- Email: [email protected]
Juglone (5-hydroxy-1,4-naphthoquinone) from Carya ovata, a deterrent to feeding by Scolytus multistriatus
J. Insect Physiol., 1967, Vol. 13, pp. 1453 to
JUGLONE CARYA
BARRY
1459. Pergamon Press Ltd. Printed in Great Britain
(SHYDROXY-1,4-NAPHTHOQUINONE) FROM OliTATA, A DETERRENT TO FEEDING BY SCOLYTUS MULTISTRIATUS”
L. GILBERT,
Department
JAMES
of Entomology,
E. BAKER,
University
and DALE
of Wisconsin,
Madison,
M. NORRIS Wisconsin
(Received 8 May 1967) Abstract-Juglone (5hydroxy-1,4-naphthoquinone) from the bark of Caryu ovata proved to be a deterrent to feeding by Scolytus multistriatus. When juglone was removed from the extracts of the hickory bark, S. multistriatus fed extensively on the remaining extracts. The beetle fed, to varying extents, on bark extracts from several other species of trees. INTRODUCTION
KORRIS and BAKER (1967) briefly summarized
our research group’s understanding of healthy host tree selection (acceptance) by Scolytus spp. bark beetles. The key points of our past findings are: (1) in-flight Scolytus disperse into the crowns of healthy host and non-host species in comparable numbers under comparable environmental conditions (MEYER and NORRIS, 1964; GOEDEN and NORRIS, 1965 ; KORRIS, 1965) and (2) the beetles are arrested and stimulated to feed in healthy host species by a combination of physical (MEYER and NORRIS, 1964; GOEDEN and NORRIS, 1964; NORRIS, 1965) and chemical (LOSCHIAVOet al., 1963; NORRIS and BAKER, 1967) stimuli. Some of our recent studies of the chemical bases for bark beetle distinction between host and non-host species of trees are reported in this paper. MATERIALS AND METHODS Bioassay
techniques
The bioassay method was similar to that of NORRIS and BAKER (1967), but the following addition was made. To further assure the maintenance of the assay disks in a flat position during the assay, a filter paper disk (chosen to fit the inside diameter of the Petri dish assay arena) was fitted over the in-place, diametrically opposed elderberry-pith assay disks. Two holes (for access of beetles to the assay disks) had been previously cut through the covering filter paper with a No. 6 cork borer; the position of each hole was such as to expose all but the margin of each of the two underlying elderberry-pith disks. Each assay disk was further secured by four pointed tips of insect-mounting pins; each tip was inserted through the overlying filter paper into the covered margin of the assay disk. * Approved for publication by the Director of the Wisconsin Agricultural Experiment Station. This study was supported in part by a research grant (GB-6580) from the National Science Foundation, and from funds supplied by the Wisconsin Conservation Department. 03
1453
1454
BARRYL. GILBERT, JAMESE. BAKER,ANDDALE M. NORRIS
Treatment of assay disks with given extracts, pure chemicals, was as described by NORRIS and BAKER (1967).
or solvent
controls
Preparation of the crude extracts Bark samples of each species of tree were taken from healthy specimens. These were weighed and ground in benzene (10 ml/g of fresh bark) using a heavy-duty, spark-proof Waring blender (see NORRIS and BAKER, 1967, for more details). The crude extract treatments, assayed with newly emerged S. multistriatus, were as follows: (1) 5 mg of benzene extracts from the bark of second to fourth-year twigs of Ulmus americana L. ; (2) 5 mg of benzene extracts from the bark of firstyear twigs of MOYUS rubra L. : (3) a 1 : 1 mixture of (1) and (2) ; (4) 5 mg of benzene extracts from the bark of first-year twigs of Malus pumila Mill; (5) a 1 : 1 mixture of twigs of (1) and (4); (6) 5 mg of b enzene extracts from the bark of first-year Magnolia acuminata L. ; (7) a 1 : 1 mixture of (1) and (6); (8) 5 mg of benzene extracts from bark of first-year twigs of Populus tremuloides Michx. ; (9) a 1 : 1 mixture of (1) and (8); (10) 5 mg of benzene extracts from the bark of first-year twigs of Fraxinus americana L. ; (11) a 1 : 1 mixture of (1) and (10); (12) 5 mg of benzene extracts from the bark of first-year twigs of Curya ovata (Mill.) K. Koch; and (13) a 1 : 1 mixture of (1) and (12). Fractionation of the crude benzene extracts from the bark of Carya ovata The crude benzene extracts from C. ovata were initially separated using thinlayer chromatography. A solvent system of Skellysolve B-diethyl ether-acetone (5 : 4 : 1) was used with a silica gel ‘H’ adsorbent. Bands were scraped from preparatory thin-layer plates, and each removed band was eluted successively with 50 ml of acetone and 50 ml of cold 95% ethyl alcohol. When thin-layer chromatography was used to compare previously separated fractions of the crude extract, one pph of acetic acid was added to the previously described solvent system. The thin-layer bands (R, O-60-0-80; and 0.0-O-60 + 0*80-1.00) were assayed in combination with 5 mg of the crude benzene extract of U. americana. The crude benzene extracts from the bark of C. ovata also were fractionated by using a Sweep co-distiller (Kontes, Franklin Park, Illinois). Columns were packed with Pyrex glass wool. Samples of 30 mg of crude extract of C. ovutu per O-5 ml chloroform were injected on a column maintained at 120°C. High-purity, dry nitrogen was forced through the column at 0.6 l/min. Each column subsequently was washed fifteen times with 0.5 ml chloroform at 3 min intervals. The distillate fraction was trapped, and then taken to dryness on a vacuum roto-evaporator at 40°C. The distillate of the crude benzene extract of C. ovnta bark was assayed in combination with 5 mg of the crude benzene extract from the bark of U. americana. The residue from the co-distiller was eluted from the glass wool with chloroform. It was dried in the same manner as the distillate fraction. The distillate fraction from the Sweep was further fractionated by adsorptioncolumn chromatography. The adsorbent was column-grade silica gel (0*05-0.20
FIG. 1. Feeding responses by S. multistriatus adults in 48 hr (extract-treated disks are in the upper row of each experiment). (A) 5 mg of crude benzene extracts from the bark of first-year twigs of C. ovata; (B) combination of 5 m g of the crude extracts from C. ovata with 5 m g of benzene extracts from the bark of second- to fourth-twigs of U. americana ; (C) 5 m g of the extracts from U. americana.
JUGLONE, A DETERRENT TO FEEDING BY SCOLYTUS
1455
MULTISTRIATLIS
mesh) with an eluting solvent system of Skellysolve B-diethyl ether-acetoneacetic acid (50 : 40 : 10 : 1). F ive ml fractions were collected. Infrared spectra of biologically active chemicals were obtained using a Beckman Infrared Spectrophotometer Model IR-5A. EXPERIMENTAL
RESULTS
Studies involving extracts from Carya ovata Beetles did not feed on disks soaked (1) in 5 mg of benzene extracts from the twig bark of C. ovata alone, or (2) in combination with 5 mg of benzene extracts from the twig bark of U. americana (Table 1, Expt. 1) (Fig. 1). The bark of firstTABLE
I-FEEDING
RESPONSE BY NEWLY
EMERGED
Scolytus
multistriatusIN 48
hr ON DISKS
SOAKED IN 5 mg OF BENZENE EXTRACTS FROM TWIG BARK OF ONE OF SEVERAL SPECIES OF TREES ALONE, OR IN COMBINATION
WITH 5 mg OF SUCH EXTRACTS FROM THE BARK OF ul?.?zUS ameriCana .4verage
mm2
of
disk eaten per Expt.
Replicates
Treatment
t
replicate
value
Rating
extract Treated
1
c. ovata
3
ovata+
N.S. N.S.
0 0
0
Non-stimulatory C.
was
ooata
in-
hibitory
americana americana americana americana U. 2
Control
3 3 3
49.8 13.6 41.9
0.6 0 0.7
11.89** N.S 32.4;**
Highly
stimulatory
Stimulatory
t was
F. americana non-inhibitory
5.6
0
N.S.
Non-to-slightly
61.9
0
12.79**
M.
rubra
stimulators
3
M. rubra+ U. americana U. americana M. pumila _M. pumila + U. americana 3
r&a
non-
wk
inhibitory
3 3 3
0 0 0
55.3 17.0 39.9
15.18** 50.18** N.S.
Highly
stimulatorp
Stimulatory
M. pumila
was non-
inhibitory
3 3
P. tremuloides P. tremuloides + U. americana U.
0 0.2
21.9 56.8
N.S. 8.91*
Stimulatoryt
P. tremuloides
was
non-inhibitory
3 3
46.4 9.2
1.1 2.5
3
44.5
0
17.68** N.S.
Highly
stimulatory
Non-to-slightly stimulatory
9-86 *
M.
was
acuminata
non-inhibitory * Significant
at the 0.05 level
t Treatment
was considered
amount replicates
of
feeding
resulted
between
; **
highly
stimulatory treated
in a non-significant
and
significant
at the 0.01 level.
because
of the large numerical
control
disks,
(0.05 level)
statistical
although
t
value.
the
difference variation
in the among
1456
BARRY L. GILBERT, JAMES E. BAKER, ANDDALE M. NORRIS
year twigs of C. ovata (collected in the fall) thus contains a factor that will prevent (deter) S. multistriatus from feeding on an otherwise highly stimulatory extract. When the components of the crude C. ovata extract were separated on preparative thin-layer plates, the inhibitory activity was located in the region between R, 0.60 to 0.80 (Table 2, Expt. 4). The major component of this region was a yellowbrown band, R,0.75. Distillate fractions of the crude benzene extract from C. ovata obtained from the Sweep also were highly inhibitory to beetle feeding (Table 2, Expt. 5). The residue fraction eluted from the glass-wool packing of the co-distiller surprisingly
TABLE ~-FEEDING RESPONSEBY NEWLY EMERGEDScolytus mdtistriatus IN 48 hr ON DISKS SOAKED IN 5 mg OF CRUDE BENZENE EXTRACTS OF i%?lUS Umc?TiLanaTWIG BARK ALONE, OR IN COMBINATION WITH 5 mg OF ONE OF SEVERAL FRACTIONS OF THE CRUDE BENEZENE EXTRACTIVES FROM
TWIG
BARK
OF CU7ya
OVUtU
Average mm2 of disk eaten per replicate Expt. ~4
5
Treatment extract or fraction Thin-layer band (Rf 0.60-0.80) of crude C.ovata 4 U. americana U. americana Thin-layer bands (& 0.0-0.60,0.80-l .OO) of crude C. oz~arn + U. americana Distillates of crude C. ovata through the Sweep + U. americana U. americana Juglone-free? residue of the crude benzene from C. extracts ovata bark, eluted from the column of the Sweep co-distiller
Replicates
Treated
Control
t value
Rating
3
0.5
0
N.S.
R, 0.60-0.80 band of C. ovata was highly inhibitory
3 3
50.2 41.1
0 0
28.69 * * N.S.
Highly stimulatory Ii, 0.0-0.60, 0.801.00 bands of C. oaata were noninhibitory
3
0.5
0
N.S.
3 3
42.8 32.7
0 2.7
14.63 * * N.S.
Distillates of crude C. ovata through Sweep were highly inhibitory Highly stimulatory Stimulatory$
t Juglone was separated from the remainder of the benzene extracts from the bark of C. ovata by co-distillation in a Sweep (Kontes, Franklin Park, Illinois). $ Treatment was considered stimulatory because of the large numerical difference in the amount of feeding between treated and control disks, although the variation among replicates resulted in a non-significant (0.05 level) statistical t value.
JUGLONE,
A DETERRENT
TO FEEDING
BY
SCOLYTL’S
1457
MULTISTRIATUS
stimulated the beetles to feed (Table 2, Expt. 5). This stimulation occurred in the absence of elm extract. The distillate from C. ovata was co-chromatographed on thin-layer with the contents of the region, R, 0.60 to 0.80, from a previous thinlayer separation of the components of the crude benzene extract of hickory. Both samples contained a predominant yellow-brown band, R, 0.75, but the fraction from the co-distillation apparatus contained fewer contaminating compounds than the thin-layer band, and thus was used for further purification of the active chemical by adsorption-column chromatography. Thin-layer chromatography of the yellow-brown fraction eluted from the silica-gel column revealed only one ‘I‘ABLE
J-FEEDING
SOAKED
IN
O’l!Uta
IN
1 mg
RESPONSE OF PURIFIED
COMBINATION
BY (1)
WITH
NEWLY
EMERGED
COMMERCIAL 5
mg
OF
Scolytus
JUGLONE
BENZENE
OR (2)
ndtistriatzu JUGLONE
EXTRACTIVES
FROM
IN
48 hr
ISOLATED THE
ON
FROM
TWIG
DISKS CaYp2
BARK
OF
Ulmus americana -.
Expt 6
.
Treatment extract, or pure chemical 1 mg pure cornmercial juglone + U. americana U. americana 1 mg pure juglone isolated from C. oerata + U. ameri-
Average mm2 of disk eaten per replicate Replicates
Treated
3
2.5
3 3
47.8 0
Control
Rating
t value
0
N.S.
1
mg juglone is highly inhibitory
0.2 0
37.28 * * N.S.
Highly stimulatory 1 mg juglone is highly inhibitory
spot when the plates were examined with U.V. light or after being charred with 500,/o sulphuric acid. An i.r. spectrum of the isolated chemical was obtained, and compared with known spectra in the Sadtler Index. These comparisons and a further review of the known chemistry of the family Juglandaceae indicated that juglone (5-hydroxy-1,4_naphthoquinone) best fit our total data on the chemical. A sample of juglone was obtained from Pierce Chemical Co., Rockford, Illinois. An assay of 1 mg of the commercial juglone mixed with 5 mg of the benzene extracts of U. americana demonstrated strong inhibition of beetle feeding (Table 3, Expt. 6). A sample of the commercial juglone was co-distilled, and then chromatographed on a silica-gel column. The purified (1) commercial juglone and (2) inhibitory chemical from C. ovutu were crystallized from benzene : Skellysolve B and analysed by i.r. spectrophotometry, and the spectra are shown in Fig. 2. The spectra showed two strong carbonyl bands, but no hydroxyl absorption band. The absence of the hydroxyl absorption band was attributed to intramolecular hydrogen bonding.
1458
BARRY
L. GILBERT, JAMES E. BAKER, AND DALE M. NORRIS
2
4
6
s MICRO$
12
14
16
FIG. 2. Scaled reproductions of the i.r. spectra obtained for (1) purified commercial juglone, and (2) purified compound isolated from the first-year twig bark of C. or&z. Note the two carbonyl absorption bands near 6 and 6.1 p.
Studies involving extracts from other tree species The extracts of Fraxinus americana, Malus pumila, and Populus tremuloides stimulated feeding by S. multistriutus (Table 1). The extracts of Mows rubra and Magnolia acuminata, at most, were very slightly stimulatory to the feeding responses of the beetle (Table 1). None of the extracts of the above tree species inhibited beetle feeding when mixed singly with the extract of Ulmus americana (Table 1). DISCUSSION
The demonstrated deterring effect of 5-hydroxy-1,4-naphthoquinone upon otherwise strong stimuli (i.e. extracts of U. americana) to S. multistriatus feeding constitutes significant new knowledge in our growing understanding of the chemical bases of the specificities between insects and their host trees. The existence of a chemical deterrent to insect feeding in non-host perennial woody tissues was proven quantitatively for the first time.
REFERENCES GOEDEN R. D. and NORRIS D. M. (1964) Some biological and ecological aspects of the dispersal fhght of Scolytus quadrispinosus (Coleoptera: Scolytidae). Ann. ent. Sot. Am.
57,743-749. GOEDEN R. D. and NORRIS D. M. (1965) The behavior of Scolytus quadrispinosus optera: Scolytidae) during the dispersal flight as related to its host specificities.
(Cole-
Ann.
ent. Sot. Am. 58, 249-252. LOSCHIAVO S. R., BECK S. D., and NORRIS D. M. (1963) Behavioral responses European elm bark beetle, Scolytus muEtistriutus (Coleoptera: Scolytidae) elm bark. Ann. ent. Sot. Am. 56, 764-768.
of the smaller to extracts of
JUGLONE,A DETERRENTTO FEEDING
BY SCOLYTUS
MULTISTRIATL’S
1459
MEYER H. J. and NORRIS D. M. (1964) Dispersal of the pathogen and vectors of Dutch elm disease. Proc. N. cent. Branch, Ent. sot. Am. 19, 26-29. NORRIS D. M. (1965) In-flight dispersal and orientation of two Scolytus species (Coleoptera) to their host plants for ovipositional purposes. Proc. 12th int. Congr. Ent. (1964), 243. NORRIS D. M. and BAKER J. E. (1967) Feeding responses of the beetle Scolytus to chemical stimuli in the bark of Ulmus. J, Insect Physiol. 13, 955-962.
JUGLONE CARYA
BARRY
1459. Pergamon Press Ltd. Printed in Great Britain
(SHYDROXY-1,4-NAPHTHOQUINONE) FROM OliTATA, A DETERRENT TO FEEDING BY SCOLYTUS MULTISTRIATUS”
L. GILBERT,
Department
JAMES
of Entomology,
E. BAKER,
University
and DALE
of Wisconsin,
Madison,
M. NORRIS Wisconsin
(Received 8 May 1967) Abstract-Juglone (5hydroxy-1,4-naphthoquinone) from the bark of Caryu ovata proved to be a deterrent to feeding by Scolytus multistriatus. When juglone was removed from the extracts of the hickory bark, S. multistriatus fed extensively on the remaining extracts. The beetle fed, to varying extents, on bark extracts from several other species of trees. INTRODUCTION
KORRIS and BAKER (1967) briefly summarized
our research group’s understanding of healthy host tree selection (acceptance) by Scolytus spp. bark beetles. The key points of our past findings are: (1) in-flight Scolytus disperse into the crowns of healthy host and non-host species in comparable numbers under comparable environmental conditions (MEYER and NORRIS, 1964; GOEDEN and NORRIS, 1965 ; KORRIS, 1965) and (2) the beetles are arrested and stimulated to feed in healthy host species by a combination of physical (MEYER and NORRIS, 1964; GOEDEN and NORRIS, 1964; NORRIS, 1965) and chemical (LOSCHIAVOet al., 1963; NORRIS and BAKER, 1967) stimuli. Some of our recent studies of the chemical bases for bark beetle distinction between host and non-host species of trees are reported in this paper. MATERIALS AND METHODS Bioassay
techniques
The bioassay method was similar to that of NORRIS and BAKER (1967), but the following addition was made. To further assure the maintenance of the assay disks in a flat position during the assay, a filter paper disk (chosen to fit the inside diameter of the Petri dish assay arena) was fitted over the in-place, diametrically opposed elderberry-pith assay disks. Two holes (for access of beetles to the assay disks) had been previously cut through the covering filter paper with a No. 6 cork borer; the position of each hole was such as to expose all but the margin of each of the two underlying elderberry-pith disks. Each assay disk was further secured by four pointed tips of insect-mounting pins; each tip was inserted through the overlying filter paper into the covered margin of the assay disk. * Approved for publication by the Director of the Wisconsin Agricultural Experiment Station. This study was supported in part by a research grant (GB-6580) from the National Science Foundation, and from funds supplied by the Wisconsin Conservation Department. 03
1453
1454
BARRYL. GILBERT, JAMESE. BAKER,ANDDALE M. NORRIS
Treatment of assay disks with given extracts, pure chemicals, was as described by NORRIS and BAKER (1967).
or solvent
controls
Preparation of the crude extracts Bark samples of each species of tree were taken from healthy specimens. These were weighed and ground in benzene (10 ml/g of fresh bark) using a heavy-duty, spark-proof Waring blender (see NORRIS and BAKER, 1967, for more details). The crude extract treatments, assayed with newly emerged S. multistriatus, were as follows: (1) 5 mg of benzene extracts from the bark of second to fourth-year twigs of Ulmus americana L. ; (2) 5 mg of benzene extracts from the bark of firstyear twigs of MOYUS rubra L. : (3) a 1 : 1 mixture of (1) and (2) ; (4) 5 mg of benzene extracts from the bark of first-year twigs of Malus pumila Mill; (5) a 1 : 1 mixture of twigs of (1) and (4); (6) 5 mg of b enzene extracts from the bark of first-year Magnolia acuminata L. ; (7) a 1 : 1 mixture of (1) and (6); (8) 5 mg of benzene extracts from bark of first-year twigs of Populus tremuloides Michx. ; (9) a 1 : 1 mixture of (1) and (8); (10) 5 mg of benzene extracts from the bark of first-year twigs of Fraxinus americana L. ; (11) a 1 : 1 mixture of (1) and (10); (12) 5 mg of benzene extracts from the bark of first-year twigs of Curya ovata (Mill.) K. Koch; and (13) a 1 : 1 mixture of (1) and (12). Fractionation of the crude benzene extracts from the bark of Carya ovata The crude benzene extracts from C. ovata were initially separated using thinlayer chromatography. A solvent system of Skellysolve B-diethyl ether-acetone (5 : 4 : 1) was used with a silica gel ‘H’ adsorbent. Bands were scraped from preparatory thin-layer plates, and each removed band was eluted successively with 50 ml of acetone and 50 ml of cold 95% ethyl alcohol. When thin-layer chromatography was used to compare previously separated fractions of the crude extract, one pph of acetic acid was added to the previously described solvent system. The thin-layer bands (R, O-60-0-80; and 0.0-O-60 + 0*80-1.00) were assayed in combination with 5 mg of the crude benzene extract of U. americana. The crude benzene extracts from the bark of C. ovata also were fractionated by using a Sweep co-distiller (Kontes, Franklin Park, Illinois). Columns were packed with Pyrex glass wool. Samples of 30 mg of crude extract of C. ovutu per O-5 ml chloroform were injected on a column maintained at 120°C. High-purity, dry nitrogen was forced through the column at 0.6 l/min. Each column subsequently was washed fifteen times with 0.5 ml chloroform at 3 min intervals. The distillate fraction was trapped, and then taken to dryness on a vacuum roto-evaporator at 40°C. The distillate of the crude benzene extract of C. ovnta bark was assayed in combination with 5 mg of the crude benzene extract from the bark of U. americana. The residue from the co-distiller was eluted from the glass wool with chloroform. It was dried in the same manner as the distillate fraction. The distillate fraction from the Sweep was further fractionated by adsorptioncolumn chromatography. The adsorbent was column-grade silica gel (0*05-0.20
FIG. 1. Feeding responses by S. multistriatus adults in 48 hr (extract-treated disks are in the upper row of each experiment). (A) 5 mg of crude benzene extracts from the bark of first-year twigs of C. ovata; (B) combination of 5 m g of the crude extracts from C. ovata with 5 m g of benzene extracts from the bark of second- to fourth-twigs of U. americana ; (C) 5 m g of the extracts from U. americana.
JUGLONE, A DETERRENT TO FEEDING BY SCOLYTUS
1455
MULTISTRIATLIS
mesh) with an eluting solvent system of Skellysolve B-diethyl ether-acetoneacetic acid (50 : 40 : 10 : 1). F ive ml fractions were collected. Infrared spectra of biologically active chemicals were obtained using a Beckman Infrared Spectrophotometer Model IR-5A. EXPERIMENTAL
RESULTS
Studies involving extracts from Carya ovata Beetles did not feed on disks soaked (1) in 5 mg of benzene extracts from the twig bark of C. ovata alone, or (2) in combination with 5 mg of benzene extracts from the twig bark of U. americana (Table 1, Expt. 1) (Fig. 1). The bark of firstTABLE
I-FEEDING
RESPONSE BY NEWLY
EMERGED
Scolytus
multistriatusIN 48
hr ON DISKS
SOAKED IN 5 mg OF BENZENE EXTRACTS FROM TWIG BARK OF ONE OF SEVERAL SPECIES OF TREES ALONE, OR IN COMBINATION
WITH 5 mg OF SUCH EXTRACTS FROM THE BARK OF ul?.?zUS ameriCana .4verage
mm2
of
disk eaten per Expt.
Replicates
Treatment
t
replicate
value
Rating
extract Treated
1
c. ovata
3
ovata+
N.S. N.S.
0 0
0
Non-stimulatory C.
was
ooata
in-
hibitory
americana americana americana americana U. 2
Control
3 3 3
49.8 13.6 41.9
0.6 0 0.7
11.89** N.S 32.4;**
Highly
stimulatory
Stimulatory
t was
F. americana non-inhibitory
5.6
0
N.S.
Non-to-slightly
61.9
0
12.79**
M.
rubra
stimulators
3
M. rubra+ U. americana U. americana M. pumila _M. pumila + U. americana 3
r&a
non-
wk
inhibitory
3 3 3
0 0 0
55.3 17.0 39.9
15.18** 50.18** N.S.
Highly
stimulatorp
Stimulatory
M. pumila
was non-
inhibitory
3 3
P. tremuloides P. tremuloides + U. americana U.
0 0.2
21.9 56.8
N.S. 8.91*
Stimulatoryt
P. tremuloides
was
non-inhibitory
3 3
46.4 9.2
1.1 2.5
3
44.5
0
17.68** N.S.
Highly
stimulatory
Non-to-slightly stimulatory
9-86 *
M.
was
acuminata
non-inhibitory * Significant
at the 0.05 level
t Treatment
was considered
amount replicates
of
feeding
resulted
between
; **
highly
stimulatory treated
in a non-significant
and
significant
at the 0.01 level.
because
of the large numerical
control
disks,
(0.05 level)
statistical
although
t
value.
the
difference variation
in the among
1456
BARRY L. GILBERT, JAMES E. BAKER, ANDDALE M. NORRIS
year twigs of C. ovata (collected in the fall) thus contains a factor that will prevent (deter) S. multistriatus from feeding on an otherwise highly stimulatory extract. When the components of the crude C. ovata extract were separated on preparative thin-layer plates, the inhibitory activity was located in the region between R, 0.60 to 0.80 (Table 2, Expt. 4). The major component of this region was a yellowbrown band, R,0.75. Distillate fractions of the crude benzene extract from C. ovata obtained from the Sweep also were highly inhibitory to beetle feeding (Table 2, Expt. 5). The residue fraction eluted from the glass-wool packing of the co-distiller surprisingly
TABLE ~-FEEDING RESPONSEBY NEWLY EMERGEDScolytus mdtistriatus IN 48 hr ON DISKS SOAKED IN 5 mg OF CRUDE BENZENE EXTRACTS OF i%?lUS Umc?TiLanaTWIG BARK ALONE, OR IN COMBINATION WITH 5 mg OF ONE OF SEVERAL FRACTIONS OF THE CRUDE BENEZENE EXTRACTIVES FROM
TWIG
BARK
OF CU7ya
OVUtU
Average mm2 of disk eaten per replicate Expt. ~4
5
Treatment extract or fraction Thin-layer band (Rf 0.60-0.80) of crude C.ovata 4 U. americana U. americana Thin-layer bands (& 0.0-0.60,0.80-l .OO) of crude C. oz~arn + U. americana Distillates of crude C. ovata through the Sweep + U. americana U. americana Juglone-free? residue of the crude benzene from C. extracts ovata bark, eluted from the column of the Sweep co-distiller
Replicates
Treated
Control
t value
Rating
3
0.5
0
N.S.
R, 0.60-0.80 band of C. ovata was highly inhibitory
3 3
50.2 41.1
0 0
28.69 * * N.S.
Highly stimulatory Ii, 0.0-0.60, 0.801.00 bands of C. oaata were noninhibitory
3
0.5
0
N.S.
3 3
42.8 32.7
0 2.7
14.63 * * N.S.
Distillates of crude C. ovata through Sweep were highly inhibitory Highly stimulatory Stimulatory$
t Juglone was separated from the remainder of the benzene extracts from the bark of C. ovata by co-distillation in a Sweep (Kontes, Franklin Park, Illinois). $ Treatment was considered stimulatory because of the large numerical difference in the amount of feeding between treated and control disks, although the variation among replicates resulted in a non-significant (0.05 level) statistical t value.
JUGLONE,
A DETERRENT
TO FEEDING
BY
SCOLYTL’S
1457
MULTISTRIATUS
stimulated the beetles to feed (Table 2, Expt. 5). This stimulation occurred in the absence of elm extract. The distillate from C. ovata was co-chromatographed on thin-layer with the contents of the region, R, 0.60 to 0.80, from a previous thinlayer separation of the components of the crude benzene extract of hickory. Both samples contained a predominant yellow-brown band, R, 0.75, but the fraction from the co-distillation apparatus contained fewer contaminating compounds than the thin-layer band, and thus was used for further purification of the active chemical by adsorption-column chromatography. Thin-layer chromatography of the yellow-brown fraction eluted from the silica-gel column revealed only one ‘I‘ABLE
J-FEEDING
SOAKED
IN
O’l!Uta
IN
1 mg
RESPONSE OF PURIFIED
COMBINATION
BY (1)
WITH
NEWLY
EMERGED
COMMERCIAL 5
mg
OF
Scolytus
JUGLONE
BENZENE
OR (2)
ndtistriatzu JUGLONE
EXTRACTIVES
FROM
IN
48 hr
ISOLATED THE
ON
FROM
TWIG
DISKS CaYp2
BARK
OF
Ulmus americana -.
Expt 6
.
Treatment extract, or pure chemical 1 mg pure cornmercial juglone + U. americana U. americana 1 mg pure juglone isolated from C. oerata + U. ameri-
Average mm2 of disk eaten per replicate Replicates
Treated
3
2.5
3 3
47.8 0
Control
Rating
t value
0
N.S.
1
mg juglone is highly inhibitory
0.2 0
37.28 * * N.S.
Highly stimulatory 1 mg juglone is highly inhibitory
spot when the plates were examined with U.V. light or after being charred with 500,/o sulphuric acid. An i.r. spectrum of the isolated chemical was obtained, and compared with known spectra in the Sadtler Index. These comparisons and a further review of the known chemistry of the family Juglandaceae indicated that juglone (5-hydroxy-1,4_naphthoquinone) best fit our total data on the chemical. A sample of juglone was obtained from Pierce Chemical Co., Rockford, Illinois. An assay of 1 mg of the commercial juglone mixed with 5 mg of the benzene extracts of U. americana demonstrated strong inhibition of beetle feeding (Table 3, Expt. 6). A sample of the commercial juglone was co-distilled, and then chromatographed on a silica-gel column. The purified (1) commercial juglone and (2) inhibitory chemical from C. ovutu were crystallized from benzene : Skellysolve B and analysed by i.r. spectrophotometry, and the spectra are shown in Fig. 2. The spectra showed two strong carbonyl bands, but no hydroxyl absorption band. The absence of the hydroxyl absorption band was attributed to intramolecular hydrogen bonding.
1458
BARRY
L. GILBERT, JAMES E. BAKER, AND DALE M. NORRIS
2
4
6
s MICRO$
12
14
16
FIG. 2. Scaled reproductions of the i.r. spectra obtained for (1) purified commercial juglone, and (2) purified compound isolated from the first-year twig bark of C. or&z. Note the two carbonyl absorption bands near 6 and 6.1 p.
Studies involving extracts from other tree species The extracts of Fraxinus americana, Malus pumila, and Populus tremuloides stimulated feeding by S. multistriutus (Table 1). The extracts of Mows rubra and Magnolia acuminata, at most, were very slightly stimulatory to the feeding responses of the beetle (Table 1). None of the extracts of the above tree species inhibited beetle feeding when mixed singly with the extract of Ulmus americana (Table 1). DISCUSSION
The demonstrated deterring effect of 5-hydroxy-1,4-naphthoquinone upon otherwise strong stimuli (i.e. extracts of U. americana) to S. multistriatus feeding constitutes significant new knowledge in our growing understanding of the chemical bases of the specificities between insects and their host trees. The existence of a chemical deterrent to insect feeding in non-host perennial woody tissues was proven quantitatively for the first time.
REFERENCES GOEDEN R. D. and NORRIS D. M. (1964) Some biological and ecological aspects of the dispersal fhght of Scolytus quadrispinosus (Coleoptera: Scolytidae). Ann. ent. Sot. Am.
57,743-749. GOEDEN R. D. and NORRIS D. M. (1965) The behavior of Scolytus quadrispinosus optera: Scolytidae) during the dispersal flight as related to its host specificities.
(Cole-
Ann.
ent. Sot. Am. 58, 249-252. LOSCHIAVO S. R., BECK S. D., and NORRIS D. M. (1963) Behavioral responses European elm bark beetle, Scolytus muEtistriutus (Coleoptera: Scolytidae) elm bark. Ann. ent. Sot. Am. 56, 764-768.
of the smaller to extracts of
JUGLONE,A DETERRENTTO FEEDING
BY SCOLYTUS
MULTISTRIATL’S
1459
MEYER H. J. and NORRIS D. M. (1964) Dispersal of the pathogen and vectors of Dutch elm disease. Proc. N. cent. Branch, Ent. sot. Am. 19, 26-29. NORRIS D. M. (1965) In-flight dispersal and orientation of two Scolytus species (Coleoptera) to their host plants for ovipositional purposes. Proc. 12th int. Congr. Ent. (1964), 243. NORRIS D. M. and BAKER J. E. (1967) Feeding responses of the beetle Scolytus to chemical stimuli in the bark of Ulmus. J, Insect Physiol. 13, 955-962.