Documente Academic
Documente Profesional
Documente Cultură
4, 1993
Department of Entomology
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061-0319
699
0998-0331/93/0400-0699507.00/0 9 1993PlentlmPublishingCorporation
700 ASCOLI-CHRISTENSEN ET AL.
INTRODUCTION
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
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).
SPONTANEOUS ACTIVITY
AIR
tG T - I F [ . . . . rr -r~
PENTANE
ENDO-BREVICOMIN
t ...... #, ...... , _ _ k _ _
TRAN~-VERBENOL 'T ........ ~ .... r l-
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
,.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.
Female Male
Stimulus (N = 74 cells) (N = 44 cells)
"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
REFERENCES
BILLINGS,R.F. 1985. Southern pine bark beetles and associated insects: Effects of rapidly released
host volatiles on response to aggregation pheromones. Z. Angew. Entomol. 99:483-491.
BIRCH,M.C. 1984. Aggregationin bark beetles, pp. 331-353, in W.J. Bell and R.T. Card6 (eds.).
Chemical Ecology of Insects. Sinauer Associates, Sunderland, Massachusetts.
710 ASCOLI-CHRISTENSEN ET AL.
BmcH, M.C., and SVlHRA, P. 1979. Novel approaches to forest insect control: Exploiting olfactory
interactions between species of Scolytidae, pp. 135-139, in W.E. Waiters (ed.). Current Topics
in Forest Entomology. John Wiley & Sons, New York.
Bmc~, M.C., SVmRA,P., PAINE, T.D., and MILLER,J.C. 1980. Influence of chemically mediated
behavior on host tree colonization by four cohabiting species of bark beetles. J. Chem. Ecol.
6:395~1&
BOECKH, J., KAISSLING,K.E., and SCHNEIDER, D. 1965. Insect olfactory receptors. Cold Spring
Harbor Syrup. Quant. Biol. 30:263-280.
BORDEN, J.H., HUNT, D.W.A., MILLER, D.R., and SLESSOR, K.N. 1986. Orientation in forest
Coleoptera: An uncertain outcome of responses by individual beetles to variable stimuli, pp.
97-109, in T.L. Payne, M.C. Birch, and C.E.J. Kennedy (eds.). Mechanisms in Insect Olfac-
tion. Clarendon Press, Oxford, UK.
BROWNE, L.E. 1972. An emergence cage and refrigerator collector for wood-boring insects and
their associates. J. Econ. Entomol. 65:1499-1501.
BYERS, J.A. 1983. Sex-specific responses to aggregation pheromone: Regulation of colonization
density in the bark beetle Ips paraconfusus. J. Chem. Ecol. 9:129-142.
BYERS, J.A. 1989. Chemical ecology of bark beetles. Experientia 45:271-283.
DELORME, J.D., and PAYNE, T.L. 1990. Olfactory responses of the black turpentine beetle, Den-
droclonus terebrans (Olivier), to beetle pheromones. J. Chem. Ecol. 16:1321-1329.
DICKENS, J.C. 1979. Electrophysiological investigations of olfaction in bark beetles. Mitt. Schwe&.
Entomol. Ges. 52:203-216.
DICKENS, LC., and PAYNE,T.L. 1977. Bark beetle olfaction: Pheromone receptor system in Den-
droctonus frontalis. J. Insect Physiol. 23:481-489.
DIXON,W.N., and PAYNE,T.L. 1979. Sequence of arrival and spatial distribution of entomophagous
and associate insects of southern pine beetle-infested trees. Texas Agriculture Experiment
Station Miscellaneous Publication 1432, 28 pp.
DIXON, W.N., and PAYNE, T.L. 1980. Attraction of entomophagous and associate insects of the
southern pine beetle to beetle- and host tree-produced volatiles. J. Ga. Entomol. Soc. 15:378-
389.
GODBEE, J.F., JR., and FRANKLIN,R.T. 1976. Attraction, attack patterns and seasonal activity of
the black turpentine beetle. Ann. EntomoL Soc. Am. 69:653-655.
HUGHES, P.R. 1974. Myrcene: A precursor of pheromones in lps beetles. J. Insect Physiol. 20:1271-
1275.
KINZER, G.W., FENTIMAN,A.F., JR., PAGE,T.F., JR., FOLTZ, R.L., VITE, J.P., and PITMAN,G.B.
1969. Bark beetle attractants: Identification, synthesis and field bioassay of a new compound
isolated from Dendroctonus. Nature 221:477-478.
LANIER, G.N. 1983. Integration of visual stimuli, host odorants, and pheromones by bark beetles
and weevils in locating and colonizing host trees, pp. 161-171, in S. Ahmad (ed.). Herbivorous
Insects: Host Selecting Behavior and Mechanisms. Academic Press, New York.
LIGHT, D.M., and BIRCH, M.C. 1979. Bark beetle enantiomeric chemoreception: Greater sensitivity
to allomone than pheromone. Naturwbsenschaften 69:243-245.
MUSTAPARTA, H. 1975. Behavioral responses of the pine weevil Hylobius abietis L. (Col.: Cur-
culionidae) to odours activating different groups of receptor cells. J. Comp. Physiol. 102:57-
63.
MUSTAPARTA, H., ANGST, M.E., and LANIER, G.N. 1977. Responses of single cells in the pine
engraver beetle, lps pini (Say) (Coleoptera: Scolitidae) to its aggregation pheromone, ipsdienol,
and the aggregation inhibitor, ipsenol. J. Comp. Physiol. 121:343-347.
MUSTAr'ARTA,H., ANGST,M.E., and LANIER,G.N. 1979. Specialization of olfactory cells to insect-
and host-produced volatiles in the bark beetle Ips pini (Say). J. Chem. Ecol. 5:109-123.
MUSTAPARTA,H., ANGST, M.E., and LANIER, G.N. 1980. Receptor discrimination of enantiomers
of the aggregation pheromone ipsdienol, in two species of lps. J. Chem. Ecol. 6:689-701.
RECEPTOR CELL RESPONSES 711
PAINE,T.D., BIRCH,M.C., and SVIHRA,P. 1981. Niche breadth and resource partitioning by four
sympatric species of bark beetles (Coleoptera: Scolytidae). Oecologia 48:1-6.
PAYNE, T.L., and DICKENS, J.C. 1976. Adaptation to determine receptor specificity in insect olfac-
tory communication. J. Insect Physiol. 22:1569-1572.
PAYNE, T.L., COSTER,J.E., RICHERSON,J.V., EDSON, L.J., and HART, E.R. 1978. Field response
of the southern pine beetle to behavioral chemicals. Environ. Entomol. 7:578-582.
PAYNE, T.L., RICHERSON,J.V., DICKENS,J.C., WEST, J.R., MORI, K., BERISFORD,C.W., HEDDEN,
R.L., VITE, J.P., and BLUM, M.S. 1982. Southern pine beetle: Olfactory receptor and behavior
discrimination of euantiomers of the attractant pheromone frontalin. J. Chem. Ecol. 8:873-
881.
PHILLIPS, T.W., NATION, J.L., WILKINSON, R.C., and FOLTZ, J.L. 1989. Secondary attraction and
field activity of beetle-produced volatiles in Dendroctonus terebrans. J. Chem. Ecol. 15:1513-
1533.
QUADE, D. 1966. On analysis of variance for the k-sample problem. Ann. Math. Star. 37:1747-
1753.
RENWICK, J.A.A. 1967. Identification of two oxygenated terpenes from the bark beetles Dendroc-
tonus frontalis and Dendroctonus brevicomis. Contrib. Boyce Thompson Inst. 23:355-360.
RENWlCK, J.A.A., and V1T~, J.P. 1972. Pheromones and host volatiles that govern aggregation of
the six-spirted engraver beetle, Ips calligraphus. J. Insect Physiol. 18:1215-1219.
RICHERSON, J.V., and PAYNE, T.L. 1979. Effects of bark beetle inhibitors on landing and attack
behavior of the southern pine beetle and beetle associates. Environ. Entomol. 8:360-364.
SCHNEIDER,D. 1969. Insect olfaction: Deciphering system for chemical messages. Science 163:1031-
1037.
SILVERSTEIN,R.M., RODIN,J.O., and WOOD, D.L. 1966. Methodology for isolation and identifi-
cation of insect pheromones with reference to studies on California five-spined lps. J. Econ.
Entomol. 60:944-949.
SMITH, M.T., BUSCH, G.R., PAYNE, T.L., and DICKENS, J.C. 1988. Antennal olfactmy respon-
siveness of three sympatric Ips species [lps avulsus (Eichhoff), lps calligraphus (Germar), lps
grandicollis (Eichhoff)], to intra- and interspecific behavioral chemicals. J. Chem. Ecol.
14:1289-1304.
SMITH, M.T., PAYNE, T.L., and BIRCH, M.C. 1990. Olfactory-based behavioral interactions among
five species in the southern pine bark beetle group. J. Chem. Ecol. 16:3317-3331.
SVIHRA, P. 1982. Influence of opposite sex on attraction produced by pioneer sex of four bark beetle
species cohabitating pine in the southern United States. J. Chem. Ecol. 8:373-378.
SVIHRA, P., PAINE, T.D., and BIRCH, M.C. 1980. Interspecific olfactory communications in southern
pine beetles. Naturwissenschafien 67:518-519.
THATCHER,R.C., SEARCY,J.L., COSTER,J.E., and HERTEL, G.D. (eds.). 1980. The Southern Pine
Beetle. USDA Forest Service Technical Bulletin 1631, 267 pp.
TCiMMER~S, B. A, MUSTAPARTA,H., and GREGOIRE, J.C. 1984. Receptor cells in Ips typographus
and Dendroctonus micans specific to pheromones of the reciprocal genus. J. Chem. Ecol.
10:759-770.
VITE, J.P., and RENWICK, J.A.A. 1971a. Inhibition of Dendroctonusfrontalis response to frontalin
by isomers of brevicomin. Naturwissenschaften 58:418.
VITt~, J.P., and RENWICK, J.A.A. 1971b. Population aggregating pheromone in the bark beetle, Ips
grandicollis. J. Insect Physiol. 17:1699-1704.
V1T~, J.P., GARA, R.I., and VON SCHELLER, H.D. 1964. Field observations on the response to
attractants of bark beetles infesting southern pines. Contrib. Boyce Thomp. Inst. 22:461-470.
VIT~, J.P., BAKKE, A., and RENWICK, J.A.A. 1972. Pheromones in lps (Coleoptera: Scolytidae):
Occurrence and production. Can. Entomol. 104:1967-1975.
WADLOW, U. 1973. Electrophysiology of a new carrion receptor and its relation to the behavior of
the carrion beetle (Necrophorus). J. Comp. Physiol. 83:415-424.
712 ASCOLI-CHRISTENSENET AL.
WERNER, R.A. 1972a. Aggregation behaviour of the beetle Ips grandicollis in response to host-
produced attraetants. J. Insect Physiol. 18:423-437.
WERNER,R.A. 1972b. Response of the beetle, lps grandicollis, to combinations of host and insect
produced attractants. J. Insect Physiol. 18:1403-1412.
WOOD, D.L. 1982. The role of pheromones, kairomones, and alomones in the host selection and
colonization behavior of bark beetles. Annu. Rev. Entomol. 27:411-446.