Journal o f Chemical Ecology, Vol. 9, No. 10, 1983
MUSHROOM CHEMICAL DEFENSE: Pungent Sesquiterpenoid Dialdehyde Antifeedant to Opossum
SCOTT
M. C A M A Z I N E , 1 J A M E S and JERROLD
F. R E S C H , 2 T H O M A S MEINWALD 2
EISNER, l
t Division of Biological Sciences, Section of Neurobiology and Behavior 2Department o f Chemistry, Cornell University Ithaca, New York 14853
(Received November 1, 1982; revised January 4, 1983) Abstract A new bioassay employing a natural fungivore, the opossum Didelphis virginiana, is described. Using this bioasssay, eighteen species of fungi were tested for palatability. Five species of mushrooms, all of which taste pungent to humans, were found to be unpalatable to the opossum. From the least palatable of these, Lentinellus ursinus, the pungent principle was isolated and identified as isovelleral, a previously described fungal metabolite. The compound was shown to be a potent antifeedant to opossums. By means of difference NOE and relaxation time NMR studies, the relative configuration and solution conformation of isovelleral were deduced. Key Words--Tricholomataceae, Lentinellus ursinus, sesquiterpene, antifeedant, bioassay, isovelleral, mushrooms, opossum, Didelphis virginiana, pungency, dialdehyde.
INTRODUCTION M u s h r o o m s are a w e l l - k n o w n s o u r c e o f t o x i n s , i n c l u d i n g e m e t i c s , c a t h a r t i c s , h a l l u c i n o g e n s , a n d liver p o i s o n s ( L i n c o f f a n d M i t c h e l , 1977). D e s p i t e t h e c o n s i d e r a b l e w o r k t h a t has b e e n u n d e r t a k e n to e l u c i d a t e t h e c h e m i s t r y a n d p h a r m a c o l o g y o f t h e s e s u b s t a n c e s , t h e i r a c t u a l f u n c t i o n in n a t u r e has r e m a i n e d u n k n o w n . W o r k r e c e n t l y d o n e o n m u s h r o o m s o f t h e f a m i l y R u s s u l a c e a e has led to t h e i s o l a t i o n o f a g r o u p o f s e s q u i t e r p e n e s , i n c l u d i n g t w o w h i c h are p u n g e n t - t a s t i n g to h u m a n s (List a n d H a c k e n b e r g , 1969; M a g n u s s o n et al., 1972; M a g n u s s o n et al., 1973). W e p r e s e n t e v i d e n c e 1439 0098-0331,83/1000-1439503,00/0 9 1983 Plenum Publishing Corporation
1440
CAMAZINE ET AL.
that the pungent Russulaceae are distasteful to a natural fungivore, the opossum Didelphis virginiana. An additional species, Lentinellus ursinus, a pungent nonpoisonous mushroom of a different family (Tricholomataceae), is also rejected by the opossum. We report here the isolation and characterization of the compound responsible for this antifeedant activity. METHODS AND MATERIALS
Opossum Bioassay. Preliminary tests with opossums in captivity has shown these animals to feed readily on commercial mushrooms (Agaricus bisporus). Presented with pieces of mushroom, they approached these, seized them in their mouths, and then, with occasional manipulative aid of the front paws, proceeded to eat them in their entirety. An opossum commonly consumed several dozen such items in quick succession. A measure of the relative palatablity of various wild mushroom species was obtained by means of a feeding protocol used previously in a bioassay with unpalatable insects and thrushes (Eisner et al., 1978). Individual opossums, tested in daily feeding sessions, were offered fresh pieces (approximately 2 • 2 • 2 cm) of a given test species of mushroom, and comparable pieces of Agaricus bisporus as the edible control. A total of 18 species of m u s h r o o m c o m m o n to the northeastern United States was collected for testing. Each species was presented in a single feeding session to several (6-10) opossums. Mushroom pieces were presented one at a time in three-item sequences consisting of two pieces of Agaricus and one randomly interspersed piece of the test mushroom. The total number of items (test plus control) presented to each opossum per session ranged from 15 to 27. Results were scored as fate of individual mushroom pieces. If an item was totally consumed, it was scored as eaten; if it was partly eaten, rejected on close inspection (tasted, sniffed, a n d / o r manipulated), or ignored from a distance, it was scored as rejected. If an item at the end of a session was rejected, it was not tallied since the negative response might have been due to satiation of the animal. For each mushroom, the scores from all the opossums were combined to calculate a palatability rating defined as the percent eaten. For Agarieus bisporus, the total number of control items from all tests was used for the calculation. For the screening of purified compounds, we utilized a bioassay similar to the preceding one except that only Agaricus bisporus mushrooms were used. These mushrooms were of two kinds: the treated items to which a topicaldosage of the compound (dissolved in pentane or hexane) was added, and control items to which the solvent alone was added. In each case the solvent was allowed to evaporate before the item was presented for feeding. Extraction and Fractionation Procedures'. A sample of the fungus was
1441
MUSHROOM CHEMICAL DEFENSE
extracted in a Soxhlet apparatus with pentane, ether, and methanol in succession. At each stage of the procedure, the extracts were tasted by one of us. The pentane extract, which was the sole pungent fraction, was partially crystalline. Recrystallization from hexane yielded a number of nonpungent, saturated fatty acids, while concentrating the pungency in the mother liquors. Subsequent fractionation of the mother liquors on a column of silica gel in hexane with increasing proportions of ether yielded a single, intensely peppery, crystalline compound (ca. 0.15% of the fresh weight of the fungus). This compound could be detected in a dosage as low as 1 #g, dissolved in hexane, placed on a 1-cm disk of filter paper applied to the tongue. RESULTS
AND DISCUSSION
It is evident from the opossum bioassay that Didelphis groups the mushrooms we tested into two distinct categories (Figure 1). The five species in the first group were generally rejected. The mean percent palatability (X ___ SD) of these mushrooms was 7 _+ 7 (range 2-18). The other group, the palatable mushrooms, comprised 13 species whose mean percent palatability was 85 _+ 13 (range 56-100). Members of nine families, the Hygrophoraceae, Paxillaceae, Boletaceae, Agaricaceae, Entolomataceae, Lycoperdaceae, Amanitaceae, Strophariaceae, and Tricholomataceae, are generally acceptable while those of the Russulaceae and a single member of the Tricholomataceae are quite unpalatable by comparison. The unpalatability of the Russulaceae, given their peppery taste and similar chemistry (Andina et al., 1980), was expected. The opossums handled Lentinellus ursinus in much the same manner. The fungi were often spat out within seconds, and the animals frequently salivated profusely, frothed at the mouth, shook their heads from side to side, and wiped their muzzles in their fur or on the bottom of the cage. When an unpalatable item was rejected, it was nearly always approached beforehand and apparently sniffed. This behavior prompted an investigation of the chemistry of Lentinellus ursinus. A single, crystalline compound was isolated (see Methods and Materials) which accounted for all of this mushroom's pungency. The physical and spectral data (melting point, optical rotation, proton and carbon-13 nuclear magnetic resonance, infrared, ultraviolet, and mass spectra) of this compound led to its unambiguous identification as isovelleral (Figure 2). Although this compound had previously been isolated from two pungent members of the Russulaceae, Lactarius vellereus and L. pergarnenus (Magnusson et al., 1972), its relative stereochemistry had not been rigorously established. While earlier workers had presented good evidence that the bridgehead protons H-4, and H-10 were cis to each other, the orientation of the cyclopropane ring with respect to these protons remained unsubstantiated.
1442
CAMAZINE ET AL.
I00 Tricholomataceae 9 Russulaceae 9 Hygrophoraceae Paxitlaceae dl~ Boletoceoe "/r Agoricoceoe I~
A 75
t~
Entolomotaceoe ~ Lycoperdoceae n Strophorioceoe ~
E 50 >_ F-
,
,
.=
!!!!ii !:iil;; ilii::;!i;; i;; i!:;i; ;:!:! i:: i :}:i:i i{ i!:ti;:il
'i:i~::!iii 'ii!ii[:!:::!=:ii:ii!!iii::':!:!i:i!;iii!iii!!:!lii!!;t:ii:i 9 9 9 9 9 iiii !~:i ii::~ :~ ii,~ :~i ~ :~:ii: ~i;=:~ :A ~ ! _~:':~: "~ . .: . . . .
..:., . . . .
:.. . . . . :.. . . . . .
-.......
-~ { .* ~ :~!:~::~ :::::: ::~:i:: :~::'~::i::. '_~ :':'.::,~ :'~. ~ .~ ~ .~ .~ :~: ~i :::::::::::::::::::::: ~::~.:~::k, :~: ~ ~ ~ ~ ~ :~-::~':'E:'~:/:'_~:I:.~:I:.~'I:%:I:~:'.~:I'-~:I:~,:I'~:I:,~
MUSHROOM CHEMICAL DEFENSE: Pungent Sesquiterpenoid Dialdehyde Antifeedant to Opossum
SCOTT
M. C A M A Z I N E , 1 J A M E S and JERROLD
F. R E S C H , 2 T H O M A S MEINWALD 2
EISNER, l
t Division of Biological Sciences, Section of Neurobiology and Behavior 2Department o f Chemistry, Cornell University Ithaca, New York 14853
(Received November 1, 1982; revised January 4, 1983) Abstract A new bioassay employing a natural fungivore, the opossum Didelphis virginiana, is described. Using this bioasssay, eighteen species of fungi were tested for palatability. Five species of mushrooms, all of which taste pungent to humans, were found to be unpalatable to the opossum. From the least palatable of these, Lentinellus ursinus, the pungent principle was isolated and identified as isovelleral, a previously described fungal metabolite. The compound was shown to be a potent antifeedant to opossums. By means of difference NOE and relaxation time NMR studies, the relative configuration and solution conformation of isovelleral were deduced. Key Words--Tricholomataceae, Lentinellus ursinus, sesquiterpene, antifeedant, bioassay, isovelleral, mushrooms, opossum, Didelphis virginiana, pungency, dialdehyde.
INTRODUCTION M u s h r o o m s are a w e l l - k n o w n s o u r c e o f t o x i n s , i n c l u d i n g e m e t i c s , c a t h a r t i c s , h a l l u c i n o g e n s , a n d liver p o i s o n s ( L i n c o f f a n d M i t c h e l , 1977). D e s p i t e t h e c o n s i d e r a b l e w o r k t h a t has b e e n u n d e r t a k e n to e l u c i d a t e t h e c h e m i s t r y a n d p h a r m a c o l o g y o f t h e s e s u b s t a n c e s , t h e i r a c t u a l f u n c t i o n in n a t u r e has r e m a i n e d u n k n o w n . W o r k r e c e n t l y d o n e o n m u s h r o o m s o f t h e f a m i l y R u s s u l a c e a e has led to t h e i s o l a t i o n o f a g r o u p o f s e s q u i t e r p e n e s , i n c l u d i n g t w o w h i c h are p u n g e n t - t a s t i n g to h u m a n s (List a n d H a c k e n b e r g , 1969; M a g n u s s o n et al., 1972; M a g n u s s o n et al., 1973). W e p r e s e n t e v i d e n c e 1439 0098-0331,83/1000-1439503,00/0 9 1983 Plenum Publishing Corporation
1440
CAMAZINE ET AL.
that the pungent Russulaceae are distasteful to a natural fungivore, the opossum Didelphis virginiana. An additional species, Lentinellus ursinus, a pungent nonpoisonous mushroom of a different family (Tricholomataceae), is also rejected by the opossum. We report here the isolation and characterization of the compound responsible for this antifeedant activity. METHODS AND MATERIALS
Opossum Bioassay. Preliminary tests with opossums in captivity has shown these animals to feed readily on commercial mushrooms (Agaricus bisporus). Presented with pieces of mushroom, they approached these, seized them in their mouths, and then, with occasional manipulative aid of the front paws, proceeded to eat them in their entirety. An opossum commonly consumed several dozen such items in quick succession. A measure of the relative palatablity of various wild mushroom species was obtained by means of a feeding protocol used previously in a bioassay with unpalatable insects and thrushes (Eisner et al., 1978). Individual opossums, tested in daily feeding sessions, were offered fresh pieces (approximately 2 • 2 • 2 cm) of a given test species of mushroom, and comparable pieces of Agaricus bisporus as the edible control. A total of 18 species of m u s h r o o m c o m m o n to the northeastern United States was collected for testing. Each species was presented in a single feeding session to several (6-10) opossums. Mushroom pieces were presented one at a time in three-item sequences consisting of two pieces of Agaricus and one randomly interspersed piece of the test mushroom. The total number of items (test plus control) presented to each opossum per session ranged from 15 to 27. Results were scored as fate of individual mushroom pieces. If an item was totally consumed, it was scored as eaten; if it was partly eaten, rejected on close inspection (tasted, sniffed, a n d / o r manipulated), or ignored from a distance, it was scored as rejected. If an item at the end of a session was rejected, it was not tallied since the negative response might have been due to satiation of the animal. For each mushroom, the scores from all the opossums were combined to calculate a palatability rating defined as the percent eaten. For Agarieus bisporus, the total number of control items from all tests was used for the calculation. For the screening of purified compounds, we utilized a bioassay similar to the preceding one except that only Agaricus bisporus mushrooms were used. These mushrooms were of two kinds: the treated items to which a topicaldosage of the compound (dissolved in pentane or hexane) was added, and control items to which the solvent alone was added. In each case the solvent was allowed to evaporate before the item was presented for feeding. Extraction and Fractionation Procedures'. A sample of the fungus was
1441
MUSHROOM CHEMICAL DEFENSE
extracted in a Soxhlet apparatus with pentane, ether, and methanol in succession. At each stage of the procedure, the extracts were tasted by one of us. The pentane extract, which was the sole pungent fraction, was partially crystalline. Recrystallization from hexane yielded a number of nonpungent, saturated fatty acids, while concentrating the pungency in the mother liquors. Subsequent fractionation of the mother liquors on a column of silica gel in hexane with increasing proportions of ether yielded a single, intensely peppery, crystalline compound (ca. 0.15% of the fresh weight of the fungus). This compound could be detected in a dosage as low as 1 #g, dissolved in hexane, placed on a 1-cm disk of filter paper applied to the tongue. RESULTS
AND DISCUSSION
It is evident from the opossum bioassay that Didelphis groups the mushrooms we tested into two distinct categories (Figure 1). The five species in the first group were generally rejected. The mean percent palatability (X ___ SD) of these mushrooms was 7 _+ 7 (range 2-18). The other group, the palatable mushrooms, comprised 13 species whose mean percent palatability was 85 _+ 13 (range 56-100). Members of nine families, the Hygrophoraceae, Paxillaceae, Boletaceae, Agaricaceae, Entolomataceae, Lycoperdaceae, Amanitaceae, Strophariaceae, and Tricholomataceae, are generally acceptable while those of the Russulaceae and a single member of the Tricholomataceae are quite unpalatable by comparison. The unpalatability of the Russulaceae, given their peppery taste and similar chemistry (Andina et al., 1980), was expected. The opossums handled Lentinellus ursinus in much the same manner. The fungi were often spat out within seconds, and the animals frequently salivated profusely, frothed at the mouth, shook their heads from side to side, and wiped their muzzles in their fur or on the bottom of the cage. When an unpalatable item was rejected, it was nearly always approached beforehand and apparently sniffed. This behavior prompted an investigation of the chemistry of Lentinellus ursinus. A single, crystalline compound was isolated (see Methods and Materials) which accounted for all of this mushroom's pungency. The physical and spectral data (melting point, optical rotation, proton and carbon-13 nuclear magnetic resonance, infrared, ultraviolet, and mass spectra) of this compound led to its unambiguous identification as isovelleral (Figure 2). Although this compound had previously been isolated from two pungent members of the Russulaceae, Lactarius vellereus and L. pergarnenus (Magnusson et al., 1972), its relative stereochemistry had not been rigorously established. While earlier workers had presented good evidence that the bridgehead protons H-4, and H-10 were cis to each other, the orientation of the cyclopropane ring with respect to these protons remained unsubstantiated.
1442
CAMAZINE ET AL.
I00 Tricholomataceae 9 Russulaceae 9 Hygrophoraceae Paxitlaceae dl~ Boletoceoe "/r Agoricoceoe I~
A 75
t~
Entolomotaceoe ~ Lycoperdoceae n Strophorioceoe ~
E 50 >_ F-
,
,
.=
!!!!ii !:iil;; ilii::;!i;; i;; i!:;i; ;:!:! i:: i :}:i:i i{ i!:ti;:il
'i:i~::!iii 'ii!ii[:!:::!=:ii:ii!!iii::':!:!i:i!;iii!iii!!:!lii!!;t:ii:i 9 9 9 9 9 iiii !~:i ii::~ :~ ii,~ :~i ~ :~:ii: ~i;=:~ :A ~ ! _~:':~: "~ . .: . . . .
..:., . . . .
:.. . . . . :.. . . . . .
-.......
-~ { .* ~ :~!:~::~ :::::: ::~:i:: :~::'~::i::. '_~ :':'.::,~ :'~. ~ .~ ~ .~ .~ :~: ~i :::::::::::::::::::::: ~::~.:~::k, :~: ~ ~ ~ ~ ~ :~-::~':'E:'~:/:'_~:I:.~:I:.~'I:%:I:~:'.~:I'-~:I:~,:I'~:I:,~
