Journal of Chemical Ecology, Vol. 17, No. 11, 1991

RESPONSE OF Dendroctonus brevicomis AND Ips paraconfusus (COLEOPTERA: SCOLYTIDAE) TO COMBINATIONS OF SYNTHETIC PHEROMONE

ATTRACTANTS AND INHIBITORS VERBENONE AND IPSDIENOL

T . D . P A I N E * and C . C . H A N L O N

Department of Entomology University of California

Riverside, California 92521

(Received February 8, 1991; accepted July 8, 1991)

Abstract--Dendroctonus brevicomis and Ips paraconfusus are sympatric bark beetle species colonizing Pinus ponderosa in western North America. Inter- specific and intraspecific competition for resources is, in part, mediated through semiochemicals. The response of D. brevicomis to its attractant pher- omone was significantly reduced by simultaneous release of either verbenone or racemic ipsdienol. Trap catch was significantly further reduced by includ- ing both inhibitors with the attractant pheromones. However, although the response of Ips paraconfusus was significantly inhibited with the addition of either verbenone or racemic ipsdienol, both compounds together did not result in a significant further reduction in trap catch. There was a trend for greater reduction in response ofD. brevicomis to attractant pheromones with increased release rates of either 69 % ( +)-/31% ( -)-verbenone or 84 % ( -)- /16 % ( +)- verbenone. Response of associates to attractants and inhibitor combinations was also determined.

Key Words--Dendroctonus brevicomis, Ips paraconfusus, bark beetle, ver- benone, ipsdienol, pheromones, inhibitors, Coleoptera, Scolytidae.

INTRODUCTION

T h e w e s t e r n p ine bee t l e , Dendroctonus brevicomis L e C o n t e , uses a c o m b i n a -

t ion o f b e e t l e - p r o d u c e d p h e r o m o n e s [ ( + ) - e x o - b r e v i c o m i n { (+)-exo-7-ethyl-5- m e t h y l - 6 , 8 - d i o x a b i c y c l o [ 3 . 2 . 1 ] o c t a n e } and ( - ) - f r o n t a l i n { ( - ) - l , 5 - d i m e t h y l -

6 , 8 - d i o x a b i o c y c l o [ 3 . 2 . 1 ] o c t a n e } ] (S i lve r s t e in et a l . , 1968; K i n z e r et a l . , 1969;

* To whom correspondence should be addressed.

2163

0098-0331/91/1100-2163506,50/0 �9 1991 Plenum Publishing Corporation

2164 PAINE AND HANLON

Wood et al., 1976) and host odors, primarily myrcene (Bedard et al., 1969), during the aggregation phase of tree colonization (Wood, 1972). The engraver beetles, Ips paraconfusus Lanier and L pini (Say), are sympatric with D. brev- icomis in California and may colonize the same host material. Ips paraconfusus pheromone consists of three components: (-)-ipsenol[(-)-2-methyl-6-meth- ylene-7-octen-4-ol], (+)-cis-verbenol, and (+)-ipsdienol[(+)-2-methyl-6- methylene-2,7-octadien-4-ol] (Silverstein et al., 1966). The pheromone of / . pini in California was initially described as a single component pheromone, (-)-ipsdienol (Birch et al., 1980a, b); however, there is some suggestion that a second component may be important to complete the pheromone blend (S.A. Teale, personal communication).

The pheromones produced have interspecific effects in addition to the intraspecific effect. Attraction ofL pini to its pheromone is inhibited by racemic ipsenol (Birch and Light, 1977) and to (+)-ipsdienol (Birch et al., 1980a, b). These compounds are components of L paraconfusus aggregation pheromone. Similarly, attraction of L paraeonfusus to its pheromone is inhibited by ( - ) - ipsdienol (Light and Birch, 1979), a component of the aggregation pheromone of L pini. Dendroctonus brevicomis was inhibited by actively boring L para- confusus (Byers and Wood, 1980). Subsequent studies by Byers and Wood (1981a) demonstrated that at least two of the constituents of L paraconfusus pheromone must be present for the inhibitory effect.

Although the constituents of D. brevicomis pheromone function to attract individuals, high release rates of exo-brevicomin or myrcene may also function to interrupt the response (Tilden et al., 1981). Oxidation of tree-produced ot-pinene by either beetles or by microorganisms results in production of ver- benone. This compound found in male D. brevicomis (Byers and Wood, 1980) interrupts the response of D. brevicomis (Bedard et al., 1980) to its attractant pheromone. In addition, verbenone similarly inhibits the attraction response of I. paraconfusus (Byers and Wood, 1980, 1981a).

Colonization of conifers usually occurs as a community process. Although there may be a temporal separation, it would be expected that each species in the community would produce the components of its pheromone with the result- ing potential for interspecific and intraspecific interactions. The objective of the study presented here was to determine how the compounds associated with inhi- bition would effect aggregation of D. brevicomis and L paraconfusus if pre- sented singly or in combination.

METHODS AND MATERIALS

The behavioral responses of D. brevicomis and L paraconfusus to inter- specific and intraspecific chemical signals were examined at two locations dur- ing July and August 1990. Each site had obvious tree mortality, and examination

RESPONSE TO ATTRACTANTS AND INHIBITORS 2165

of the recently killed trees indicated that populations of both D. brevicomis and L paraconfusus were high. While both sites were located within Yellow Pine Forest ecosystems (Munz and Keck, 1973), one site (Julian, San Diego County) was at 1300 m elevation, dominated by Pinus couheri D. Don. and Q. agrifolia Nee near the interface with the Chaparral ecosystem, while the other site (Pal- omar Mountain, San Diego County) was at 1500 m elevation and dominated by mixed conifers.

One of 12 combinations of D. brevicomis attractant [Db = (+)-exo-brev- icomin (released at 2.6 mg/24 hr at 25~ (-)-frontalin (2.0 mg/24 hr), and myrcene (300 mg/24 hr)], L paraconfusus pheromone lip = 50% (+)-/50% (-)-ipsenol (0.2 mg/24 hr), cis-verbenol (2 mg/24 hr), and 95% (+)-/5% ( - ) - ipsdienol (0.2 mg/24 hr)], 16% (+)-/84% (-)-verbenone (10 rag/24 hr), and/ or 50% (+)-/50% (-)-ipsdienol (10 mg/24 hr) was placed on a Lindgren funnel trap (trap and all synthetic compounds from Phero Tech Inc., Delta, British Columbia, Canada). Inhibitors were always tested in combination with one or more of the attractant pheromones. An unbaited trap was also tested as a con- trol. A 3-cm z piece of DDVP-impregnated plastic was placed in the collection cup of each trap to kill any captured beetles. A line of traps spaced at 20-m intervals containing one of each of the 13 treatments constituted a block. Three lines of traps were located in the Julian site and two lines of traps were located at Mount Palomar in two separate tests. Beetles were collected from the traps daily for 12 days, each day constituting a replicate. After each collection, the chemical baits were shifted in a predetermined sequence to a new position within each line. The trapped insects were brought into the laboratory and the numbers of L paraconfusus and D. brevicomis were counted. We also counted Den- droctonus valens LeConte and the predators Enocleris lecontii (Wolcott) (Coleoptera: Cleridae) and Temnochila chlorodia (Mannerheim) (Coleoptera: Trogositidae).

Verbenone has been reported as an inhibitor of both L paraconfusus and D. brevicomis. However it is not clear whether either or both enantiomers of verbenone are effective at inhibiting the response to the attractant pheromones. We tested a mixture of 16% (+)-/84% (-)-verbenone released at five elution rates at the Mount Palomar site during August 1990. Funnel traps were baited with one of six treatments: D. brevicomis pheromone [exo-brevicomin (released at 2.6 mg/24 hr at 24~ frontalin (2.0 mg/24 hr), and myrcene (300 mg/24 hr)] alone and D. brevicomis pheromone plus the verbenone enantiomers eluted at 0.01 mg/24 hr (at 25~ 0.18 mg/24 hr, 1.8 mg/24 hr, 5 mg/24 hr, and 20 mg/24 hr. As in the previous test, an unbaited trap served as a control. Each of the seven treatments was replicated 10 times. Beetles were collected from the traps three times at 72-hr intervals. An identical test was subsequently con- ducted using 69 % (+)-/31% (-)-verbenone in the same location.

Differences in trap catches among the 13 treatments at each location for

2166 PAINE AND HANLON

each beetle species, as well as the differences in the response of D. brevicomis among release rate treatments for an enantiomeric blend were determined using General Linear Modeling procedures (PROC GLM) and Duncan's new multi- pie-range test on SAS for microcomputers (SAS Institute, 1988).

RESULTS

At both study sites, the traps baited with D. brevicomis pheromone (Db) alone caught significantly more of that species than any of the other treatments [Julian: F = 18.41, d f = 12, P = 0.0001 (Table 1); Palomar: F = 13.99, d f = 12, P = 0.0001 (Table 2)]. Four treatments [Db and verbenone, Db and ipsdienol, Db and Ips paraconfusus pheromone (Ip) and Db and Ip and ips- dienol] at both sites inhibited the response of D. brevicomis to the attractant pheromone, but still caught more beetles than the blank control traps. The three remaining treatments (Db and Ip and verbenone, Db and verbenone and ips- dienol, and Db and Ip and verbenone and ipsdienol) further reduced catches of D. brevicomis to levels not significantly different from either the unbaited con- trol or to those treatments lacking the aggregation pheromone (Db) of this spe- cies.

The arrival of L paraconfusus at traps at the Mount Palomar location was significantly inhibited (F = 8.34, d f = 12, P = 0.0001) by the addition of verbenone and ipsdienol to treatments that included the aggregation pheromones (Table 2). The aggregation pheromones of this species alone and in combination with the aggregation pheromones from D. brevicomis were the only two treat- ments that were significantly different from the responses to the unbaited con- trol. Although the response of L paraconfusus was significantly greater (F = 6.87, dd = 12, P = 0.0001) to the aggregation pheromones of both L para- confusus and D. brevicomis together, the response to the aggregation phero- mone of L paraconfusus alone was not different from the unbaited control or any of the inhibitor treatments at the Julian location (Table 1).

While there were some beetles caught in the traps, there were no significant differences in the arrival of E. lecontii (F = 0.76, d f = 12, P = 0.2799) to any of the 13 treatments tested in the first study at Julian, but the response of both D. valens (F = 4.27, d f = 12, P = 0.0001) and T. chlorodia (F = 3.25, d f = 12, P = 0.0002) was significantly greater to the E-M-F and [50% (+)- / 50 % ( -)- ipsenol , cis-verbenol, and 95 % (+)- /5 % ( -)-ipsdienol] and ipsdienol treatment (Table 1). The response of D. valens at the Mount Palomar location was not significantly different to any treatment (F = 0.90, d f = 12, P = 0.5519). However, there were significant differences observed for the response of the other two associates to the same treatments at the Mount Palomar site [E. lecontii (F = 2.06, d f = 12, P = 0.0197), T. chlorodia (F = 2.12, d f =

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RESPONSE TO ATTRACTANTS AND INHIBITORS 2169

12, P = 0.0159), Table 2]. Individuals of these three species were also caught in traps baited with D. brevicomis aggregation pheromone and different release rates of verbenone. The response of D. valens was significantly less (F = 3.60, df = 6, P = 0.0021) when any 69% (+)-/31% (-) -verbenone treatment was included, but there were no significant differences among treatments for E. lecontii (F = 0.96, df = 6, P = 0.4519) or T. chlorodia (F = 1.71, df = 6, P = 0.1199) (Table 3). In contrast, when 16% (+)- /84% (-) -verbenone was tested, catch of D. valens was significantly greater than the unbaited control (F = 2.37, df = 6, P = 0.0311) for only the lowest release rate (Table 4). Response of E. lecontii was significantly different from the control (F = 2.72, df = 6, P = 0.0147) at the highest release rate, and response of T. chlorodia was not different to any verbenone treatment (F = 1.37, df = 6, P = 0.2287).

The response of D. brevicomis to the aggregation pheromone alone was significantly greater (F = 15.06, df = 6, P = 0.0001) than to any of the treatments containing the 69 % (+)- /31% (- ) -verbenone (Table 3). There was no clear dose-response relationship, but all treatments attracted more beetles than did the unbaited control. In contrast, the responses ofD. brevicomis to the two lowest release rates of 16% (+)- /84% (-) -verbenone were not signifi- cantly different (F = 17.57, df = 6, P = 0.0001) than the response to the aggregation pheromone alone (Table 4). However, traps baited with the lowest release rate of this enantiomeric blend of verbenone plus aggregation phero- mone caught significantly more beetles than did the treatment with the next lowest release rate. As in the previous test of enantiomers, all treatments attracted more beetles than did the unbaited control traps.

D I S C U S S I O N

Our results confirm that the response of D. brevicomis to its aggregation pheromone is reduced by the presence of L paraconfusus pheromone, racemic ipsdienol, or verbenone, but also demonstrate that the presence of multiple inhibitor compounds further reduces the response. While it is not clear why the response of L paraconfusus to its aggregation pheromone was not different (P = 0.05) from the control treatment at the Julian site, the reduction in the response at the Mt. Palomar location was not significantly enhanced by the addition of more than one inhibitory compound. However, at the Julian loca- tion, there was a tendency for the treatments with either D. brevicomis or L paraconfusus pheromones alone or both pheromones together plus only a single inhibitor to have higher trap catches. The response of the associates was gen- erally very low to any of the treatments. However, Wood et al. (1968) dem- onstrated that E. lecontii responds to pheromone constituents of L paraconfusus, while Vit6 and Pitman (1969) found that T. chlorodia responded to D. brevi- comis pheromone.

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2172 PAINE AND HANLON

The process of tree colonization by D. brevicomis takes place within a very short time and is mediated by attractant semiochemicals (Wood, 1972). How- ever, there is the potential for competition among the arriving beetles for the limited amount of available resource (Alcock, 1982; Berryman et al., 1985). One of the mechanisms used by the beetles appears to be a complex coordina- tion of timing in release of attractants and production of inhibitory compounds (Byers et al., 1984). Consequently, the arriving beetles are spaced to maximize individual fitness (Schlyter et al., 1987; DeJong and Sabelis, 1988).

The recognition of verbenone as an inhibitor occurs in several species within the Dendroctonus and Ips genera, Pitman et al. (1969) identified ver- benone from D. brevicomis, D. frontalis Zimmermann, and D. ponderosae Hopkins. Subsequent investigations demonstrated that the compound inhibits arrival of beetles to attractive sources (Bedard et al., 1980; Ryker and Yandell, 1983; Payne and Billings, 1988). Ips paraconfusus and L typographus (L.) were also shown to be inhibited by verbenone (Byers and Wood, 1980, 1981a; Bakke, 1981).

It is not common to observe individuals of a single bark beetle species colonizing a tree. Rather it is usually a community of species (Paine et al., 1980). The interactions among the members of the community may be mediated by the pheromone communication system (Birch et al., 1980a, b; Smith et al., 1990). However, several beetle species may produce the same chemical com- pounds and exploit the inhibitor message to reduce competition. Male D. brev- icomis and male D. ponderosae produce (+)-ipsdienol from myrcene (Renwick et al., 1976; Byers, 1982; Hunt et al., 1986). This enantiomer inhibits conspe- cifics (Byers, 1982; Hunt and Borden, 1988) as well as L pini (Birch et al., 1980a, b), a potential competitor of either species. Thus, D. brevicomis and D. ponderosae release two compounds (verbenone and ipsdienol) that have inter- specific and intraspecific inhibitory effects. The effect of releasing the inhibitors simultaneously has not been observed previously for a Dendroctonus species. There was a suggestion from the studies of Bakke (1981) that the release of two inhibitory compounds was more effective for inhibiting the response of L typo- graphus than the release of either compound alone, but subsequent work could not confirm those results (Schlyter et al., 1988).

The chemical components of the pheromone blends have multifunctional effects; they may function as attractant pheromones, inhibitory pheromones, or as synomones. The insects may produce the compounds, or production may be the result of microbial action on either ingested substrate or exposure to volatile substrates. Brand et al. (1975) isolated a bacterium from L paraconfusus that utilized ot-pinene to synthesize cis- and trans-verbenol. Byers and Wood (1981b) reported that streptomycin treatments of L paraconfusus prevented the conver- sion of myrcene to ipsenol and ipsdienol. In contrast, L typographus produces cis- and trans-verbenol but requires associated yeasts to convert these com- pounds to verbenone (Leufven et al., 1984; Leufven and Birgersson, 1987).

RESPONSE TO A T T R A C T A N T S AND INHIBITORS 2173

The yeasts were unable to produce either the verbenols or verbenone directly from a-pinene (Leufven et al., 1988). Similarly, D. ponderosae requires a gut microorganism to produce verbenone (Hunt and Borden, 1989).

There is an obvious ecological advantage to both arriving and colonizing beetles to avoid either marginal resources or intense competition. Verbenone, derived from ot-pinene through the intermediate verbenols by microbial action in several systems, appears to be a common signal that the resource may be attacked and potentially unsuitable for further colonization (Schlyter et al., 1988). The terpene alcohols, ipsenol and ipsdienol, may also be interspecific and intraspecific inhibitors that serve to reduce detrimental competition. Light et al. (1983) demonstrated that interspecific competition between L paracon- fusus and L pini produced greater negative effects than intraspecific competition and speculated that these competitive interactions may be the selective force that resulted in mutual pheromone inhibition between the species. The geo- graphic range of D. brevicomis is within the range of Pinus ponderosa Lawson. Ips pini and D. brevicomis are sympatric in much of that range, and the enan- tiomer of ipsdienol produced by D. brevicomis is an effective inhibitor of I. pini where sympatry occurs (Birch et al., 1980a, b). It is, however, a constituent of the attractant pheromone of L paraconfusus that overlaps with D. brevicomis in only a portion of the geographic range.

While parallel studies have not been completed for L paraconfusus and D. brevicomis, the competitive interactions may be similarly detrimental. How- ever, it is not clear why the presence of multiple inhibitors produced a greater effect on D. brevicomis than L paraconfusus. It is possible that this Ips species is more sensitive to the presence of the inhibitors or the described pheromone blend for this species is incomplete, and as a result, the maximum reduction was observed to any single compound. There is a difference between L para- confusus and L typographus. The results of Bakke (1981) suggest that attraction of L typographus is inhibited more by a combination of ipsenol plus verbenone than by either compound alone. However, in the European spruce system, this species may be more of an ecological homologue to D. brevicomis than to L paraconfusus in westem North America.

Acknowledgments--We thank J. Trumble, R. Redak, P. Luft, T. Grasswitz, P. Leddy, S. Bertram, and M. Moratorio for helpful reviews of the manuscript; Phero Tech Inc. (Delta, British Columbia, Canada) for the release devices used in the enantiomeric dose test; and D. Messore, S. Gilbert, M. Goeden, and S. Bertram for data collection. This study was supported in part by a grant from the California Department of Forestry and Fire Protection.

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2174 PAINE AND HANLON

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