Synergistic pheromones and monoterpenes enable aggregation and host recognition by a bark beetle, Pityogenes chalcographus

Byers, J.A., Birgersson, G., Löfqvist, J., and Bergström, G. 1988. Synergistic pheromones and monoterpenes enable aggregation and host recognition by a bark beetle. Naturwissenschaften 75:153- I55. pdf

Pityogenes chalcographus male looking for place to begin boring into bark of Norway spruce, Picea abies. Only males produce aggregation pheromone.

We report the strong synergism between two pheromone components in causing the attraction of the six-spined spruce bark beetle or "Kupferstecher", Pityogenes chalcographus L., in the field. Our field results also suggest that several monoterpenes of Norway spruce, Picea abies (L.) Karst, when presented with the pheromone components, play a role in host recognition and colonization by the beetle because they caused relatively more beetles to enter holes in artificial hosts. Furthermore, sexual differences in strategies of host resource competition are indicated because of the lower proportions of males than females that entered the holes as well as were attracted to higher release rates of pheromone.

In Europe, P. chalcographus is a serious pest of Norway spruce upon which the tiny adults (2 mm long) aggregate in response to a male-produced pheromone that earlier was reported to consist of at least one component, chalcogran, a unique dioxaspiro acetal [1]. However, the release of 15 mg h^-1 of synthetic chalcogran was not as attractive as a host log infested with 30 males, suggesting that a second component may be necessary for maximum response [1]. Recently, we used two-dimensional gas chromatography and subtractive-combination bioassay to isolate a second male-produced component, methyl-E,Z-2,4-decadienoate (E,Z-MD), from odors of beetle-infested logs [2]. E,Z-MD and chalcogran were further shown to be synergistically attractive in the laboratory bioassay, while an additional several monoterpenes in mixture caused a slight enhancement of the response [2].

We tested E,Z-MD and chalcogran for their attractivities alone and in combination in the forest during the spring dispersal/host-seeking flight of P. chalrographus (Table 1). Traps with the pheromone components or blanks were placed 10 m apart in a line in clear-cut areas of Norway spruce forest. Each trap consisted of a vertically oriented black plastic cylinder (1.35 m x l2cm diam. with 900, 2-mm diam. holes uniformly spaced over the surface [3]) that was centered over a 33-cm diam. funnel. Beetles were collected either during landing by this funnel (by striking the cylinder and falling or by flying downward) or after they landed and entered one of the holes in the cylinder by a smaller 12-cm diam. funnel fixed at the bottom (by slipping inside the cylinder).

In test 1 (Table 1), the combination of the pheromone components was clearly synergistic, catching 34 times more than chalcogran, while E,Z-MD had no activity alone (19 replicates, Wilcoxon P< 0.01).

	Attraction to cylinder trap
Table 1. Attraction of Pityogenes chalcographus to perforated cylinder traps releasing synthetic host ((+ )-alpha-pinene = (+ A), (- )-alpha-pinene = (- A), (- )-(B-pinene = (- B), and camphene = Cp) and pheromone components (methyl-E,Z-2,4-decadienoate = E,Z-MD and chalcogran = CH) in the forest (June 1-15,1986, Grib skov, Denmark).
	Landing		Entering holes
Chemicals released^a	Total	Male proportion	Total	Male proportion	[ % ]
test 1
Blank	1	-	0	-	0
CH	39	0.26	8	-	17.0
E,Z-MD	0	-	0	-	-
E,Z-MD + CH	1376	0.42	240	0.35	14.9
test 2
E,Z-MD + CH	123	0.46	7	-	5.4^b
E,Z-MD + CH + (+A) + (-A) + (-B) + Cp	482	0.40	338	0.35	41.2
test 3^c
E,Z-MD + CH	763	0.33	103	0.32	11.9^b
E,Z-MD + CH + (+A)	720	0.33	318	0.29	30.6
E,Z-MD + CH + (-A)	1476	0.33	815	0.28^d	35.6
E,Z-MD + CH + (-B)	1077	0.35	618	0.27^d	36.5
E,Z-MD + CH + Cp	749	0.33	208	0.24^d	21.7
test 4
Blank	4	-	0	-	0
0.1 x (E,Z-MD + CH)	665	0.43^e	139	0.26	17.3
1 x (E,Z-MD + CH)	1968	0.37	534	0.33	21.3
10 x (E,Z-MD + CH)	2772	0.34	507	0.30	15.5

^aExcept as indicated in test 4, chemicals inside each cylinder were released (per day) at 18 µg E,Z-MD, > 99.5% GC pure; 1 mg CH, 46 % E: 54 % Z, > 98 % pure from W. Francke, Univ. of Hamburg, West Germany; l4mg (+A), [alpha]²²_D = +48^o; 14mg (-A), [alpha]²²_D = - 42^o; 10 mg (- B), [alpha]²²_D = - 21^o ; and 40 mg Cp (all monoterpenes < 98 % pure, Fluka and Carl Roth).
^bPercentages of males or females entering holes were significantly different from those entering treatments releasing monoterpenes (P< 0.001, X², except for males to Cp, P= 0.11). ^cWilcoxon tests indicated the catch was significantly greater on pheromone (E,Z-MD + CH) plus (-A) baits than on pheromone alone (N=10, P< 0.01).
^dMale proportions were significantly different from corresponding proportions of landing (P< 0.01, X²).
^eMale proportion was significantly different from male proportion entering holes or from male proportions of landing at the higher release rates (P< 0.01, X²).

In the second test (Table 1), a mixture of several host monoterpenes that were released with the pheromone components caused a significant increase in the proportion of both sexes that entered holes in the cylinder trap. Each of the various monoterpenes when released with the pheromone components also caused a greater percentage of both sexes to enter holes (test 3, Table 1). These results indicate that the monoterpenes, especially a mixture, when released from a 12-cm diam. cylinder can simulate a host tree and induce hole-entering and thigmo- tactic behavior in both sexes. (±)-alpha-Pinene, (-)-B-pinene, and camphene, major volatiles of host oleoresin [2], are thus used by P. chalcographus to recognize its host during colonization. The release rates used here are comparable to those released from infested host material [2].

If possible, it would be to the advantage of a male to enter holes releasing monoterpene vapors because this would indicate a direct route to the phloem tissue without the necessary energy and time expenditure of boring through the outer bark. On the other hand, females normally enter holes with monoterpenes and pheromone component vapors because this indicates the host tissue needed for egg laying as well as a male for mating and possible brood protection (the male guards the entrance to the gallery system). Monoterpenes either alone [4] or usually in combination with pheromone components [5] have been shown to be attractive to bark beetles, but the effect on hole-entering has not been reported earlier.

Increasing release rates over a 100-fold range of E,Z-MD and chalcogran were tested to observe response relationships in both sexes (test 4, Table 1). Catch increased in a logarithmic relationship, Y[%] = 66.88 + 16.39 x lnX (where X = 0.1 to 10 and maximum catch = 100 %), but the highest release rate may have eaused some disorientation (confusion) near the source because the increase in catch was proportionately less than the increase between the lowest and middle doses. While there was no apparent effect of release rate on the percentage entering holes, there was an effect on the male proportions that landed or entered holes.

The proportion of males in the landing catch decreased as the release rate was increased (test 4, Table 1). This effect of relative male inhibition by higher releases of pheromone has also been observed in other polygynous bark beetles, Ips paraconfusus [6] and I. typographus [7]. Furthermore, the relative proportion of males caught decreases between landing and entering holes in all cases either with or without monoterpenes, and at all release rates (Table 1). In several cases, the differences were significant (P< 0.01, X²), as were the differences between the pooled results for the male proportions of landing (0.384) and entering (0.335) in response to the pheromone components alone (P< 0.01) as well as between the male proportions of landing (0.340) and entering (0.286) in response to the pheromone with monoterpenes (P< 0.001). This again indicates that males may be proportionately less attracted by higher release rates encountered while proceeding up the pheromone concentration gradient.

It has been proposed that males of the polygynous bark beetle species (e.g., Ips, Pityogenes) which produce the pheromone and establish territories of resource utilization will likely be relatively less attracted by higher release rates of their pheromone than are females [6]. Higher release rates would indicate to arriving males that the emanating areas are fully colonized and are thus to be avoided in order to avoid competition for food and space resources. Females, on the other hand, require male-initiated galleries and thus the male-produced aggregation pheromone serves additionally as a sex pheromone.

The highly synergistic properties of E,Z-MD and chalcogran in attracting P. chalcographus indicate that a potent pheromone blend has been identified. Host monoterpenes may enhance slightly the attraction and further serve to increase catch through hole-entering in certain kinds of traps. Bark beetle control methods using pheromone-baited traps have recently been undertaken [8]. This method removes the most vigorous beetles, those that are flying and responding and are potentially the most damaging of the population. Trap-out methods are selective, environmentally safe, and do not compete significantly with other natural mortality agents during the dispersal flight when probably most adult mortality occurs. Based on our results E,Z-MD and chalcogran have recently been marketed as a commercial lure ("Chalcoprax", Shell Agrar GmbH & Co. KG, P.O. Box 200, D-6507 Ingelheim am Rhein) for trapping of P. chalcographus [9].

Received November 10 and December 17, 1987

J. A. BYERS¹, G. BIRGERSSON², J. LÖFQVIST¹, and G. BERGSTRÖM² ¹Department of Animal Ecology, Lund University, SE-223 62 Lund, Sweden Present address: ²Chemical Ecology, Göteborg University, SE-405 30 Göteborg, Sweden

1. Francke, W., et al.: Naturwissenschaften 64, 590 ( 1977)
2. Byers, J. A., et al.: J. Chem. Ecol. (submitted)
3. Bakke, A., Saether, T., Kvamme, T.: Medd. Nor. inst. skogforsk. 38, 1 (1983)
4. Byers, J. A., et al.: Naturwissenschaften 72, 324 (1985)
5. Wood, D. L.: Ann. Rev. Entomol. 27, 411 (1982)
6. Byers, J. A.: J. Chem. Ecol. 9,129 ( l983)
7. Schlyter, F., Löfqvist, J., Byers, J. A.: Physiol. Entomol. I2, 185 (1987)
8. Bakke, A.: Z. angew. Ent. 99, 33 (1985)
9. Baader, V. E., Vité, J. P.: Allg. Forstz. 41, 1008 ( 1986)