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Toxicity and antifeedant activity of a sesquiterpene lactone from Encelia against Spodoptera littoralis
Phytochemistry,
0031-9422/90$3.00+0.00 0 1990Pergamon Press plc
Vol. 29, No. 11,pp. 3445-3448,1990 Printed in Great Britain.
TOXICITY AND ANTIFEEDANT ACTIVITY OF A SESQUITERPENE LACTONE FROM ENCELIA AGAINST SPODOPTERA LITTORALIS RAJESH P. SRIVASTAVA,* PETER PRoKsCHt
and VICTOR WRAY$
Institut fiir Pharmazeutische Biologic der TU Braunschweig, Mendelssohnstrasse 1, D-3300 Braunschweig, F.R.G.; JGesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig, F.R.G. (Received 23 March 1990) Key Word Index-Enceiia;
Asteraceae;
Spodoptera
iitmdis; sesquiterpene iactones; toxicity; antifeedants.
Abstract-Three major sesquiterpenes (including an acid derivative as well as the two eudesmanolide lactones encelin and farinosin) from Encelia actoni and E. asperijblia were studied for toxicity and antifeedant activity against larvae of the polyphagous Spodoptera littoralis (Noctuidae). The sesquiterpene lactone encelin that contained an a-methylene-ylactone moiety was the most active compound encountered; the other two compounds showed only weak activity. When neonate larvae were kept on an artificial diet spiked with those concentrations of encelin that are present in the plant, high mortality of the larvae as well as significant growth reduction and a pronounced increase in the duration of the larval period were observed. The deleterious effects of encelin were due to toxicity as well as to antifeedant activities. Toxicity of encelin against larvae of S. littoralis was demonstrated by injecting the lactone into the to 111 mg kg- ‘. When haemolymph of final instar larvae. The LD,, was observed at 60 pg per larva corresponding encelin was incorporated into artificial diet at a concentration of 1 pmol g -I fr. wt and offered to second instar larvae, larval weight gain as well as the amount of diet consumed were significantly reduced compared to controls indicating antifeedant activity.
larval growth relative to controls was observed [6]. These data suggested that biologically active coristituents different from the chromenes were present in the crude extracts of these two Encelia species. We have now identified a sesquiterpene lactone as an active constituent of the latter species and report on its toxicity and antifeedant activity against larvae of the polyphagous Spodoptera littoralis (Noctuidae).
INTRODUCTION
The genus Encelia Adans. comprises some 18-20 shrubby perennial species which inhabit the Sonoran and Mojave deserts of the southwestern United States and adjacent Mexico [l]. Several Encelia species such as E. farinosa A. Gray in Torr contribute to the dominant elements of the respective desert flora. Chromenes and benzofurans are prominent natural nroducts in several of ~~____~~~~~~~.~~~~~ ~~the Encelia species as for example in E. farinosa and can be used to identify related species within the genus [2]. Several of the chromenes found in Encelia such as encecalin (2,2-dimethyl-6-acetyl-7methoxychromene) accumulate in high concentrations in the leaves and flowering heads (up to 5% of the dry weight) [2]. Encecalin and some of its derivatives exhibit marked contact toxicity and antifeedant activity against a plethora of herbivorous insects [3-51 and are thought to be an important factor in the chemical defence of Encelia as well as of other species from the Asteraceae [S]. Several Encelia species such as E. actoni Elmer or E. asperijblia (S. F. Blake) Clark & Kyhos either lack chromenes or accumulate only minute concentrations when E. ,-.. furinosa r21. However: ..__.. Lag ., ..“__ mmnnred __-- _r”--- fnr --- exmmle r-- tn -- -when a crude extract from leaves of Encelia asperifolia or E. actoni was incorporated in artificial diets and sub-
RESULTS AND DISCUSSION
Three major sesquiterpenes including a sesquiterpene acid methyl ester (1) as well as the sesquiterpene lactones encelin (2) and farinosin (3) were isolated from leaves of E. asperifolia and E. actoni. The two lactones are cis-
lactonized eudesmanolides whereas the acid derivative is probably a biogenetic precursor of the lactones. The natural concentrations of the three sesquiterpenes in leaves of E. asperifolia or E. actoni were found to vary from approximately 0.5 to 5.0 pmol g- I fr. wt as shown by HPLC analysis. Therefore the compounds were incorporated into artificial diets at concentrations ranging from 0.5 to 5.0 pmol g- l fr. wt and offered to neonate I___..._ _P 0. P I‘LL”ru“J I.‘.._“_l.‘” 11, :_ an ti,,I”111ti ,-J.___;,. +---A;“” ,arvac “1 ,-L&11.5 &,;T\Q.En., “~“UUOU,.1After . ..“I six days the survival of the larvae on the treated diet was monitored and compared to controls (Fig. 1). Presence of encelin (2) in the diet caused a strong dose-dependent mortality of the larvae; the LC,, of encelin was ca
sequently offered to neonate larvae of the polyphagous Peridroma saucia (Noctuidae)
a significant
reduction
in
*Permanent address: Department of Entomology, College of Agriculture, G. B. Pant University of Agriculture SCTechnology, Pantnagar 263 145, Nainital (U.P.), India. t Author to whom correspondence should be addressed.
1.8 pmol g- 1fr. wt. The LC,, of the sesquiterpene lactone encelin is thus virtually identical to that of the chromene encecalin reported earlier [S] which is the most active chromene of chromene-accumulating species of Encelia. In comparison
3445
to 2, the sesquiterpene
lactone 3 as well as
3446
R. P.
SRIVASTAVA
al.
prolonged larval period there was a high mortality of the encelin-treated larvae. For example, the number of surviving larvae reared for the first six days on a diet, containing 1.0 pmol 2 g- 1fr. wt dropped from 28 (Fig. 1)
0CH3
0
et
14
0 0
8 0.5
/
I
I
I
1
2
3
4
I
5
PMOL
3
G DIET I
the acid derivative 1 had almost no influence on the survival of the larvae (Fig. 1). Encelin (2) was further found to significantly reduce larval growth. At the lowest concentration of 0.5 pmol encelin g- t fr. wt, the’ growth of surviving larvae was reduced to less than 20% compared to the controls (set at 100%) (Fig. 2). The effects of 3 and the sesquiterpene acid derivative 1 were far less pronounced in comparison to encelin. The ED,,s (doses at which larval growth is reduced by half) of both compounds (1 and 3) were almost identical and observed at ca 1.7 pmol g- ’ fr. wt. The surviving larvae of the chronic feeding experiment were subsequently transfered to untreated diet and reared until pupation. The mean larval period (expressed as number of days before pupation) was 24 days for control larvae (Fig. 3) but was found to increase strongly in a dose-dependent manner for those larvae that had been exposed to diet treated with 2. The larval period of larvae treated with 3-5 pmol of encelin g- ’ fr. wt thus increased almost two-fold compared to controls. In addition to a
0
0.5
1.0
Fig. 1. Chronic feeding bioassay. Survival of neonate larvae (n = 30) of S. littoralis on diet spiked with different concentrations of compounds 1-3 (survival of controls was set at 100%). Larvae were kept on the diet for six days. 100
0.5
3
4
5
,tJMOL
G DIET I
Fig. 2. Growth of neonate larvae of S. littoralis on diet spiked with different concentrations of compounds l-3. Larvae used for weighing were the survivors from the chronic feeding bioassay (Fig. 1). Growth (= weight) of control larvae was set at 100%.
2.0 /JMOL/G
2
1
3.0 FRESH
4.0
5.0
WEIGHT
Fig. 3. Duration of larval period (number of days needed from hatching to pupation) of treated larvae and of controls (n = 30). Larvae used for this experiment were the survivors of the chronic feeding bioassay (Fig. 1) that had been transferred to control diet after feeding on treated diet for the first six days. Vertical bars represent standard deviations, numbers adjacent to the symbols represent the larvae that survived until1 pupation.
Toxicity and antifeedant activity from Encelia to merely 12 at pupation (Fig. 3). The effect of the other two compounds on the larval growth period was far less pronounced (Fig. 3) Only the highest concentration of the acid derivative 1 (5 pmol g-’ fr. wt) was found to cause a marked prolongation of the larval period when compared to controls. At this concentration of 1 there was also a strong decrease in survival of the larvae compared to the first observation (11 compared to 23 survivors) (Figs 1 and 3). The pronounced delayed mortality of encelin-treated larvae observed after the larvae had been transferred to control diet suggested delayed toxic effects of this sesquiterpene lactone. In order to determine the toxicity of encelin as well as of the other two sesquiterpenes, without possible interference of feeding deterrence, each compound was injected in known amounts ranging from 50 to 100 pg per larva into the haemolymph of final instar larvae (Fig. 4). Toxicity was monitored after 48 hr. Doses of 2 higher than 50 pg per larva were found to cause pronounced mortality. The LD,, was calculated to be 60 pg per larva which is equivalent to 111 mg kg- ’ body wt The other two sesquiterpenes had no toxic effects to the larvae at these concentrations. The chromene ence-
80 60
OJ,
I
50
60
I
I
I
70
00
90
I
100
pG/LARVA
Fig. 4. Toxicity of sesquiterpenes l-3 to last instar larvae of S. littoralis. Each compound was injected at known doses into the haemolymph of the larvae (n = lo), controls were injected with the carrier only. Survival of the larvae was monitored after 48 hr (survival of control larvae was set at 100%).
3441
calin, even though showing contact toxicity against second instar larvae of S. littoralis [S], is non-toxic to final instar larvae when injected into the haemolymph at a dose of 100 ,ug per larva (unpublished results). The injection experiment clearly reveals the importance of the exocyclic methylene group at the lactone ring (cc-methylene-y-lactone moiety) for the toxicity of encelin. The exocyclic methylene group at C-4, which is present in all three sesquiterpenes analysed, has no effect on the toxicity of the compounds because 3 as well as the sesquiterpene acid 1 showed no toxicity when injected into the larvae. The importance of the a-methylene-yla&one moiety for the toxicity of sesquiterpene lactones against insects has for example also been demonstrated with the grasshopper Melanoplus sanguinipes [7, 81. Encelin (2) besides being toxic also has pronounced antifeedant activity against larvae of S. littoralis. Encelin was incorporated into artificial diet at a concentration of 1 pmol g- 1fr. wt. Second instar larvae of S. littoralis were released on the diet. After three days the net weight gain of the larvae, the total amount of diet consumed as well as the relative growth rate (RGR), relative consumption rate (RCR) and efficiency of conversion of ingested food (ECI) were determined and compared to controls (Table 1). None of the larvae feeding on treated diet died during the experiment. However, the presence of encelin significantly reduced feeding as well as dietary utilization (ECI) of the larvae and caused significant reduction of the net weight gain as well as of the RGR relative to the controls. The sesquiterpene acid 1 as well as the la&one 3 were not analysed for antifeedant and growth retarding activity because their effect on survival of neonate larvae in the chronic feeding bioassay (Fig. 1) was only marginal. Farinosin had previously been shown to have no antifeedant activity against larvae of Heliothis zea (Noctuidae) [9]. A significant growth reduction due to feeding inhibition is also observed when second instar larvae are allowed to feed on diet containing 1 ,umol encecalin (unpublished results). The present study demonstrates that 2 is both toxic and antifeedant to larvae of the polyphagous S. littoralis at concentrations that correspond to the natural concentration of this sesquiterpene lactone in leaves of E. actoni and E. asperijblia. The two structurally related sesquiterpenes (1 and 3) that co-occur with encelin have no pronounced biological activity against S. littoralis. The antifeedant
Table 1. Feeding, growth and dietary utilization of compound 2 by second instar larvae of S. littoralis Control (f k s.d.) Net weight gain (mg fr. wt) Diet consumed (mg dry wt) Relative growth rate (mg dry wt mg-’ dry wt day-‘) Relative consumption rate (mg dry wt mg-’ dry wt day-‘) Efficiency of conversion of ingested food (mg dry wt mg-‘dry wt day-’ x 100)
Encelin (n + s.d.)
t-value
P
io.001
27.86 + 8.49
6.62 k 1.56
11.79
16.88+ 5.67
5.96 + 2.58
8.39
0.88 + 0.08
0.49 * 0.05
18.13
l&I&O.37
2.22* 1.23
2.87
2.14
< 0.05
24.43 _+5.96
19.36k9.67
n = 24 for each group; larvae were kept individually on diet (treated with 1 pmol en&in g- ’ fr. wt or control diet) for three days.
R. P. SRIVASTAVA et al.
3448
activity of 2 is comparable to that of encecalin whereas the toxic properties of 2 (at least to last instar larvae of S. littoralis) are even more pronounced than observed for encecalin. It is therefore suggested that the presence of the sesquiterpene la&one 2 found in two Encelia species that 1._-.-.-.-- .___~ 1.. -r-_~~-l _3_ ._‘_ ._~~~ I art: poor m cnrwnenes may oe 01 equal auaprlve Imporrante for the chemical defence of these species as the presence of encecalin in the chromene-rich taxa of this genus. EXPERIMENTAL
Isolation and quantijcation of compounds. Plant material of E. actoni and E. asperifblia was collected in spring and summer of 1984 and 1988 in California and Baja California (Mexico). Dates and localities can be obtained from one of us (P.P). Voucher specimens are deposited in the herbarium of P.P. Identification of the plants was secured by comparison with voucher specimens that are on file in the herbarium of the _ ,.P Lmnornia State Poiytechnic University, Pomona, U.S.A. Leaves were dried, ground and extracted with MeOH. The extracts were taken to dryness and chromatographed on a column filled with silica gel with CH,Cl,-MeOH (49:l) as eluent. Fractions (20 ml) were monitored by TLC on silica gel (Fzs4) using the same solvent. Detection was under UV,,,. Similar fractions were combined and purified by CC on Sephadex LH-20. Final purification of the sesquiterpenes was achieved by recrystallization from petrol-EtOAc. ‘H and 13C’NMR spectra were recorded in CDCI, on 300 or 400 MHz spectrometers. Mass spectra of compounds l-3 were obtained by CC-MS. CC-MS: Carlo Erba 5160 GC (equipped with a 30 m x 0.32 mm id. fused silica quartz capillary column coated with DB-1, temp. program 100-300” at 6‘ min..‘, quadrupole mass spcctromeier. The spectrdi data obiained were ideniicai to those published previously [lo]. Quantification of the sesquiterpcnes in the crude extracts from E. actoni and E. asperijolia was achieved by HPLC (UV detection at 254 nm) by the external standard method using the purified compounds. The crude extracts were separated by gradient elution as described previously [I 11. Experiments with insects. Larvae of S. [irtoralis were from a laboratory colony reared on artificial diet under controlled conditions as described previously [S]. The chronic feeding bioassays were conducted with neonate larvae that were kept on diet spiked with different concentrations (0.5-5.0 pmol gg ’ fr. wt) of the compounds studied. After 6 days survival and larval weight (=growth) of the surviving larvae were monitored and compared io conirois. Surviving iarvae were subsequentiy transferred to control diet and reared further until1 all larvae had pupated. Number of surviving larvae and duration of larval period (number of days required to pupate) were recorded.
Known amounts of each compound (S~lOO~g) were dissolved in 2 ~12-ethoxyethanol and injected into the haemolymph of last instar larvae of S. littoralis with the help of a microliter syringe. Controls were injected with the carrier only. After 48 hr mortality was recorded and compared to controls. cnceun \r, was mcorporarea uuo rne artificiai diet at a concentration of 1 pmol go’ fr. wt. Second instar larvae of S. littoralis were weighed individually and transferred individually to treated diet cubes of known weight. Control larvae were weighed and transferred to the control diet. After 3 days larvae and the remaining diet were weighed again to determine the net larval weight gain and the amount of diet consumed. RGR, RCR and EC1 were calculated according to previously described methods 112, 131. Analysis of variance and r-test were performed according to standard procedures 1141. Acknowledgements-Financial support of this project by a grant of the DFG (Schwerpunkt ‘Chemische akologie’) to P.P. is gratefully acknowledged. R.P.S. wishes to thank the DAAD for a schoiarship. We are indebted to Dr T. Vieregge (insi. fiir Pharmakologie und Toxikologie, TU Braunschweig) for statistical analysis and to MS Claudia Wahrenburg for skillful technical assistance. REFERNCES
1. Blake, S. F. (1913) Proc. Am. Acad. Arts 49, 346. 2. Proksch, P. and Clark, C. (1987) Phytochemistry 26, 171. 3. Isman, M. B., Proksch, P. and Yan, J.-Y. (1987) Entomol. Exp. Appl. 43, 87. P. (1986) Nat4. Isman, M. B., Yan, J.-Y. and Proksch, urwissenschaften 13, 500. 5. Srivastava, R. P. and Proksch, P. (1990) EntomoLogia Gen. . ,. eraus (m pressj. 6. Isman, M. B., Proksch, P. and Clark, C. (1990) Rec. Ado. Phytochem. 24, (in press). 7. Isman, M. B. (1985) Pest. Biochem. Physiol. 24, 348. 8. Arnason, J. T., Isman, M. B., Philogene, B. J. R. and Waddell, T. G. (1987) J. Nat. Prod. 50, 690. 9. Wisdom, C. S., Smiley, J. T. and Rodriguez, E. (1983)5. Econ. Entomol. 76, 993. 10. Fischer, N. H., Olivier, E. J. and Fischer, H. D. (1979) Fortschr. Chem. Org. Naturst. 38, 1. 11. Strack. D., Proksch, P. and Giilz, P.-G. (1980) Z. Naturforsch. 3!5c, 915. 12. Scriber, J. M. and Slansky, F. (1981) Ann. Rev. Entomol. 26, 183. __ ,_ ^^,.. ii ,_ _... i3. Kogan, M. (IYX6) m ‘insect-Piantinteractions (Miller, .i. R. and Miller, T. A., eds), p. 155. Springer, Heidelberg. 14. Weber, E. (1967) Grundriss der Biologischen Statistik. Gustav Fischer, Stuttgart.
0031-9422/90$3.00+0.00 0 1990Pergamon Press plc
Vol. 29, No. 11,pp. 3445-3448,1990 Printed in Great Britain.
TOXICITY AND ANTIFEEDANT ACTIVITY OF A SESQUITERPENE LACTONE FROM ENCELIA AGAINST SPODOPTERA LITTORALIS RAJESH P. SRIVASTAVA,* PETER PRoKsCHt
and VICTOR WRAY$
Institut fiir Pharmazeutische Biologic der TU Braunschweig, Mendelssohnstrasse 1, D-3300 Braunschweig, F.R.G.; JGesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig, F.R.G. (Received 23 March 1990) Key Word Index-Enceiia;
Asteraceae;
Spodoptera
iitmdis; sesquiterpene iactones; toxicity; antifeedants.
Abstract-Three major sesquiterpenes (including an acid derivative as well as the two eudesmanolide lactones encelin and farinosin) from Encelia actoni and E. asperijblia were studied for toxicity and antifeedant activity against larvae of the polyphagous Spodoptera littoralis (Noctuidae). The sesquiterpene lactone encelin that contained an a-methylene-ylactone moiety was the most active compound encountered; the other two compounds showed only weak activity. When neonate larvae were kept on an artificial diet spiked with those concentrations of encelin that are present in the plant, high mortality of the larvae as well as significant growth reduction and a pronounced increase in the duration of the larval period were observed. The deleterious effects of encelin were due to toxicity as well as to antifeedant activities. Toxicity of encelin against larvae of S. littoralis was demonstrated by injecting the lactone into the to 111 mg kg- ‘. When haemolymph of final instar larvae. The LD,, was observed at 60 pg per larva corresponding encelin was incorporated into artificial diet at a concentration of 1 pmol g -I fr. wt and offered to second instar larvae, larval weight gain as well as the amount of diet consumed were significantly reduced compared to controls indicating antifeedant activity.
larval growth relative to controls was observed [6]. These data suggested that biologically active coristituents different from the chromenes were present in the crude extracts of these two Encelia species. We have now identified a sesquiterpene lactone as an active constituent of the latter species and report on its toxicity and antifeedant activity against larvae of the polyphagous Spodoptera littoralis (Noctuidae).
INTRODUCTION
The genus Encelia Adans. comprises some 18-20 shrubby perennial species which inhabit the Sonoran and Mojave deserts of the southwestern United States and adjacent Mexico [l]. Several Encelia species such as E. farinosa A. Gray in Torr contribute to the dominant elements of the respective desert flora. Chromenes and benzofurans are prominent natural nroducts in several of ~~____~~~~~~~.~~~~~ ~~the Encelia species as for example in E. farinosa and can be used to identify related species within the genus [2]. Several of the chromenes found in Encelia such as encecalin (2,2-dimethyl-6-acetyl-7methoxychromene) accumulate in high concentrations in the leaves and flowering heads (up to 5% of the dry weight) [2]. Encecalin and some of its derivatives exhibit marked contact toxicity and antifeedant activity against a plethora of herbivorous insects [3-51 and are thought to be an important factor in the chemical defence of Encelia as well as of other species from the Asteraceae [S]. Several Encelia species such as E. actoni Elmer or E. asperijblia (S. F. Blake) Clark & Kyhos either lack chromenes or accumulate only minute concentrations when E. ,-.. furinosa r21. However: ..__.. Lag ., ..“__ mmnnred __-- _r”--- fnr --- exmmle r-- tn -- -when a crude extract from leaves of Encelia asperifolia or E. actoni was incorporated in artificial diets and sub-
RESULTS AND DISCUSSION
Three major sesquiterpenes including a sesquiterpene acid methyl ester (1) as well as the sesquiterpene lactones encelin (2) and farinosin (3) were isolated from leaves of E. asperifolia and E. actoni. The two lactones are cis-
lactonized eudesmanolides whereas the acid derivative is probably a biogenetic precursor of the lactones. The natural concentrations of the three sesquiterpenes in leaves of E. asperifolia or E. actoni were found to vary from approximately 0.5 to 5.0 pmol g- I fr. wt as shown by HPLC analysis. Therefore the compounds were incorporated into artificial diets at concentrations ranging from 0.5 to 5.0 pmol g- l fr. wt and offered to neonate I___..._ _P 0. P I‘LL”ru“J I.‘.._“_l.‘” 11, :_ an ti,,I”111ti ,-J.___;,. +---A;“” ,arvac “1 ,-L&11.5 &,;T\Q.En., “~“UUOU,.1After . ..“I six days the survival of the larvae on the treated diet was monitored and compared to controls (Fig. 1). Presence of encelin (2) in the diet caused a strong dose-dependent mortality of the larvae; the LC,, of encelin was ca
sequently offered to neonate larvae of the polyphagous Peridroma saucia (Noctuidae)
a significant
reduction
in
*Permanent address: Department of Entomology, College of Agriculture, G. B. Pant University of Agriculture SCTechnology, Pantnagar 263 145, Nainital (U.P.), India. t Author to whom correspondence should be addressed.
1.8 pmol g- 1fr. wt. The LC,, of the sesquiterpene lactone encelin is thus virtually identical to that of the chromene encecalin reported earlier [S] which is the most active chromene of chromene-accumulating species of Encelia. In comparison
3445
to 2, the sesquiterpene
lactone 3 as well as
3446
R. P.
SRIVASTAVA
al.
prolonged larval period there was a high mortality of the encelin-treated larvae. For example, the number of surviving larvae reared for the first six days on a diet, containing 1.0 pmol 2 g- 1fr. wt dropped from 28 (Fig. 1)
0CH3
0
et
14
0 0
8 0.5
/
I
I
I
1
2
3
4
I
5
PMOL
3
G DIET I
the acid derivative 1 had almost no influence on the survival of the larvae (Fig. 1). Encelin (2) was further found to significantly reduce larval growth. At the lowest concentration of 0.5 pmol encelin g- t fr. wt, the’ growth of surviving larvae was reduced to less than 20% compared to the controls (set at 100%) (Fig. 2). The effects of 3 and the sesquiterpene acid derivative 1 were far less pronounced in comparison to encelin. The ED,,s (doses at which larval growth is reduced by half) of both compounds (1 and 3) were almost identical and observed at ca 1.7 pmol g- ’ fr. wt. The surviving larvae of the chronic feeding experiment were subsequently transfered to untreated diet and reared until pupation. The mean larval period (expressed as number of days before pupation) was 24 days for control larvae (Fig. 3) but was found to increase strongly in a dose-dependent manner for those larvae that had been exposed to diet treated with 2. The larval period of larvae treated with 3-5 pmol of encelin g- ’ fr. wt thus increased almost two-fold compared to controls. In addition to a
0
0.5
1.0
Fig. 1. Chronic feeding bioassay. Survival of neonate larvae (n = 30) of S. littoralis on diet spiked with different concentrations of compounds 1-3 (survival of controls was set at 100%). Larvae were kept on the diet for six days. 100
0.5
3
4
5
,tJMOL
G DIET I
Fig. 2. Growth of neonate larvae of S. littoralis on diet spiked with different concentrations of compounds l-3. Larvae used for weighing were the survivors from the chronic feeding bioassay (Fig. 1). Growth (= weight) of control larvae was set at 100%.
2.0 /JMOL/G
2
1
3.0 FRESH
4.0
5.0
WEIGHT
Fig. 3. Duration of larval period (number of days needed from hatching to pupation) of treated larvae and of controls (n = 30). Larvae used for this experiment were the survivors of the chronic feeding bioassay (Fig. 1) that had been transferred to control diet after feeding on treated diet for the first six days. Vertical bars represent standard deviations, numbers adjacent to the symbols represent the larvae that survived until1 pupation.
Toxicity and antifeedant activity from Encelia to merely 12 at pupation (Fig. 3). The effect of the other two compounds on the larval growth period was far less pronounced (Fig. 3) Only the highest concentration of the acid derivative 1 (5 pmol g-’ fr. wt) was found to cause a marked prolongation of the larval period when compared to controls. At this concentration of 1 there was also a strong decrease in survival of the larvae compared to the first observation (11 compared to 23 survivors) (Figs 1 and 3). The pronounced delayed mortality of encelin-treated larvae observed after the larvae had been transferred to control diet suggested delayed toxic effects of this sesquiterpene lactone. In order to determine the toxicity of encelin as well as of the other two sesquiterpenes, without possible interference of feeding deterrence, each compound was injected in known amounts ranging from 50 to 100 pg per larva into the haemolymph of final instar larvae (Fig. 4). Toxicity was monitored after 48 hr. Doses of 2 higher than 50 pg per larva were found to cause pronounced mortality. The LD,, was calculated to be 60 pg per larva which is equivalent to 111 mg kg- ’ body wt The other two sesquiterpenes had no toxic effects to the larvae at these concentrations. The chromene ence-
80 60
OJ,
I
50
60
I
I
I
70
00
90
I
100
pG/LARVA
Fig. 4. Toxicity of sesquiterpenes l-3 to last instar larvae of S. littoralis. Each compound was injected at known doses into the haemolymph of the larvae (n = lo), controls were injected with the carrier only. Survival of the larvae was monitored after 48 hr (survival of control larvae was set at 100%).
3441
calin, even though showing contact toxicity against second instar larvae of S. littoralis [S], is non-toxic to final instar larvae when injected into the haemolymph at a dose of 100 ,ug per larva (unpublished results). The injection experiment clearly reveals the importance of the exocyclic methylene group at the lactone ring (cc-methylene-y-lactone moiety) for the toxicity of encelin. The exocyclic methylene group at C-4, which is present in all three sesquiterpenes analysed, has no effect on the toxicity of the compounds because 3 as well as the sesquiterpene acid 1 showed no toxicity when injected into the larvae. The importance of the a-methylene-yla&one moiety for the toxicity of sesquiterpene lactones against insects has for example also been demonstrated with the grasshopper Melanoplus sanguinipes [7, 81. Encelin (2) besides being toxic also has pronounced antifeedant activity against larvae of S. littoralis. Encelin was incorporated into artificial diet at a concentration of 1 pmol g- 1fr. wt. Second instar larvae of S. littoralis were released on the diet. After three days the net weight gain of the larvae, the total amount of diet consumed as well as the relative growth rate (RGR), relative consumption rate (RCR) and efficiency of conversion of ingested food (ECI) were determined and compared to controls (Table 1). None of the larvae feeding on treated diet died during the experiment. However, the presence of encelin significantly reduced feeding as well as dietary utilization (ECI) of the larvae and caused significant reduction of the net weight gain as well as of the RGR relative to the controls. The sesquiterpene acid 1 as well as the la&one 3 were not analysed for antifeedant and growth retarding activity because their effect on survival of neonate larvae in the chronic feeding bioassay (Fig. 1) was only marginal. Farinosin had previously been shown to have no antifeedant activity against larvae of Heliothis zea (Noctuidae) [9]. A significant growth reduction due to feeding inhibition is also observed when second instar larvae are allowed to feed on diet containing 1 ,umol encecalin (unpublished results). The present study demonstrates that 2 is both toxic and antifeedant to larvae of the polyphagous S. littoralis at concentrations that correspond to the natural concentration of this sesquiterpene lactone in leaves of E. actoni and E. asperijblia. The two structurally related sesquiterpenes (1 and 3) that co-occur with encelin have no pronounced biological activity against S. littoralis. The antifeedant
Table 1. Feeding, growth and dietary utilization of compound 2 by second instar larvae of S. littoralis Control (f k s.d.) Net weight gain (mg fr. wt) Diet consumed (mg dry wt) Relative growth rate (mg dry wt mg-’ dry wt day-‘) Relative consumption rate (mg dry wt mg-’ dry wt day-‘) Efficiency of conversion of ingested food (mg dry wt mg-‘dry wt day-’ x 100)
Encelin (n + s.d.)
t-value
P
io.001
27.86 + 8.49
6.62 k 1.56
11.79
16.88+ 5.67
5.96 + 2.58
8.39
0.88 + 0.08
0.49 * 0.05
18.13
l&I&O.37
2.22* 1.23
2.87
2.14
< 0.05
24.43 _+5.96
19.36k9.67
n = 24 for each group; larvae were kept individually on diet (treated with 1 pmol en&in g- ’ fr. wt or control diet) for three days.
R. P. SRIVASTAVA et al.
3448
activity of 2 is comparable to that of encecalin whereas the toxic properties of 2 (at least to last instar larvae of S. littoralis) are even more pronounced than observed for encecalin. It is therefore suggested that the presence of the sesquiterpene la&one 2 found in two Encelia species that 1._-.-.-.-- .___~ 1.. -r-_~~-l _3_ ._‘_ ._~~~ I art: poor m cnrwnenes may oe 01 equal auaprlve Imporrante for the chemical defence of these species as the presence of encecalin in the chromene-rich taxa of this genus. EXPERIMENTAL
Isolation and quantijcation of compounds. Plant material of E. actoni and E. asperifblia was collected in spring and summer of 1984 and 1988 in California and Baja California (Mexico). Dates and localities can be obtained from one of us (P.P). Voucher specimens are deposited in the herbarium of P.P. Identification of the plants was secured by comparison with voucher specimens that are on file in the herbarium of the _ ,.P Lmnornia State Poiytechnic University, Pomona, U.S.A. Leaves were dried, ground and extracted with MeOH. The extracts were taken to dryness and chromatographed on a column filled with silica gel with CH,Cl,-MeOH (49:l) as eluent. Fractions (20 ml) were monitored by TLC on silica gel (Fzs4) using the same solvent. Detection was under UV,,,. Similar fractions were combined and purified by CC on Sephadex LH-20. Final purification of the sesquiterpenes was achieved by recrystallization from petrol-EtOAc. ‘H and 13C’NMR spectra were recorded in CDCI, on 300 or 400 MHz spectrometers. Mass spectra of compounds l-3 were obtained by CC-MS. CC-MS: Carlo Erba 5160 GC (equipped with a 30 m x 0.32 mm id. fused silica quartz capillary column coated with DB-1, temp. program 100-300” at 6‘ min..‘, quadrupole mass spcctromeier. The spectrdi data obiained were ideniicai to those published previously [lo]. Quantification of the sesquiterpcnes in the crude extracts from E. actoni and E. asperijolia was achieved by HPLC (UV detection at 254 nm) by the external standard method using the purified compounds. The crude extracts were separated by gradient elution as described previously [I 11. Experiments with insects. Larvae of S. [irtoralis were from a laboratory colony reared on artificial diet under controlled conditions as described previously [S]. The chronic feeding bioassays were conducted with neonate larvae that were kept on diet spiked with different concentrations (0.5-5.0 pmol gg ’ fr. wt) of the compounds studied. After 6 days survival and larval weight (=growth) of the surviving larvae were monitored and compared io conirois. Surviving iarvae were subsequentiy transferred to control diet and reared further until1 all larvae had pupated. Number of surviving larvae and duration of larval period (number of days required to pupate) were recorded.
Known amounts of each compound (S~lOO~g) were dissolved in 2 ~12-ethoxyethanol and injected into the haemolymph of last instar larvae of S. littoralis with the help of a microliter syringe. Controls were injected with the carrier only. After 48 hr mortality was recorded and compared to controls. cnceun \r, was mcorporarea uuo rne artificiai diet at a concentration of 1 pmol go’ fr. wt. Second instar larvae of S. littoralis were weighed individually and transferred individually to treated diet cubes of known weight. Control larvae were weighed and transferred to the control diet. After 3 days larvae and the remaining diet were weighed again to determine the net larval weight gain and the amount of diet consumed. RGR, RCR and EC1 were calculated according to previously described methods 112, 131. Analysis of variance and r-test were performed according to standard procedures 1141. Acknowledgements-Financial support of this project by a grant of the DFG (Schwerpunkt ‘Chemische akologie’) to P.P. is gratefully acknowledged. R.P.S. wishes to thank the DAAD for a schoiarship. We are indebted to Dr T. Vieregge (insi. fiir Pharmakologie und Toxikologie, TU Braunschweig) for statistical analysis and to MS Claudia Wahrenburg for skillful technical assistance. REFERNCES
1. Blake, S. F. (1913) Proc. Am. Acad. Arts 49, 346. 2. Proksch, P. and Clark, C. (1987) Phytochemistry 26, 171. 3. Isman, M. B., Proksch, P. and Yan, J.-Y. (1987) Entomol. Exp. Appl. 43, 87. P. (1986) Nat4. Isman, M. B., Yan, J.-Y. and Proksch, urwissenschaften 13, 500. 5. Srivastava, R. P. and Proksch, P. (1990) EntomoLogia Gen. . ,. eraus (m pressj. 6. Isman, M. B., Proksch, P. and Clark, C. (1990) Rec. Ado. Phytochem. 24, (in press). 7. Isman, M. B. (1985) Pest. Biochem. Physiol. 24, 348. 8. Arnason, J. T., Isman, M. B., Philogene, B. J. R. and Waddell, T. G. (1987) J. Nat. Prod. 50, 690. 9. Wisdom, C. S., Smiley, J. T. and Rodriguez, E. (1983)5. Econ. Entomol. 76, 993. 10. Fischer, N. H., Olivier, E. J. and Fischer, H. D. (1979) Fortschr. Chem. Org. Naturst. 38, 1. 11. Strack. D., Proksch, P. and Giilz, P.-G. (1980) Z. Naturforsch. 3!5c, 915. 12. Scriber, J. M. and Slansky, F. (1981) Ann. Rev. Entomol. 26, 183. __ ,_ ^^,.. ii ,_ _... i3. Kogan, M. (IYX6) m ‘insect-Piantinteractions (Miller, .i. R. and Miller, T. A., eds), p. 155. Springer, Heidelberg. 14. Weber, E. (1967) Grundriss der Biologischen Statistik. Gustav Fischer, Stuttgart.