Journal of Chemical Ecology, Vol. 19, No.

4, 1993

OLFACTORY RECEPTOR CELL RESPONSES OF Ips

grandicollis (EICHHOFF) (COLEOPTERA:
SCOLYTIDAE) TO INTRA- AND INTERSPECIFIC
BEHAVIORAL CHEMICALS

A. ASCOLI-CHRISTENSEN, I S.M. SALOM,* and T.L. PAYNE

Department of Entomology
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061-0319

(Received September 21, 1992; accepted November 23, 1992)

Abstract--Electrophysiological recordings from antennal olfactory receptor

cells were obtained from Ips grandicollis. Recordings were made from olfac-
tory receptor cells from nine regions of the antennae in response to stimulation
with the semiochemicals ~-pinene, frontalin, endo-brevicomin, verbenone,
trans-verbenol, cis-verbenol, ipsdienol, and ipsenol. In many cases, up to two
cells were recorded concurrently from the same location. When compared to
males, females had a greater percentage of cells responsive to the primary
pheromones of Dendroctonus frontalis, frontalin and trans-verbenol, and of
lps spp., ipsdienol and ipsenol. Among females, more cells responded to
trans-verbenol and the lps-produced volatiles than to host or other D. frontalis-
produced compounds. Olfactory cells of males responded mostly to cis-ver-
benol, followed by ~-pinene, verbenone, trans-verbenol, and endo-brevi-
comin. Of those cells responsive primarily to one compound, the greatest
percentage were responsive to trans-verbenol in females and to verbenone in
males. The response of the antennal olfactory receptor cells to semiochemicals
used by male and female L grandicollis is consistent with the presence of
these compounds during the host colonization period for each sex. Our results,
which show a lack of specificity in most pheromone and host odor receptor
cells, is in contrast with previously published accounts of olfactory receptor
cell specificity in other Ips species.

Key Words--lps, Dendroctonus, Coleoptera, Scolytidae, interspecific attrac-

* TO whom correspondence should be addressed.

1Present Address: Department of Natural Sciences, Pima Community College, Tucson, Arizona
85745.

699

0998-0331/93/0400-0699507.00/0 9 1993PlentlmPublishingCorporation
700 ASCOLI-CHRISTENSEN ET AL.

tion, behavioral chemicals, single olfactory cells, electrophysiology, phero-

mones.

INTRODUCTION

Ips grandicollis (Eichhoff) (Coleoptera: Scolytidae) is one of five species of the

southern pine bark beetle guild, the most destructive group of softwood forest
insects in the southern United States (Thatcher et al., 1980). During epidemic
phases of infestation, L grandicollis is found cohabiting pine trees with Den-
droctonus frontalis (Zimmerman), D. terebrans (Oliver), L avulsus (Eichhoff),
and L calligraphus (Germar). Ips grandicollis is not a pioneering species and
relies on the presence of other more aggressive species, typically D. frontalis,
to weaken defenses of the tree prior to colonization (Vit6 et al., 1972).
Host tree selection, aggregation, and colonization by these bark beetles is
mediated by a complex array of olfactory cues emanating from both hosts and
insects (see reviews by Wood, 1982; Lanier, 1983; Birch, 1984; Borden et al.,
1986; Byers, 1989). Behavioral investigations have confirmed the existence of
intra- and interspecific olfactory communication between conspecific or sym-
patric beetle species (Vit6 et al., 1964; Godbee and Franklin, 1976; Payne et
al., 1978; Richerson and Payne, 1979; Dixon and Payne, 1980; Birch et al.,
1980; Svihra, 1982; Billings, 1985; Phillips et al., 1989; Smith et al., 1990).
Svihra et al. (1980) suggested that the temporal and spatial organization of
bark beetles on the host may be determined, in part, by olfactory communication
among the species. Of the five primary guild members, L grandicoUis has the
smallest overlap of its niche, occupying primarily branches in the lower portion
of the tree canopy. The other species colonize the lower to upper tree bole (Birch
and Svihra, 1979; Dixon and Payne, 1979; Birch et al., 1980; Svihra et al.,
1980; Paine et al., 1981). Peak arrival of L grandicollis on the host tree follows
arrival of other guild members (Dixon and Payne, 1979; Svihra et al., 1980).
Smith et al. (1990) showed that L grandicollis also exhibits the greatest degree
of cross-attractancy to semiochemicals produced by the sympatric species, most
notably to those produced by D. frontalis. They suggested the ability of L
grandicollis to locate suitable hosts, yet not compete with sympatric species,
may be a result of interspecific olfactory cues.
Electrophysiological experiments support the hypothesis of interspecific
olfactory communication, i.e., semiochemicals produced by lps and Dendroc-
tonus spp. elicit electroantennograms (EAGs) from all guild members (Smith et
al., 1988; Delorme and Payne, 1990). Individual olfactory cell responses to
conspecific pheromones and to host volatiles have been recorded from the anten-
nae of D. frontalis (Payne and Dickens, 1976; Dickens and Payne, 1977; Payne
et al., 1982). However, characterization of the single olfactory cells of other
guild members to the chemical stimuli has not been reported. We investigated
RECEPTOR CELL RESPONSES 701

single olfactory cell response to interspecific olfactory signals, using L gran-

dicollis, in an effort to elucidate the specificity of the receptor cells and improve
our understanding of the chemical ecology of the insect.

METHODS AND MATERIALS

Insects. Ips grandicollis from East Texas were used to infest loblolly pine,
Pinus taeda L., bolts from southwestern Virginia. The bolts were placed in a
ventilated rearing chamber at 20-25~ Bolts containing mostly pupae and cal-
low adults were placed in an emergence chamber (Browne, 1972). Emerged
beetles were placed on moistened filter paper and stored individually at 5~ in
3.7-ml vials until use (one to two days after emergence). Recordings of each
antennal location described below were made from five beetles of each sex.
Electrophysiology. Single olfactory cell recording techniques were modified
from Dickens (1979). Tungsten electrodes were sharpened to a tip of ca. 1 /~m.
The reference electrode was inserted into the body of the insect through the oral
cavity. The recording electrode was positioned in one of nine regiolas on the
antenna (Figure 1).
Electrophysiological signals were passed through an automatic baseline-
restoring preamplifier (G. Johnson) to a Tektronix 122 low-level preamplifier,
through a 60-Hz notch filter, and then displayed on a Tektronix R561B oscil-
loscope. Records were stored on cassette tape with a TEAC R-61 data recorder
and displayed on Polaroid film with a Tektronix C-27 oscilloscope camera.
Bioassay. Stimuli used included a terpene of loblolly pine: c~-pinene; com-
pounds identified from the frass of D. frontalis: frontalin (Kinzer et al., 1969),
endo-brevicomin (Vit6 and Renwick, 1971a), trans-verbenol, and verbenone
(Renwick, 1967); and compounds identified from lps species: cis-verbenol, ips-
dienol, and the major pheromone component of L grandicollis, ipsenol (Silver-
stein et al., 1966). Sources and purity of all compounds are listed in Table 1.
All compounds were racemic. The compounds were prepared as 10/zg//~l solu-
tions in nanograde pentane. Stimuli were delivered as 5-/zl aliquots on a piece
of Whatman No. 2 filter paper, inserted into glass cartridges (75 mm long, 5
mm ID), and oriented toward the antennal preparation from ca. 1 cm. Stimuli
were presented in random order, following presentation of clean air and a pen-
tane control. Stimulus duration was 800 msec and air flow was 1 liter/rain. A
3-min interval separated each stimulation to allow for recovery of the action
potential frequency in the cells. To monitor the viability of the preparation,
stimulation with the first compound to elicit a response was repeated as a stan-
dard once stimulation by all other compounds had been completed.
Data Analysis. Often, more than one amplitude of action potential was
observed in a record (e.g., Figure 2). The two largest amplitude spikes were
702 ASCOLI-CHRISTENSEN ET AL.

8. Cd

ta f _..-'~ 9 Ma

4. tdb

FIG. 1. Diagram of left Ips grandicollis antennal flagellum showing regions of the club
from which recordings were obtained: position 1. Lpb, lateral proximal band; 2. Cpb,
central proximal band; 3. Mpb, medial proximal band; 4. Ldb, lateral distal band; 5.
Cdb, central distal band; 6. Mdb, medial distal band; 7. Ld, lateral distal portion; 8.
Cd, central distal portion; 9. Md, medial distal portion.

used for separate counts of action potential frequencies. Each of the two spike
patterns was considered to be from separate cells. It was impossible to resolve
smaller amplitude potentials from background noise. These potentials were
ignored, since previous single cell studies with Ips spp. have not found more
than two cells per record (Mustaparta et al., 1977, 1979). To obtain the change
in action potential frequency of each cell due to application of a stimulus, the
action potential frequency from the first 500 msec following stimulation was
subtracted from two times the number of spontaneously occurring action poten-
RECEPTOR CELL RESPONSES 703

TABLE 1. SOURCE AND PURITY OF STIMULUS COMPOUNDS USED IN

ELECTROPHYSIOLOGICAL EXPERIMENTS ON Ips grandicollis

Chemical
Compound purity ( %)~' Source
c~-Pinene 97 Aldrich Chem. Co.
Frontalin 99 Chem. Samp. Co.
endo-Brevicomin 99 Chem. Samp. Co.
Verbenone 98 Chem. Samp. Co.
trans-Verbenol 95 Borregard Industries
cis-Verbenol 95 Borregard Industries
Ipsdienol 81 Borregard Industries
Ipsenol 89 Borregard Industries

"Via GLC.

tials in the 250 msec preceding stimulation. The difference was subjected to
analysis. For each cell, mean spontaneous activity prior to stimulation was
computed. If a stimulus caused a change in frequency that exceeded the 95 %
confidence interval of the mean spontaneous activity for that cell, the stimulus
was said to have caused a response. Cells for which only one stimulus caused
an increased frequency of action potentials that exceeded the upper 95 % con-
fidence limit of the mean change in action potential frequency for that cell were
said to be primarily responsive to that one stimulus. The percentages of respond-
ing cells were ranked and compared using the nonparametric Kruskal-Wallis k-
sample test (Quade, 1966).

RESULTS AND DISCUSSION

Sexual Dimorphism. Seventy-four cells were recorded from five females,

whereas only 44 cells were recorded from the same number of males. This
suggests the possibility of fewer olfactory receptors in males compared to
females. A comparison between sex and location of the responding cells showed
that in females, the percentage of responding cells in the distal portion of the
club and in the two sensory bands did not differ significantly (F2,96 = 0.9; P
> 0.05), whereas in males, the distal and proximal sensory bands contained a
greater percentage of responding cells than the middle band (F2,96 -~- 3.5; P _<
0.05) (Figure 3). Single cell recordings of different antennal regions have been
reported by Wadlow (1973) for the carrion beetle, Necrophorus vespilloides
Herbst and by Mustaparta (1975) for the pine weevil, Hylobius abietis L. In
both cases, responses were associated with different types of olfactory receptors,
704 ASCOLI-CHRISTENSEN ET AL.

SPONTANEOUS ACTIVITY

AIR
tG T - I F [ . . . . rr -r~
PENTANE

ALPHA-PINENE _,,.J. ~ . J u l i l t l . l t .... mi

-"i ...... rlllll rl .......
i,,~..l._.~JLIJJ.t i_L J . . . . .
FRONTALIN --"r 'Tll]'F] r I. . . . . . . .

ENDO-BREVICOMIN

.,,.Lt .... ~ Li __~.....

VERBENONE Tl r-lT-t .... "

t ...... #, ...... , _ _ k _ _
TRAN~-VERBENOL 'T ........ ~ .... r l-

C/S-VERBENOL "T'I'--Pl ...... I r ,--ip

IPSDIENOL

_ ._ J . . . . . . . IlillLi,tln,nhultlh, ltl
IPSENOL -~---I]lllll I,.I ....... I,1,'.,

FIG. 2. Representative traces of two cells from the antenna of [ps grandicollis. The
smaller spike is from a cell that responds primarily to ipsenol, and to a lesser extent to
(x-pinene, frontalin, and ipsdienol. The firing rate of the larger action potential is not
dependent on the application of any of the behavioral chemicals tested.

which were usually localized to specific areas of the antennae. In this study, no
attempt was made to differentiate among the different types of receptors sampled.
A significantly greater percentage of cells responded to frontalin, trans-
verbenot, ipsdienol, and ipsenol in females than in males (Table 2). This finding
does not correspond with EAG data of L grandicollis, where differences in
 POS9

'=" 1 ~ ~~ fl,~ l~n :i ici o. ott 1_=_1_

me ~:, :vo~ :1/",,.~

,~.
,ol/
Vn.nDD~]nn
0~

,.il /~ ~'Vnn
,oo| /rl~]nrl]ln ,n~l
o\o ,~, '~.~n ]] n ,; n n g ~ n '::
I~ IoOoO~/
1 o

fln 1~ 1~ I] fl 1] fl ~ n ~ ~] FI [1
160 ~ 0

so l]. ~ I] n n rl ,o 1] n fl l] ~ ~ I] r~
1oo 1

,o nnrlnInan ~ n,no .
0 0
a ap cv eb f id ie p sa Iv v
:U a ap cv eb f id ie p sa Iv v
STIMULUS STIMULUS

FEMALES
MALES

Fio. 3. Percentage of responding cells for each antenna/ region to the following stimuli: a = air; ap = ~-pinene; cv = c i s - v e r b e n o l ; eb
= e n d o - b r e v i c o m i n ; f = frontalin; id = ipsdienol; ie = ipsenol; p = pentane; sa = spontaneous activity; tv = t r a n s - v e r b e n o l ; v =
verbenone. Total bar height represents the total percentage of cells responding, speckled bar height represents the percentage of cells
responding only to that stimulus.
706 ASCOLI-CHRISTENSEN ET AL.

TABLE 2. COMPARISON BETWEEN FEMALE AND MALE Ips grandicollis OF MEAN

PERCENT OF CELLS RESPONDING TO A GIVEN STIMULUS

Female Male
Stimulus (N = 74 cells) (N = 44 cells)

Spontaneous activity 10.4 4.3

Air 13.2 7.1
Pentane 28.7 11.0
e~-Pinene 43.7 43.4
Frontalin 45.9 21.7 **"
endo-Brevicomin 36.2 32.6
Verbenone 46.7 36.0
trans-Verbenol 63.4 36.0**
cis-Verbenol 52.9 49.6
Ipsdienol 55.4 23.1 **
Ipsenol 48.4 28.2*

"Significant differences between females and males were determined using Friedman's two-way
analysis for block designs (*P _< 0.05; **P < 0.01).

response were not found between sexes (Smith et al., 1988). The LAG, how-
ever, may represent more cells (Boeckh et al., 1965; Schneider, 1969) and thus,
could be a more accurate measure of total antennal olfactory response. For L
paraconfusus Lanier, equal LAGs were recorded for both sexes in response to
ipsenol (Light and Birch, 1979), while in the field, females were more attracted
to this pheromone than were males (Byers, 1983). Byers concluded that the
behavioral difference between the sexes must be the result of integration within
the central nervous system. However, one must not exclude the possibility of
the activity of a few cells being responsible for behavioral differences. Such
may be the case with L grandicollis, in which more females than males are
caught in traps baited with ipsenol and host volatile blends (Smith et al., 1990).
Responses by Females. In females, trans-verbenol elicited responses from
the greatest percentage of cells, followed by ipsdienol, cis-verbenol, and ipsenol
(Figure 4), compounds that are all produced by Ips species (Vit6 et al., 1972).
Vit6 and Renwick (1971b) found that ipsenol and trans-verbenol both attract a
greater number of female than male L grandicollis. The addition of c~-pinene,
however, increased the ratio of males caught. Smith et al. (1990) showed that
female L grandicollis are greatly attracted to traps baited with a blend of racemic
ipsdienol, ipsenol, and cis-verbenol. They also showed that females were more
responsive to a blend of synthetic pheromones of both sexes of D. frontalis than
to blends produced by either sex alone. In nature, the presence of pheromones
of both sexes may be a sign stimulus to I. grandicollis of a susceptible host that
was successfully attacked by D. frontalis. The pheromones of other more aggres-
RECEPTOR CELL RESPONSES 707

females
80

-=
"0
60
c
0
e~
r
m

~ 40
o
0

~ 20
ID
E

iiiii!il
tv id cv ie v f ap eb p a sa

stimulus

F[6. 4. Mean percent of cells from females responding to a given stimulus. Bar height
represents total percentage of cells responding to a compound; speckled bar height rep-
resents percentage of cells responding primarily to that stimulus. Means of total per-
centage of cells responding to a compound grouped under one line are not significantly
different (P -< 0.05) as determined by least significant differences (LSD) computed from
Friedman's two-way analysis for block designs. For all stimuli, N = 74 cells, tv =
trans-verbenol; id = ipsdienol; cv = cis-verbenol; ie = ipsenol; v = verbenone; f =
frontalin; ap = a-pinene; eb = endo-brevicomin; p = pentane; a = air; sa = sponta-
neous activity.

sive Ips species may also serve as similar sign stimuli for I. grandicollis (Smith
et al., 1990).
The percentage o f cells responding primarily to one stimuli was low (Figure
4). Of all the stimuli tested, only cells responding primarily to trans-verbenol
and ipsdienol were found more frequently than cells responding primarily to the
controls. (Fj0,8 s = 1.99; P _< 0.05). These results differ from those obtained
for L pini (Say), L paraconfusus (Mustaparta et al., 1977, 1979, 1980), and L
typographus (T~bmmer~s et al., 1984). Cells from those species were responsive
mostly to a single compound or enantiomer, rather than several compounds.
Responses by Males. cis-Verbenol elicited responses from a significantly
greater percentage o f cells in males than did ipsenol, ipsdienol, or frontalin
(Figure 5). Since I. grandicollis produce only trace amounts o f cis-verbenol
708 A S C O L I - C H R I S T E N S E N E T AL.

males

50

p.
40
"0
O
30

0
20

C
m
@ 10

IDn
E

E
0
cv ap v tv ~ ie id f p a sa
stimulus

FIG. 5o Mean percent of cells from males responding to a given stimulus. Bar height
represents total percentage of cells responding to a compound; speckled bar height rep-
resents percentage of cells responding primarily to that stimulus. Means of total per-
centage of cells responding to a compound grouped under one line are not significantly
different (P _ 0.05) as determined by least significant differences (LSD) computed from
Friedman's two-way analysis for block designs. For all stimuli, N = 44 cells, cv = cis-
verbenol; ap = a-pinene; v = verbenone; tv = trans-verbenol; eb = endo-brevicomin;
ie = ipsenol; id = ipsdienol; f = frontalin; p = pentane; a = air; sa = spontaneous
activity.

(Vit6 et al., 1972), it appears that males may use cis-verbenol produced by L
calligraphus (Renwick and Vit6, 1972; Hughes, 1974). c~-Pinene, verbenone,
trans-verbenol, and endo-brevicomin elicited responses from significantly more
cells than did the pentane or air controls, whereas ipsenol, ipsdienol, and fron-
talin did not. Very few cells must be needed to respond in order to elicit a
behavioral response to ipsenol, as males are known to aggregate to their own
pheromone, ipsenol (Vit6 and Renwick, 1971b). It is interesting to note that
there was no difference in response to o~-pinene between males and females
(Table 2). Werner (1972a,b) found that males were more than twice as respon-
sive to ot-pinene as females, and in general males were more attracted than
females to host terpenes in both laboratory and field tests, Presumably, in addi-
tion to interspecific olfactory cues, pioneering males can use host compounds
as primary attractants for host tree selection.
RECEPTOR CELL RESPONSES 709

In males, the percentage of cells responding primarily to one compound

was also low. However, o f those that did, verbenone was the most frequent
stimulus eliciting such a response (Figure 5). Verbenone, an antiaggregation
pheromone of D. frontalis, may function as a kairomone, aiding male L gran-
dicoUis in the location of weakened trees. In contrast to females, no cells
responded primarily to ipsdienol.
Implications to Bark Beetle Ecology. Both sexes of L grandicollis have
antennal olfactory cells that are sensitive to pheromones of sympatric species.
Some of these kairomones stimulated receptor cells that were also stimulated by
host chemicals or by the insect's own pheromone. For some kairomones, how-
ever, cells were found that responded to only one of the kairomones tested, i.e.,
verbenone for males, and trans-verbenol and ipsdienol for females.
In a pedestrian laboratory bioassay, L grandicollis oriented toward several
host volatiles; however, the attraction was masked by the D. frontalis phero-
mones frontalin or trans-verbenol (Werner, 1972b). In contrast, frontalin and
trans-verbenol, in combination, enhanced attraction of L grandicollis to host
compounds in the field. Therefore, it appears that some compounds can elicit
both avoidance and attraction of L grandicollis to a source. This being the case,
one would expect to find such plastic behavior to be mediated, at least in part,
by an across-fiber type of receptor cell patterning, unless combinations of com-
pounds are able to antagonize one another's stimulatory activity on the receptor
cell. Smith et al. (1990) demonstrated that ipsenol, specifically the ( S ) - ( - )
enantiomer, attracted L grandicollis. (R)-(+)-Ipsenol masked the effect of its
optical enantiomer. Information regarding the optical specificity of ipsenol
receptors in this species is unknown.
Both sexes o f L grandicollis are attracted to L calligraphus-infested bolts;
however, L calligraphus attack and colonization o f L grandicoUis-infested bolts
will reduce the attraction of the bolts to host-searching L grandicollis (Birch et
al., 1980; Byers, 1989). Synthetic blends of the pheromones of I. calligraphus
and L avulsus do attract L grandicoUis (Smith et al., 1990). The presence of
sympatric species may therefore aid L grandicollis in locating suitable habitats
while signaling them to stay away from parts of the host already colonized by
I. avulsus and L calligraphus.
Acknowledgments--The authors with to thank J.C. Dickens and M.T. Smith of USDA. ARS,
and two anonymousreviewers for critical review of the manuscript. The research was supported in
part by NATO Grant 0710/86 to T.L. Payne.

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