Acknowledgements--We thank the Insectory staff in the Unit for rearing the insects, Dr A. F. WHITE for the radiolabelled substances and Dr F. A. MELLONfor mass spectra. We are grateful to Professor A. W. JOHNSON for his advice and encouragement during the course of this work.
REFERENCES BLIGHT M. M. and WENHAM M. J. (1976) Juvenile hormone activity in larvae and adult females of Schistocerca gregaria. J. Insect. Physiol. 22 141-145. JuDY K. J., SCHOOLEYD. A., DUNHAM L. L., HALL M. S., BERGOT B. J., and SIDDALL J. B. (19738) Isolation, structure and absolute configuration of a new natural insect juvenile hormone from Manduca sexta. Proc. nat. Acad. Sci. U.S.A. 70, 1509-1513. JUDYK. J., SCHOOLEYD. A., HALL M. S., BERGOTB.J., and SIDDALL J. B. (1973b) Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro. Life Sci. 13, 1511-1516. JUDY K. J., SCHOOLEY D. A., TROETSCHLER R. G., JENNINGSR. C., BERGOTB.J., and HALLM. S. (1975) Juvenile hormone production by corpora allata of Tenebrio molitor in vitro. Life Sci. 16, 1059-1066. MULLERP. J., MASNERP., TRAUTMANNK. H., SUCH~M., and WIPE H-K. (1974) The isolation and identification of juvenile hormone from cockroach corpora allata in vitro. Life Sci. 15. 915-921. PERRIN D. D., ARMAREOOW. L. F., and PERmN D. R. (1966) Purification of Laboratory Chemicals. Pergamon Press, Oxford. PaATT G. E. and Tone S. S. (1974) Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586. SNYDEa L. R. (1971) Modern Practice of Liquid Chromatography (Ed. by KInKLANI)J. J.), pp. 223-225, 227228. Wiley-Interscience, New York. TRAUTMANNK. H., SCHULERA., SUCH# M., and WIPF H-K. (1974a) Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen yon Insekten. Nachweis yon 10, ll-epoxy-3,7,11trimethyl-2-tram-6-trans-dodecadiensiiuremethylester in Melolontha melolontha. Z. Naturf. 29c, 161-168. TRAUTMANNK. H., MASNERP., SCHULERA., SUCH? M., and WIPE H-K. (1974b) Evidence of the juvenile hormone methyl (2E,6E)-10, ll-epoxy-3,7,11-trimethyl-2,6-dodecadienoate (JH-3) in insects of four orders. Z. Naturf. 29c 757-759. WIOOLmWORTHV. B. (1970) Insect Hormones. Oliver & Boyd, Edinburgh.
IDENTIFICATION OF JH III IN HAEMOLYMPH FROM ADULTS AND LARVAE OF SCHISTOCERCA GREGARDI MAae~a~'r M. BLIGHT and MICHAELJ. Wm'~H~Vt Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, University of Sussex, Falmer, Brighton, BN1 9QJ, England
(Received 19 ffune 1975) Abstract--An analytical method for the separation and identification of JH III and (E,E)-methyl farnesoate from locust haemolymph is described. Using this method, JH III was identified in haemolymph from adults, but no (E,E)-methyl farnesoate was found. JH III was also identified in fourth instar hopper haemolymph fractions, which had previously been shown to be active in the Galleria pupal wax assay. INTRODUCTION RECENTLY (BLIGHT and WENHAM, 1975) we described the separation of three fractions, 'AI', 'A2', and 'BI', with juvenile hormone activity from Schistocerca gregaria. These fractions, which were purified with the aid of the Galleria bioassay, were isolated from whole animal extracts of third instar hoppers, and from haemolymph of fourth instar hoppers and adult females. Fractions ' A I ' and 'A2' did not contain any of the known natural insect juvenile hormones. The chromatographic behaviour of fraction ' B I ' was consistent with its containing JH III. This paper describes an analytical investigation of haemolymph from adults, undertaken when bioassay facilities were no longer available. Its purpose was to discover if (E,E)-methyl famesoate (MF), (1) and JH I I I [(E,E)-methyl-10, l l epoxy-farnesoate], (2), were present, and by analogy,
[
]
H
(3s) R= OH OMe
(31)) R= OCOCF3
R
JH It
Scintillation counting The procedure used was described (BLIGHTand WENHAM,1975).
O
•" ' O ~ ~ ~ O M
" R-...[
MF rE,E)
OMe
• {2)
Preparation of the diol ester of ~ H I I l and its derivatisation JH III was converted to the diol ester (3a) on the nanogram scale, using the method of JODYet aL (1973a). The diol ester was then converted to the bis-trifluoroacetyl derivative (3b) with trifluoroacetic anhydride+ pyridine in dioxan at 25°C for 24 hr. (The reagent and
solvents used were freshly distilled before use). The reaction mixture was taken up in acetone and purified by preparative thin-layer chromatography on Merck silica gel H (0.75 mm layer), 25% ether in benzene being used for development. Marker spots of the Merck lipophilic dye mixture were used to delineate the position of the derivative on the plate. The derivative was eluted from the silica with ether.
o (1]
(P~aaxr¢ et aL, 1966). The preparation of most of the chemicals used has been described previously (BLIGHT and WF2qHAM,1975).
(E,E)
e
to show whether activity ' B I ' was JH I I I . T h e analysis, for JH III, of hopper haemolyrnph ' B I ' fractions, which had previously been shown to be active (BLIGHTand WENHAM, 1975) in the bioassay, was then performed.
previously
Chromatography (1) The Sephadex LH-20 chmmatographie method was the same as that used previously (BLIGHTand W~'qHAM, 1975). (2) Preparative thin-layer chromatographic (TLC) purification of Sephadex column fractions from haemolymph extracts of adults containing putative JH III and EXPERIMENTAL (E,E)-MF were separated on Merck silica gel H, The insect material used and the collection and 750/an layer, pre-run in purified methanol. The extraction of the haemolymph is described by BLIGHT loading was 1 mg material]13 cm sample streak, and the plate was developed with 20% ether in light petroleum and Wm~rH~V[(1975). As before, glassware was heated at 200 to 250°C at 5°C. The fractions which should contain JH III and before use, and all solvents were rigorously purified (E,E).MF were delineated with reference to marker 35
36
MARGARETM. BLIGHTAND MICHAELJ. WENHAM
spots of the 2,4-dinitrophenyl hydrazones of acetone, ethyl methyl ketone and tetradecylaldehyde. They were extracted from the silica with ether. Combined hopper haemolymph 'BI' fractions (see below) were purified by preparative thin layer chromatography in the same manner. (3) High pressure liquid chromatographic (HPLC) separations were obtained using a Perkin-Elmer 1220 instrument equipped with a variable wavelength UV detector (Cecil Instruments CE-212), set at 230 nm. The column used (40 cm×4.6 mm i.d.), was packed using a Waters Model 6000 pump. A slurry of 6# Partisil (Reeve Angel Scientific Ltd.) in a balanced density solvent (tetrabromoethane+tetrachloroethane; 4 5 : 5 5 v/v) was placed in a pre-column, and covered with a layer of water. The slurry was pumped into the column using heptane, at 6000 psi. The column was conditioned by pumping through consecutively methanol, acetone, ethyl acetate, and finally ether, which was 75% saturated with water. HPLC separations were performed using ether-pentane mixtures (75% saturated with water), or pentane containing iso-propanol and acetonitrile. (4) Gas-liquid chromatography (GLC) using wallcoated glass capillary columns and flame ionization detection, was performed in a Pye 104 gas chromatograph, with nitrogen as the carrier gas. The columns used were a 20m × 0.32 mm i.d. OV-101 (Phase Separations Ltd., Flintshire, Wales), a 20m×0"32 mm i.d. Ucon oil 50HB-2000 (H. Jaeggi, CH-9043, Trogen, Switzerland) and a 26m× 0"5 mm i.d. Carbowax 20M (Phase Separations Ltd.). Samples were injected using an aU-glass splitless injector (Phase Separations Ltd.). Electron capture gas chromatography (EC-GC) was performed using a Pye GCV gas chromatograph with nitrogen as the carrier gas. The glass columns (5 ft × 4ram i.d.) were packed with 3% OV-225 and 3% OV-17 on 80[100 mesh Gas Chrom. Q.
Analysis procedurefor the identification offill III and (E,E)-MF in haemolymph from adults Haemolymph was collected from 500 mature adults 14 to 20 days old. The extracts obtained were combined and a one-sixth aliquot was taken. To this aliquot, c. 1.4/tg [3H]-JH III (3.6 × 105 dis/ rain) and c. 1.3/tg [3H]-(E,E)-MF (2-3× 105 dis/rain) were added. The aliquot was then used to design the analytical method to be employed for the detection of any endogenous (E,E)-MF and JH III in the remaining (unlabelled), five-sixths of the extract.
RESULTS
The analysis of haemolymph from adults for ffH I l l and (E,E)-MF Th e method used to separate endogenous (E,E)M F and J H I I I from haemolymph is outlined in Fig. 1. As previously stated (BLIGHT and WENHAM, 1975) J H I I I and (E,E)-MF elute in the same Sephadex fractions, 'B' (Fig. 2). These were combined and separated by T L C to give 'BI ', which should contain any J H I I I and 'B2', which should contain any (E,E)-MF. EXTRACT OF HAEMOLYMPH FROM ADULTS Sephadex LH-20 chromatography
COMBINED FRACTIONS 'B' TLC 202' ether in light petroleum
I FRACTION 'B 1'
FRACTION 'B2'
(i) HPLC
(i) HPLC
(ii) glass capillary
(ii) glass capillary G LC
JH "111"
no MF
Fig. 1. Scheme for the purification of the haemolymph extract from adults.
Before the H P L C separation of ' B I ' and 'B2' was performed, an H P L C study showed that only a partial separation of the (Z,E)- and (E,E)-isomers of J H I I I was obtained on a 40 cm 61~ partisil column, when ether-pentane mixtures, 75% saturated with water, were used as eluants. Deactivation of the stationary phase with water was necessary (SNYD~, 1971) to prevent degradation of J H III. However, complete resolution of the isomers was obtained when pentane containing iso-propanol and acetoAnalysis procedure for the identification of fill 111 in nitrile was used as the eluant. This system also has hopper haemolymph ' B I ' fractions practical advantages because it does not contain Haemolymph had been collected, over a period of water. Gas liquid chromatographic and mass spectrometric analyses of the J H I I I eluted from the 8 months, from fourth instar hoppers 0 to 3 days old. Extracts were prepared immediately and separated by column with the latter eluant mixture confirmed thin-layer chromatography (BLIGHT and WEN'HAM, that no degradation took place. 1975). Seven fractions, which were active in the Fraction ' B I ' (Fig. 1) was therefore separated Galleria assay, and which, according to their thin-layer by H P L C using 1"5% acetonitrile+0"3% isochromatographic behaviour contained 'BI' activity, propanol in pentane at 1 ml/min as the eluant were obtained and stored at --20°C. These fractions were combined and separated by preparative thin-layer mixture. N o peak was seen at the retention time of ] H I I I during replicate H P L C separations, because chromatography to obtain a purified 'BI' fraction.
37
JH III in haemolymph from adults and larvae of Schistocerca gregaria 45--
35--
25--
15--
5--
/ I 5
I 15
Elution volume,
I 25
I 35
rnl
Fig. 2. Sephadex LH-20 chromatography of the haemolymph extract from adults. 'B' denotes the zone where (E,E)-MF and JH III elute. the quantity present was below the detection limit. T h e H P L C fraction which should contain any JH I I I was therefore delineated using other peaks in the chromatogram as internal standards. These fractions from replicate separations were combined. Eight per cent of the combined fraction, when analysed on the 20m OV-101 G L C column at 155°C and 4"5 ml/min flowrate, gave a major peak eluting, and co-chromatographing with J H I I I , at 20.7 min. There was only one other major peak in the chromatogram, which eluted at 25.6 min.
Another 8% aliquot of this fraction, when analysed on the 20 m Ucon oil 50HB-2000 column at 165°C and a 1"5 ml/min flowrate, gave the ehromatogram shown in Fig. 3, the peak at 13"2 rain eluting at the retention time of J H I I I . Confirmation of the identification of J H I I I as one of the two major components of this fraction was obtained by conversion of the remainder to the b i s - T F A derivative (3b) of the diol ester (3a). E C - G C analysis of this derivative on the 3% OV-225 column at 165°C, with a nitrogen carrier flowrate of 30 ml/min gave a peak eluting, and co-chromatographing, with the authentic derivative of J H I I I at 7-5 rain. On the 3% OV-17 column at 170°C and 30 ml/rnin nitrogen flowrate, a peak eluted and co-chromatographed with the authentic JH I I I derivative at 5"8 min. This result was confirmed by G L C analysis (and co-chromatography) on the 20m OV-101 column, using flame ionization detection. F r o m these G L C analyses, and the analysis procedure with labelled JH I I I , which allowed experimental losses during the workup to be estimated, the endogenous JH I I I titre was estimated to be of the order of 500 to 600 pg per adult insect. Fraction 'B2' was separated by H P L C , eluting with 0"75% aeetonitrile+0.15% iso-propanol in pentane at 2 ml/min. No peak was detected by the UV monitor at the retention time of (E,E)-MF b u t the fractions which might contain it were examined by G L C on the 20m OV-101 glass capillary column at 155°C and a nitrogen flowrate of 3 ml/min. N o (E,E)-MF was detected. Allowing for losses during work-up, it would have been possible to detect 20 pg (E,E)-MF per locust, if present.
The analysis of hopper haemolymph fractions which contained 'BI ' activity The purified T L C fraction containing ' B I ' activity was fractionated further by H P L C using the system employed during the analysis of the J H ~a,
I 25
f 20
I 15
I 10
I 5
I 0
Time, minutee
Fig. 3. Gas chromatograrn of HPLC fraction separated from the haemolymph extract from adults. JH I I I eluted at 13.2 min. Column: 20mx0-32 mm i.d. glass capillary, wall-coated with Ucon oil 50HB-2000. Operating conditions: Isothermal at 165°C, with 1.5 ml/min carrier gas, and flame ionization detection.
38
MARGARETM. BLIGHTAND MICHAELJ. WENHAM
haemolymph extract from adults. T h e quantity of J H I I I present was again below the detection limit under the conditions used, and other peaks in the chromatogram had to be used to delineate the fraction containing it. J H I I I was identified in this fraction by G L C analysis on the OV-101 and Carbowax 20M capillary columns. From the G L C results, and making an estimate of the experimental losses, the J H I I I content per fourth instar hopper was of the order of 150 pg.
DISCUSSION This study has confirmed the presence of J H I I I in S. gregaria adults (TRAUTMANe t al., 1974b) and, in addition, has shown that it is present in S. gregaria hoppers. T h e quantity found in adult blood (0"6 ng/insect) is of the same order as that found in older adults (whole bodies) by TRAUTMAN et al. (1974b) (0"5 ng/g body weight, i.e. 1"4 ng/ animal approx.) Th e estimates of the titres may, however, be too low, since the aim of this study was primarily one of identification. At least some of the juvenile hormone activity in fraction ' B I ' (BLIGHT and WENHAM, 1975) from hoppers and adults is due to J H III, but the presence of other juvenilising substances in this fraction cannot be excluded since it was not possible to obtain the desired bioassay data. A search was made for (E,E)-methyl farnesoate in haemolymph from adults because it is probably a biosynthetic precursor, in vivo, of J H III. Failure to detect it in haemolymph may indicate that it is synthesised, hut not released from the corpus aUatum, in vivo. PRATTand TOBE (1974) found that corpora allata from adult female S. gregaria, incubated in vitro with [3H]-(E,E)-farnesenic acid, synthesised (E,E)-MF but it was not released into the medium. T h e presence of J H I I I in many sexually mature adult insects (JUDY et al., 1973a, b; 1975; MULLER et al., 1974; TRAUTMANNet al., 1974a, b; this study) suggests to us that it may contribute primarily to the gonadotropic action of the corpus allatum (WIGGLESWORTH,1970); and that J H I, J H II and other unknown juvenilising substances (e.g. ' A I ' and 'A2' in S. gregaria; BLIGHT and W~HAM, 1975) may be mainly concerned with larval development. On the other hand, lack of evidence so far for the presence of J H I I I in larvae may simply reflect the greater difficulties involved in the isolation of juvenilising substances from larvae.
Note added in proof--JH I, II and III have now been identified in the haemolymph of larvae of the cockroach,
Nauphoeta cinema [LANZREIN B., HASHIMOTO M., PARMAKOVlCH V., NAKANISHI m., WILHELM R., and Li)SCHER M. (1975). Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nauphoeta cinema. Life Sci. 16, 12711284]. Acknowledgements--We thank the Insectory staff in the Unit for rearing the insects, Dr A. F. WHITE for the radiolabelled substances and Dr F. A. MELLONfor mass spectra. We are grateful to Professor A. W. JOHNSON for his advice and encouragement during the course of this work.
REFERENCES BLIGHT M. M. and WENHAM M. J. (1976) Juvenile hormone activity in larvae and adult females of Schistocerca gregaria. J. Insect. Physiol. 22 141-145. JuDY K. J., SCHOOLEYD. A., DUNHAM L. L., HALL M. S., BERGOT B. J., and SIDDALL J. B. (19738) Isolation, structure and absolute configuration of a new natural insect juvenile hormone from Manduca sexta. Proc. nat. Acad. Sci. U.S.A. 70, 1509-1513. JUDYK. J., SCHOOLEYD. A., HALL M. S., BERGOTB.J., and SIDDALL J. B. (1973b) Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro. Life Sci. 13, 1511-1516. JUDY K. J., SCHOOLEY D. A., TROETSCHLER R. G., JENNINGSR. C., BERGOTB.J., and HALLM. S. (1975) Juvenile hormone production by corpora allata of Tenebrio molitor in vitro. Life Sci. 16, 1059-1066. MULLERP. J., MASNERP., TRAUTMANNK. H., SUCH~M., and WIPE H-K. (1974) The isolation and identification of juvenile hormone from cockroach corpora allata in vitro. Life Sci. 15. 915-921. PERRIN D. D., ARMAREOOW. L. F., and PERmN D. R. (1966) Purification of Laboratory Chemicals. Pergamon Press, Oxford. PaATT G. E. and Tone S. S. (1974) Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586. SNYDEa L. R. (1971) Modern Practice of Liquid Chromatography (Ed. by KInKLANI)J. J.), pp. 223-225, 227228. Wiley-Interscience, New York. TRAUTMANNK. H., SCHULERA., SUCH# M., and WIPF H-K. (1974a) Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen yon Insekten. Nachweis yon 10, ll-epoxy-3,7,11trimethyl-2-tram-6-trans-dodecadiensiiuremethylester in Melolontha melolontha. Z. Naturf. 29c, 161-168. TRAUTMANNK. H., MASNERP., SCHULERA., SUCH? M., and WIPE H-K. (1974b) Evidence of the juvenile hormone methyl (2E,6E)-10, ll-epoxy-3,7,11-trimethyl-2,6-dodecadienoate (JH-3) in insects of four orders. Z. Naturf. 29c 757-759. WIOOLmWORTHV. B. (1970) Insect Hormones. Oliver & Boyd, Edinburgh.