Interspecific effects of pheromones on the attraction of the bark beetles, Dendroctonus brevicomis and Ips paraconfusus in the laboratory

Byers, J.A., and Wood, D.L. 1981. Interspecific effects of pheromones on the attraction of the bark beetles, Dendroctonus brevicomis and Ips paraconfusus in the laboratory. Journal of Chemical Ecology 7:9-18. pdf

Bark beetles (small spots on paper in center) responding to pheromone from feeding male Ips paraconfusus in the laboratory olfactometer developed at Univ. of Calif. at Berkeley.

Abstract--Dendroctonus brevicomis was attracted to a mixture of the Ips paraconfusus pheromones, ipsenol, cis-verbenol, and ipsdienol at 10^-9 g each/µl but was not attracted to these pheromones at higher and lower release rates. I. paraconfusus was not attracted to the D. brevicomis pheromones exo-brevicomin, frontalin, and myrcene at any release rate tested. Increased release rates of a mixture of the three pheromones of l. paraconfusus inhibited the attraction of D. brevicomis to its synthetic pheromones. A mixture of ipsenol + ipsdienol or cis-verbenol alone failed to cause inhibition indicating that at least two of the I. paraconfusus pheromones are required to inhibit the response of D. brevicomis. The pheromones of D. brevicomis did not inhibit the attraction of I. paraconfusus to its pheromones; however, verbenone was a potent inhibitor.

Key Words-Dendroctonus brevicomis, Ips paraconfusus, Coleoptera, Scolytidae, Pinus ponderosa, bark beetle, exo-brevicomin, frontalin, myrcene, verbenone, ipsenol, ipsdienol, cis-verbenol, attractants, inhibition, semiochemicals, pheromones.

INTRODUCTION
In California D. brevicomis LeConte and I. paraconfusus Lanier colonize the phloem-cambium tissues of ponderosa pine, Pinus ponderosa Laws., during the same period of time. Byers and Wood (1980) found that the attraction of both species to their naturally produced pheromones was inhibited by the presence of the other species feeding in logs of the host tree. The function of this mutual inhibition is believed to reduce interspecific competition for food and space. They showed that the response of D. brevicomis to its synthetic pheromones, exo-brevicomin (E) and frontalin (F) and the host terpene, myrcene (M) (Silverstein et al.,1968; Kinzer et al.,1969) was inhibited by the presence of logs infested with male I. paraconfusus. Byers and Wood (1980) also found that E + F + M had no effect on the attraction of I. paraconfusus to its natural pheromone. However, they established that synthetic verbenone inhibited the response of I. paraconfusus to its pheromone. Furthermore, they showed that verbenone was present in male D. brevicomis feeding in logs cut from pheromone-baited trees that had inhibited the response of I. paraconfusus to its pheromone. However, they did not test the pheromones of I. paraconfusus, ipsenol (I), cis-verbenol (II), and ipsdienol (III) (Silverstein and Rodin, 1965; Silverstein et al., 1966; Wood et al., 1966) as possible inhibitors of the response of D. brevicomis to its pheromone or as attractants for D. brevicomis, nor did they test for inhibition of I. paraconfusus attraction to I + II + III by verbenone. The objectives of this study were to investigate the responses of these beetles to semiochemicals, both intra- and interspecific, under laboratory conditions.

METHODS AND MATERIALS
Both I. paraconfusus and D. brevicomis were collected from the Sierra National Forest near Bass Lake, Madera County, California, at an approximate elevation of 1000 m. The D. brevicomis were obtained by removing bark from naturally attacked ponderosa pines, while I. paraconfusus were obtained from infested logging debris. The rearing, preparation, and determination of sex were as described in Byers and Wood (1980).

The responses of D. brevicomis and I. paraconfusus to semiochemicals were tested in the laboratory olfactometer developed by Browne et al (1974) for I. pini Say. However, several modifications were used. The polyurethane foam was removed from the plexiglass manifold to maintain the air speed at 0.9 m/ sec at the semiochemical source and 0.6 m/ sec where the beetles were released (21 ą 2 cm "downwind"). A positive response was recorded when a beetle arrived within 1 cm of the attractive source in the time required for various mixtures of the semiochemicals in diethyl ether to elute from a 5-µl capillary tube (126 ą 10 sec). The release rate of semiochemicals in the bioassay (g/min) from the 5-µl capillary pipette was estimated to be 2.2 times the concentration of the starting solution (g/µl), assuming that the compounds were released in proportion to the volume reduction of the solvent. However, the actual release rates of compounds from the pipette probably was not linear due to chemical interactions and different rates of distillation (vapor pressures) of the semiochemicals and the solvent. At least 30 beetles of each sex were tested for each release rate of the compounds. Differences in the percent responding between various release rates were determined by a chi-square test.

Both sexes of D. brevicomis and I. paraconfusus was tested for interspecific attraction to the appropriate synthetic pheromones: I + II + III (each > 98%) (Figure 1) or E (> 95%) + F (> 95%) + M (> 99%) (Figure 2). I. paraconfusus were also tested for their response to verbenone (> 99.8%, GLC purified) (Figure 2). I, III, E, and F were all racemic, the enantiomeric composition of II was not known, and verbenone was [a]_D²⁵ = +90^o. All compounds were obtained from Chemical Samples Co., Cleveland, Ohio.

The three pheromones of each species must be released simultaneously for maximum attraction in the laboratory assay (Wood et al., 1967, 1968, 1976; Silverstein et al., 1968; Wood, 1970; Byers et al., 1979). Each sex of D. brevicomis was tested for attraction to E + F + M to determine a release rate that elicited an approximate 50% response (Figure 3). D. brevicomis then were tested at this concentration (10^-9 g E + F + M / µl) for inhibition of their response by increasing the release rate (g/µl) of either I + II + III (Figure 4), I + III, or II (Table 1). Similarly, each sex of l. paraconfusus was tested for inhibition of their response to 10^-9 g I + II + III / µl by increasing the release rate of either E + F + M (Figure 2) or verbenone (Figure 5).

Since verbenone is structurally related to II and thus might compete for acceptor sites of II on the antennae of I. paraconfusus, we wanted to know how much II was required to significantly enhance the beetles' attraction to I + III. This was determined by testing female response to mixtures of a 10-fold concentration series of II from 10^-13 to 10^-9 g/µl with 5 X 10^{- 9} g each I + III / µl.

RESULTS
Both sexes of D. brevicomis responded in significantly greater proportions to I + II + III at 10^-9 g each/µl than to these pheromones released at either higher or lower rates (Figure 1).

FIG. 1. Response of male and female Dendroctonus brevicomis to ipsenol (I), cis-verbenol (II), and ipsdienol (III) at 10^-11 to 10^-6 g each/&181l (October 5-7, 1976). Brackets represent 95% binomial confidence limits.

The percent of males responding at 10^-9 g/µl was different than the percent responding at 10^-11 ", 10^-7 ', or 10^-6 g I + II + III/µl (P < 0.05). The percent of females responding at 10^-9 g/µl was different than at either 10^-11, 10^-10, or 10^-6 g I + II + III/µl (P < 0.05). In contrast, I. paraconfusus was not attracted to E + F + M or verbenone at any concentration tested (P > 0.1) (Figure 2).

FIG. 2. Response of male and female Ips paraconfusus to exo-brevicomin (E), frontalin (F), and myrcene (M) at 10^-11 to 10^-8 g each /µI; ipsenol (I), cis-verbenol (II), and ipsdienol (III) at 10^-9 g each /µI in mixtures with E, F, M at 10^-11 to 10^-6 g each/µl; and verbenone alone at 10^-11 to 10^-7 g/µl (July 28-29, 1976).

Both sexes of D. brevicomis responded similarly to each release rate of E + F + M. The percent of males and females responding to 10^-10 and 10^-9 g E + F + M/µl, respectively, and all higher rates were significantly greater than the percent responding to solvent controls (P < 0.05). There were no significant differences in percent responding between the sexes at any concentration of E + F + M (P > 0.1) (Figure 3).

FIG. 3. Response of male and female Dendroctonus brevicomis to exo-brevicomin (E), frontalin (F), and myrcene (M) at 0 and 10^-11 to 10^-6 g each/µl (October 8,1976). Brackets represent 95% binomial confidence limits.

The response of both sexes of D. brevicomis to E + F + M was inhibited by I + II + III released at 10^-6 and 10^-5 g each/µl compared to 0, 10^-10, and 10^-9 g each I + II + III/µl (P < 0.05) (Figure 4).

FIG. 4. Response of male and female Dendroctonus brevicomis to exo-brevicomin (E), frontalin (F), and myrcene (M) at 10^-9 g each/µl in mixtures with ipsenol (I), cis-verbenol (II), and ipsdienol (III) at 0 and 10^-10 to 10^-5 g each/µl (October 11-12, 1976). Brackets represent 95% binomial confidence limits.

Neither I + III nor II inhibited the response of D. brevicomis to E + F + M (Table 1) which indicates a mixture of at least two (II + I or II + III) and possibly all three pheromones of I. paraconfusus are necessary.

Compounds tested [Dose (g/µl)]	Sex	Number tested	Percent responding	Confidence interval (95%0
TABLE 1. Response of male and female Dendroctonus brevicomis to exo brevicomin (E), frontalin (F), and myrcene (M) in various mixtures with ipsenol (I), cis-verbenol (II), and ipsdienol (III) at 0 and 10^-10 to 10^-5 g each / µl (October 11-12, 1976). Brackets represent 95% binomial confidence limits.
E, F, M [10^-9]	females	90	63	52 - 73
E, F, M [10^-9]	males	120	68	59 - 76
E, F, M [10^-9] + I, III [10^-6]	females	30	87	69 - 97
E, F, M [10^-9] + I, III [10^-6]	males	30	83	64 - 94
E, F, M [10^-9] + II [10^-6]	females	60	53	40 - 68
E, F, M [10^-9] + II [10^-6]	males	60	60	46 - 72
E, F, M [10^-9] + I, III [10^-6]	females	90	30	20 - 41^a
E, F, M [10^-9] + I, II, III [10^-6]	males	90	31	21 - 42^a

^aSignificantly different from above treatments (P < 0.05).

The response of either sex of I. paraconfusus to 10^-9 g I + II + III/µl was unaffected by E + F + M at concentrations of 10^-10 g to 10^-6 g/µl (Figure 2). However, verbenone at 10^-7 to 10^-6 g/µl was effective in inhibiting the response of both sexes of I. paraconfusus to I + II + III (Figure 5).

FIG. 5. Response of male and female Ips paraconfusus to ipsenol (I), cis-verbenol (II), and ipsdienol (III) at 10^-9 g each/µI in mixtures with verbenone at 10^-11 to 10^-6 6 g/µl (July 27-28, 1976). Brackets represent 95% binomial confidence limits.

The percent of males responding to I + II + III at 10^-6 g verbenone/µl was significantly less than the percent responding at 10^-7 or 10^-6 g/µl (P < 0.05). The percent of females responding to I + II + III at 10^-7 or 10^-6 g verbenone/µl was significantly less than the percent responding at 10^-11 or 10^-10 g/µl (P < 0.05) (Figure 5).

The response of female I. paraconfusus to 5 X 10^-9 g each I + III/µl was increased from 25% (95% binomial confidence limits, BCL,16% and 38%) to 57% (BCL 38% and 75%) (different at P < 0.05) by adding as little as 10^-10 g II/µl to the I + III mixture.

DISCUSSION
Dethier ( 1947) and Dethier et al. (1952) described a phenomenon in which a gustatory response to an optimum dosage of a stimulant could be lowered by either increasing or decreasing the dosage. A similar relationship has been shown for D. frontalis Zimm. and D. pseudotsugae Hopk., where response to a certain release rate of a pheromone was lowered by both increasing and decreasing the release rate of the intraspecific compounds, verbenone or 3-methyl-2-cyclohexen-1-one (MCH), for each species, respectively (Rudinsky 1973a,b). We report the flrst instance of this type of response curve for interspecifically active compounds, i.e., I + II + III at 10^-9 g each/µl was attractive to D. brevicomis but either lower or higher release rates were not (Figure 1). Further experiments with various mixtures of I, II, and III are needed to elucidate which compounds are involved. In contrast, I. paraconfusus was not attracted to either E + F + M or to verbenone (Figure 2). There have been no reports of I. paraconfusus trapped in the field at sources of naturally produced or synthetic pheromones of D. brevicomis. The attraction of D. brevicomis to I + II + III in our laboratory studies, but apparently not to natural or synthetic pheromones in the field (Wood et al. 1968, 1976), may be explained by a high release rate of the pheromones thus inhibiting the beetle before encountering the trap or by differences in the walking and flight response to I + II + III.

Struble and Hall (1955) and Miller and Keen (1960) have summarized several reports that I. paraconfusus may precede D. brevicomis in the successful colonization of a ponderosa pine. The attraction of D. brevicomis to trees that were top-killed by I. paraconfusus also appeared to be greater than to trees that had their tops removed (Miller and Keen, 1960). D. brevicomis may exploit weakened and more susceptible hosts by responding to the pheromone produced by I. paraconfusus which is believed to be a less aggressive tree-killer. However, field experiments utilizing various release rates of synthetic and naturally produced pheromones are required before this sequence of host selection can be attributed to interspecific attraction.

The proportion of D. brevicomis responding to a constant dose of E + F + M was reduced as the release rate of I + II + III was increased (Figure 4). The inhibition occured only at the two higher release rates tested, which suggests that D. brevicomis would be inhibited in the field only at close range to substrates containing a mixture of these species. The inhibition of attraction of D. brevicomis to naturally infested substrates and synthetic pheromones in the field by logs infested with I. paraconfusus males (Byers and Wood, 1980) may be due, at least in part, to the release of I + II + III from these logs. In this regard, the laboratory assay has provided the first evidence that a mixture of at least two compounds is necessary to cause this interspecific inhibition.

The response of I. paraconfusus to male-produced pheromone was not inhibited by E + F + M in the laboratory. This supports the field observation where E + F + M did not inhibit the response of I. paraconfusus to male-infested logs (Byers and Wood, 1980). However, in the present study, verbenone at relatively high release rates inhibited the response of I. paraconfusus to I + II + III (Figure 5), which complements the field results where verbenone inhibited the attraction of I. paraconfusus to naturally produced pheromone (Byers and Wood, 1980). I. paraconfusus probably would be attracted in flight to a tree under colonization by both species but inhibition of I. paraconfusus would increase as the beetles approached the D. brevicomis-infested areas where the concentration of verbenone is highest.

Certain sensory cells on the antennae of I. paraconfusus and I. pini are known to be sensitive to several monoterpenes (verbenone, II, and trans-verbenol) that are structurally related (Mustaparta, 1979). It is possible that less evolutionary change in D. brevicomis and I. paraconfusus would be necessary to acquire sensory systems responsive to interspecific compounds, if each species utilized compounds structurally similar to their own pheromones. Thus, the acceptor sites for myrcene (2-methyl-6-methylene-2,7-octadiene) on the antennae of D. brevicomis may have evolved into new sites capable of accepting the structurally similar I (2-methyl-6-methylene-7-octene-4-ol) and/or III (2-methyl-6-methylene-2,7-octadiene-4-ol). Similarly, the D. brevicomis acceptor site for the intraspecific inhibitor verbenone (4,6,6-trimethyl-bicyclo[3.1.1]hept-3-en-2-one) (Bedard et al., 1980) may have evolved into new sites capable of accepting structurally similar II (cis-verbenol = 4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-ol). I. paraconfusus may have evolved acceptor sites for verbenone from acceptor sites for II.

On the other hand, verbenone may compete with II for the acceptor sites of II in I. paraconfusus so that the beetle effectively can respond only to I + III which has been shown to be essentially unattractive in the field (Wood et al., 1967, 1968). However, in competitive interactions verbenone would probably have to have a much stronger affinity for the acceptor site than II to cause the observed inhibition of the behavioral response since as little as 10^-10 g II/ul significantly increased the response of female I. paraconfusus to 5 X 10^-9 g/ul each of I + III. Further, the enhancement of attraction by adding II to I + III does not support the hypothesis of Kikuchi and Ogura ( 1976), based on molecular binding-site models, that II interacts with acceptor sites in a similar conformational way as III or by the conversion of III into II.

Verbenone primarily from male D. brevicomis (Renwick, 1967; Byers and Wood, l980), I + II + III from male, and II from female (Renwick et al., 1976) I. paraconfusus appear to cause, at least in part, the observed interspecific inhibition of the responses of these species to their pheromones. We do not know how closely the release rates of I, II, III, and verbenone reported in our study represent the release rates from infested pine substrates in nature. However, Browne et al. (1979) have quantified the release rates of E, F, and M per beetle per day in a tree as 4.1 X 10^-6 g E, 8.6 X 10^-7 g F, and 4.1 X l0^-4 g M. Our release rates were estimated to range from 3 X 10^-8 to 3 X 10^-3 g/day (Figure 3). Inhibition of the response of D. brevicomis to naturally produced pheromone by I + II + III has not been tested in the laboratory or field, nor has the test of inhibition of response to E + F + M by I + II + III been conducted in the field. Further work would be necessary to elucidate the role or each semiochemical at the enantiomeric level (Wood et al., 1976; Borden et al., 1976). Verbenone and I + II + III may prove useful in inhibiting aggregation and host colonization by I. paraconfusus and D. brevicomis and thus function to reduce tree mortality caused by these bark beetles.

Acknowledgements- Arknowledgments-We would like to thank L.E. Browne, W.J. Loher, P.A. Rauch, and E. Zavarin of the University of California at Berkeley: M.C. Birch and D. M. Light of the University of California at Davis; and W.D. Bedard of the Forest Service, USDA, ior their helpful suggestions and review of the manuscript. We also thank K.Q. Lindahl, Jr., University of California at Berkeley, for review of statistical methods.

REFERENCES
BEDARD, W.D., TILDEN, P.E., LINDAHL, K.Q., JR., WOOD, D.L., and RAUCH, P.A. 1980a. Effects of verbenone and trans-verbenol on the response of Dendroctonus brevicomis to natural and synthetic attractant in the field. J. Chem. Ecol. 6:997-1013.

BORDEN, J.H., CHONG, L., McLEAN, J.A., SLESSOR, K.N., and MORl, K. 1976. Gnathotrichus sulcatus. Synergistic response to enantiomers of the aggregation pheromone sulcatol. Science 192:894-896.

BROWNE, L.E., BIRCH, M.C., and WOOD, D.L. 1974. Novel trapping and delivery systems for airborne insect pheromones. J. lnsect Physiol. 20:183-193.

BROWNE, L.E., WOOD, D.L., BEDARD, W.D., SILVERSTEIN, R.M., and WEST, J.R. 1979. Quantitative estimates of the Western pine beetle attractive pheromone components, exo-brevicomin, frontalin, and myrcene in nature. J. Chem. Ecol. 5:397-414.

BYERS, J.A., and WOOD, D.L. 1980. Interspecific inhibition of the response of the bark beetles, Dendroctonus brevicomis and Ips pararonfusus to their pheromones in the feld. J. Chem. Ecol. 6:149-164.

BYERS, J.A., WOOD, D.L., BROWNE, L.E., FISH, R.H., PIATEK, B. HENDRY, L.B. 1979. Relationship between a host plant compound, myrcene, and pheromone production in the bark beetle Ips paraconfusus. J. Insect Physrol. 25:477-482.

DETHIER, V.G. 1947. Chemical lnsect Attractants and Repellents. Blakiston, Philadelphia. 289 pp.

DETHIER, V.G., HACKLEY, B.D., and WAGNER-JAUREGG, T. 1952. Attraction of flies by isovaleraldehyde. Science 115:141-142.

KINZER, G. W., FENTIMAN, A. F., JR., PAGE, T. F., JR., FOLTZ, R. L., VITÉ, J. P., and PITMAN, G. B. 1969. Bark beetle attractants and field bioassay of a new compound isolated from Dendroctonus. Nature 22:475-476.

KIKUCHI, T., and OGURA, K. 1976. A three-binding site model for aggregation pheromone activities of the bark beetle, Ips confusus. Insect Biochem. 6:115-122.

MILLER, J. M., and KEEN, F. P. 1960. Biology and control of the western pine beetle. USDA Misc. Pub. No. 800, 381 pp.

MUSTAPARTA, H. 1979. Chemoreception in bark beetles of the genus Ips: Synergism, inhibition and discrimination of enantiomers, pp 147-158, in F.J. Ritter (ed.). Chemical Ecology: Odour Communication in Animals. Elsevier/North-Holland Biomedical Press, Oxford.

RENWICK, J.A.A. 1967. Identification of two oxygenated terpenes from the bark beetle, Dendrortonus frontalis and Dendroctonus brevicomis. Contrib. Boyce Thompson Inst. 23:355-360.

RENWICK, J.A.A., HUGHES, P.R., and KRULL, I.S. 1976. Selective production of cis- and trans-verbenol from (-) and (+) alpha-pinene by a bark beetle. Science 191:199-201.

RUDINSKY, J.A. 1973a. Multiple functions of the southern pine beetle pheromone verbenone. Environ. Entomol. 2:511-514.

RUDINSKY, J.A. 1973b. Multiple functions of the Douglas fIr beetle pheromone 3-methyl-2-cyclohexen-1-one. Environ. Entomol. 2:579-585.

SILVERSTEIN, R. M., and RODIN, J.O. 1965. Spectrometric identification of organic compounds on a milligram scale. The use of complementary information. Microchem. J. 9:301-308.

SILYERSTEIN, R.M., RODIN, J.O., and WOOD, D.L. 1966. Sex attractants in frass produced by male Ips confusus in ponderosa pine. Science 154:509-510.

SILVERSTEIN, R.M., BROWNLEE, R.G., BELLAS, T.E., WOOD, D.L., and BROWNE, L.E. 1968. Brevicomin: Principal sex attractant in the frass of the female western pine beetle. Science 159:889-891.

STRUBLE, G.R., and HALL, R.C. 1955. The California five-spined engraver, its biology and control. USDA Circ. No. 964, 21 pp.

WOOD, D.L. 1970. Pheromones of bark beetles, pp. 301-316, in D.L. Wood, R.M. Silverstein, and M. Nakajima (eds.). Control of Insect Behavior by Natural Products. Academic Press, New York.

WOOD, D.L., BROWNE, L.E., BEDARD, W.D., TILDEN, P.E., SILYERSTEIN, R.M., and RODlN, J.O. 1968. Response of Ips confusus to synthetic sex pheromones in nature. Science. 159:1373-1374.

WOOD, D.L., BROWNE, L.E., EWING, B., LINDAHL, K., BEDARD, W.D., TILDEN, P.E., MORI, K., PITMAN, G.B., and HUGHES, P.R. 1976. Western pine beetle: specificity among enantiomers of male and female components of an attractant pheromone. Science. 192:896-898.

WOOD, D.L., BROWNE, L.E., SILVERSTEIN, R.M., and RODIN, J.O. 1966. Sex pheromones of bark beetles-I. mass production, bioassay, source, and isolation of the sex pheromone of Ips confusus (LeC.). J. Insect Physiol. 12:523-536.

WOOD, D.L., STARK, R. W., SILVERSTEIN, R.M., and RODIN, J.O. 1967. Unique synergistic effects produced by the principal sex attractant compounds of Ips confusus (LeConte) (Coleoptera: Scolytidae). Nature. 215:206.