The role of semiochemicals in tritrophic interactions between the spruce bark beetle Ips typographus, its predators and infested spruce

Abstract:  Semiochemical interactions between the spruce bark beetle Ips typographus , its predators Medetera setiventris , Thanasimus formicarius and Thanasimus femoralis , and the host Norway spruce, Picea abies , were studied in the field. The chemicals S - cis -verbenol, 2-methyl-3-buten-2-ol, ipsdienol, (+)- α -pinene, (−)- α -pinene, (±)- α -pinene, limonene, camphor and their naturally occuring mixtures were used as trap baits in a multiple-choice design that allowed for comparison of their attractivity for the focal species. Medetera was attracted to both the prey aggregation pheromone and its multifunctional component, ipsdienol. On the contrary, both Thanasimus species responded predominantly to ipsdienol and less to the prey aggregation pheromone. In the case of I. typographus , the attractivity of aggregation pheromone seems to be increased by the addition of a mixture of monoterpenic tree volatiles, and by addition of ipsdienol. Bark beetles and predators showed species-specific responses to volatile mixtures representing different stages of tree decay and different stages of bark beetle colony establishment. These responses correlates with the optimal foraging habitat of each species. None of the predator species responded to 2-methyl-3-buten-2-ol, a substantial component of I. typographus pheromonal bouquet, thus it is hypothesized that only substances of monoterpenic origin attract predators.     

Aggregation response of European engraver beetles of the genus Ips mediated by terpenoid pheromones

Upon exposure to vapours of the host tree's monoterpene (–)--pinene, Ips acuminatus produces the terpene alcohol cis-verbenol which, besides ipsdienol and ipsenol, is a component of its aggregation pheromone. I. cembrae transforms the same monoterpene predominantly into myrtenol and trans-verbenol but little into cis-verbenol, which interrupts pheromone response under field conditions.On the other hand, I. cembrae releases, upon feeding in the bark, the terpene alcohol amitinol in major amounts. Amitinol significantly enhances field response to the aggregation pheromone that also includes ipsdienol, ipsenol, and 3-methyl-3-buten-1-ol. However, amitinol reduces pheromone response in I. acuminatus and I. erosus. Also, males of I. sexdentatus release amitinol which appears to increase response to its attractive principle pheromone component, racemic ipsdienol. There is some evidence that present knowledge of the chemical communication systems among European Ips spp. still lacks satisfactory explanation of the naturally occuring aggregation en masse, perhaps with the exception of I. erosus and I. typographus.     

An operative kairomonal lure for managing pine sawyer beetle Monochamus galloprovincialis (Coleoptera: Cerymbycidae)

Abstract:  Monochamus galloprovincialis Olivier (Col., Cerambycidae) is a vector of the pine wood nematode, Bursaphelenchus xylophilus , causing the destructive pine wilt disease. An effective lure for monitoring and/or mass-trapping would be of great interest in the management of this pine sawyer. Males and females of this species show an attractive kairomonal response to blends composed of four pheromone compounds used by Ips spp. bark beetles and two host volatiles from pines. This six-component lure is highly attractive but may to be too complex and costly for practical use as each component is released from a separate lure. The role of each component, ipsdienol, ipsenol, cis -verbenol, methyl butenol, α -pinene and ethanol as attractants for M. galloprovincialis was field tested in Spain to obtain a simpler but equally effective bait. Ipsenol was confirmed as the strongest kairomonal signal to M. galloprovincialis synergizing response to α -pinene by 95 times. The addition of methyl butenol to this blend doubled the number of males and females trapped. On the other hand, neither ipsdienol, cis -verbenol nor ethanol improved the results when incorporated into the above three-component blend. A lure consisting of ipsenol, methyl butenol and α -pinene may be very cost-efficient in operational monitoring or mass trapping of M. galloprovincialis . Three potentially repellent candidates, (−)verbenone, methyl cyclohexenone and trans -conophthorin, were also tested against the attractive three-component bait. trans -Conophthorin significantly reduced male catches of M. galloprovincialis ; methyl cyclohexenone had no effect. Verbenone significantly enhanced the response of females to the attractive combination of α -pinene, ipsenol and methyl butenol.     

Neural and hormonal control of pheromone biosynthesis in the bark beetle, Ips paraconfusus

ABSTRACT. Synthetic juvenile hormone (JH) induced the synthesis of the three pheromones, ipsenol, ipsdienol and 2-phenylethanol, by Ips paraconfusus males and enhanced the conversion of (–)-α-pinene to a fourth pheromome, cis -verbenol. Synthetic JH also mimicked the stimulatory effect of feeding on the metabolism of myrcene to ipsdienol and ipsenol. Implantations of corpora allata and/or corpora cardiaca from fed or newly-emerged males into intact or decapitated males were performed. The results indicated that JH acts indirectly by stimulating the release of brain hormone, and the release of JH in unfed males is prevented by neural inhibition which is removed by the stretching of the gut during normal feeding. Failure of any hormonal treatment or gland implantation to stimulate cis -verbenol synthesis in the absence of exogenous α-pinene is presented as evidence that no endogenous precursor for this pheromone is present. An apparent seasonal variation in the ability of males to produce ipsdienol and ipsenol when treated with JH was observed.     

Host tree resistance influencing pheromone production in Ips typographus (Coleoptera: Scolytidae)

Analyses of volatiles in hindguts of Ips typgraphus males from different spruce trees and attack phases are reviewed. The composition of monoterpenes, and the chirality of α-pinene, have been determined in phloem samples. Relationships between compounds emanating from spruce trees and bark beetles, respectively, have been studied. Male beetles depend on their host tree for the production of pinene alcohols. The ratio between the pinene alcohols is almost constant in males boring in the same tree but can vary widely between males from different spruce trees. Very good correlations were found between some host tree monoterpene hydrocarbons and bark beetle produced pinene alcohols. The production of the essential pheromone component 2-methyl-3-buten-2-ol, was not correlated with the monoterpene content in the host trees, while the production of the other essential pheromone component, cis-verbenol, depends on the amount of the precursor, (−)-α-pinene in the phloem. Male beetles boring in a resistant spruce tree will continue to produce the pinene alcohols, including cis -verbenol, as long as the tree defends itself with resin.     

Influence of the proportion of (−) α-pinene in pheromone bait on Ips typographus (Col., Scolytidae) catch in pheromone trap barriers and in single traps

Abstract:  Further possibilities of increasing catch efficiency of Ips typographus L. in pheromone trap barriers with two different levels of release of (4S)- cis -verbenol (cV) and in single traps were tested. Pheromone mixtures containing cV, 2-methyl-3-buten-2-ol (MB) and 0, 1, 10, 35, 60 and 90% (−) α -pinene were prepared. Basically, MB was successively replaced by (−) α -pinene in the mixtures. Tests were performed on the edge of a spruce forest when outbreaks of I. typographus occurred. The substitution of MB with (−) α -pinene had different effects in different types of pheromone trap barriers and in single traps. It is not possible to increase the attractiveness of the bait having a higher percentage of cV by supplementing a certain percentage of MB with (−) α -pinene. The catch of I. typographus decreased with an increase in the percentage of (−) α -pinene in the bait. It is possible to increase the attractiveness of the bait having a lower percentage of cV by supplementing certain percentages of MB with (−) α -pinene. A slight synergistic effect of (−) α -pinene with MB/cV mixture is visible in this type of bait. In the case of single traps, supplementing MB with (−) α -pinene possibly causes a slight synergistic effect. An increase in the percentage of (−) α -pinene increases the percentage of males in the catch.     

Behavioural sequence in the attraction of the bark beetle Ips typographus to pheromone sources

ABSTRACT. The functions of the two synergistic pheromone components, (4S)- cis -verbenol (cV) and 2-methyl-3-buten-2-01 (MB), and the role of ipsdienol in the attraction of Ips typographus (L.) (Scolytidae) to pheromone sources were studied in the field. Absolute and relative beetle catches were compared between several traps placed at and nearby a central pheromone source: a pipe trap containing the source, a surrounding sticky trap, a nearby window trap, and four distant window traps. A higher catch in the outer down-wind distant traps indicated an up-wind anemotaxis to the source. Increased MB release, with cV constant, increased the proportion caught in the central pipe trap, indicating MB as a landing stimulus. Release of MB alone gave a very small catch. Ipsdienol could not substitute for cV in the synergism with MB. An increase of cV, with MB constant, increased the number of beetles caught, but not the proportion caught in the pipe trap. The sex ratio was equal in the window traps, but fell to 30% males in both sticky and pipe traps, showing that a large proportion of the males attracted to the source did not land. The proportion of males in the pipe trap was reduced at the highest cV dose. The results support the idea of each pheromone component having a different relative importance in releasing different steps in the behavioural chain.     

Influence of sex,maturity and host substances on pheromones in the guts of the bark beetles, Ips paraconfusus and Dendroctonus brevicomis

Pheromones and metabolites of host (ponderosa pine) compounds were found in association with the hindgut of both naturally fed and of non-fed, host vapour-exposed bark beetles, Ips paraconfusus and Dendroctonus brevicomis. Much smaller amounts were found in the corresponding heads and mid guts. Sex-specific differences in content of pheromones were observed as in earlier studies. Exposure of I. paraconfusus to vapours of a pheromone component, ipsenol and other monoterpene alcohols resulted in their accumulation in the hindgut but relatively very low amounts in the head. The possible sites of pheromone biosynthesis are discussed. Exposure of male I. paraconfusus to vapours of host compounds, myrcene and α-pinene, revealed that immature adults do not produce the pheromone components, ipsenol and ipsdienol, as mature adults do while both immature and mature sexes produced another pheromone component, cis-verbenol, as well as trans-verbenol and myrtenol. Immature D. brevicomis adults did not contain pheromones until their exposure to vapours of (?)-α-pinene which caused production of trans-verbenol but only about 10% that of mature adults treated similarly. Verbenone, a male-produced inhibitory pheromone of D. brevicomis, apparently was not synthesized from (?)-α-pinene in females nor was its synthesis in males enhanced by exposure to this host compound.     

Sulcatol: population aggregation pheromone in the scolytid beetle, Gnathotrichus sulcatus

The population aggregation pheromone produced by males of Gnathotrichus sulcatus, a timber pest, has been identified from boring dust as a $\text{65}\text{35}$ mixture of the (S)-(+) and the (R)-(?) enantiomers of 6-methyl-5-hepten-2-ol. In field studies beetles were attracted in a 2·65 female: 1 male ratio by racemic synthetic pheromone.     

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway.     

Biosynthesis of 2-methyl-3-buten-2-ol,a pheromone component of Ips typographus (Coleoptera: Scolytidae)

Exposure of beetles to ¹⁴C-labelled mevalonate by injection, resulted in significant incorporation of radioactivity in 2-methyl-3-buten-2-ol. Radiolabelled 2-methyl-3-buten-2-ol was obtained both by organic solvent extraction of beetle hindguts and by entrainment of volatiles in air surrounding logs with boring beetles. It is suggested that 2-methyl-3-buten-2-ol, an essential component of the aggregation pheromone of Ips typographus, can be synthesized from mevalonate.     

Host resistance elicited by methyl jasmonate reduces emission of aggregation pheromones by the spruce bark beetle, Ips typographus

We treated Norway spruce (Picea abies) stems with methyl jasmonate (MeJA) to determine possible quantitative and qualitative effects of induced tree defenses on pheromone emission by the spruce bark beetle Ips typographus. We measured the amounts of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two main components of the beetle's aggregation pheromone, released from beetle entrance holes, along with phloem terpene content and beetle performance in MeJA-treated and untreated Norway spruce logs. As expected, phloem terpene levels were higher and beetle tunnel length was shorter (an indication of poor performance) in MeJA-treated logs relative to untreated logs. Parallel to the higher phloem terpene content and poorer beetle performance, beetles in MeJA-treated logs released significantly less 2-methyl-3-buten-2-ol and (S)-cis-verbenol, and the ratio between the two pheromone components was significantly altered. These results suggest that host resistance elicited by MeJA application reduces pheromone emission by I. typographus and alters the critical ratio between the two main pheromone components needed to elicit aggregation. The results also provide a mechanistic explanation for the reduced performance and attractivity observed in earlier studies when bark beetles colonize trees with elicited host defenses, and extend our understanding of the ecological functions of conifer resistance against bark beetles.     

Identification of sulcatol, a potential pheromone of the ambrosia beetle Gnathotrichus materiarius (Col., Scolytidae)

Abstract: We report the identification of a potential pheromone for Gnathotrichus materiarius (Fitch) (Col., Scolytidae). The population sex ratio is close to 1 : 1, and males initiate attacks on host trees. Headspace and hindgut samples from single males showed the presence of the putative pheromone 6-methyl-5-hepten-2-ol, sulcatol. Unmated males released sulcatol for at least 12 days, and ceased producing the pheromone after 20 days. The peak sulcatol release occurred after 2 days. Males cease production of sulcatol 24 h after being paired with females. Single females were unable to initiate galleries, and no sulcatol was detected from their headspace and hindgut samples. The chiral ratio of the pheromone, observed from headspace samples, was 31% (S)-(+)- and 69% (R)-(−)-sulcatol.     

Myrcene: A precursor of pheromones in Ips beetles

Males of Ips spp. produced the pheromones ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and/or ipsenol (2-methyl-6-methylene-7-octen-4-ol) when exposed to vapours of myrcene, a monoterpene present in their hosts (Pinus spp.). Ips grandicollis and Ips calligraphus require feeding before metabolizing the myrcene, whereas Ips avulsus and Ips paraconfusus produce some pheromone without prior feeding. Topical treatment with ipsdienol results in ipsenol production in both fed and unfed I. paraconfusus males but only in fed I. gradicollis males. I. calligraphus males, which do not produce ipsenol in nature, did not produce any with the topical treatment regardless of prior conditioning. It is concluded that myrcene can serve as a precursor for these terpene alcohols and suggested that ipsenol is produced by the reduction of ipsdienol. Furthermore, the biosynthesis of these pheromones appears to be under some form of control in certain species, with the stimulus for production occurring upon feeding.     

Field and laboratory evidence for a volatile pheromone produced by parous females of the Scottish biting midge, Culicoides impunctatus

Abstract. Electrophysiological, behavioural and field evidence is presented for a volatile attractant produced by host-seeking, parous female Culicoides impunctatus Goetghebuer. Attraction of host-seeking females to other live parous females or to extracts of parous females was demonstrated in Y-tube bioassays, as well as in field trials using baited sticky traps. Threshold numbers were 100–200 females, both in the laboratory and field. Antennal receptors to this pheromone were revealed by recording electroantennograms (EAGs) in response to odour from live parous females and to the extracts of female midges. The female-produced attractant did not alter field catches of male C. impunctatus.     

Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations

Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (?)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (?)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure–function relationships.     

Synthesis of Optically Pure (1S, 4S, 5S)-2-Pinen-4-ol (cis-Verbenol) and Its Antipode,the Pheromone of Ips Bark Beetles

The enantiomers of cis-verbenol (4a and 4a′) were first synthesized in optically pure state. (1S, 4S, 5S)-2-Pinen-4-ol (4a′) was dextrorotatory in acetone or in methanol but it was levorotatory in chloroform; cis-verbenols are indistinguishable by a prefix (+) or (?). The designation of the Ips pheromone as (+)-cis-verbenol is therefore ambiguous and it should be called as (1S, 4S, 5S)-2-pinen-4-ol (4a′) or (S)-cis-verbenol.     

Influence of proportion of (4S)-cis-verbenol in pheromone bait on Ips typographus (Col., Scolytidae) catch in pheromone trap barrier and in single traps

Abstract:   The possibilities of increasing catching efficiency of male Ips typographus L in pheromone traps barriers and in single traps were tested. Pheromone dispensers with different percentages of (4S)- cis -verbenol (cV) in pheromone mixtures were used. The results show that the different male catching efficiencies of pheromone trap barriers and single traps were related to the different percentages of cV in the pheromone bait. The proportions of males were higher in pheromone trap barriers than in single traps. Thus increasing the catch of males in pheromone trap barriers is possible by using a combination of pheromone traps containing pheromones giving a higher release of cV with others containing pheromones giving a lower release of cV .