Smelling your way to food: can bed bugs use our odour?

The resurgence in developed countries of the common bed bug, Cimex lectularius, has led to a search for new sustainable methods to monitor and control this human ectoparasite. Because of increased resistance to insecticides, traps baited with attractive cues are considered a promising method to be developed into efficient monitoring tools for bed bugs. Despite their potential as attractants, only a few studies have investigated the odorant cues implicated in the attraction of bed bugs to human hosts. In this study, we used aeration extracts from human volunteers to assess the role of olfaction in host searching by bed bugs. By coupled gas chromatography and single sensillum recordings on all the antennal sensilla, we measured the electrophysiological response elicited by the compounds present in our human odour extracts. Only five compounds were clearly detected by the olfactory receptor neurons housed in the smooth-peg sensilla of the bed bugs. We tested the behavioural effect of these extracts in a still-air arena and showed a gradient of repellence linked to the dose, as well as a higher propensity of local search behaviour associated with human odours containing a lower ratio of 6-methyl-5-hepten-2-one to C(7)-C(10) aldehydes. We conclude that human odour alone has a weak influence on the behaviour of C. lectularius and we propose that human kairomones may have a significant impact on bed bug behaviour in combination with heat and carbon dioxide, the only two currently known attractive vertebrate cues used by bed bugs for host seeking.

Non-host volatiles mediate associational resistance to the pine processionary moth.

An increasing body of evidence indicates that the association between different plant species may lead to a reduction in insect herbivory, i.e. associational resistance. This might be due to a top-down regulation of herbivores by increased numbers of natural enemies or to a disruptive bottom-up influence of lower host plant accessibility. In particular, the richer plant communities release more diverse plant odours that may disturb olfactory-guided host choice and mating behaviour of insect herbivores, i.e. the "semiochemical diversity hypothesis". However, this hypothesis has been rarely tested experimentally in natural habitats, notably forest ecosystems. We tested the effects of non-host volatiles (NHV) on mate and host location by the pine processionary moth (PPM) at the scale of individual pine trees with branches of non-host tree (birch) at their base. Pheromone trap catches and the numbers of larval nests were both reduced by non-host presence under treated pine trees, confirming an associational resistance mediated by NHV. In both males and females, the antenna could detect several birch volatiles, including methyl salicylate (MeSa). MeSa inhibited the attraction of the PPM male to pheromone traps, as did bark and leaf chips from birch trees. Our test of three doses of MeSa at the habitat scale (50 m forest edges) showed that the reduction in the numbers of male PPM captured in traps and in larval nests was MeSa dose-dependent. These results show that odours released by deciduous non-host trees can reduce herbivory by a forest defoliator in conifers, providing support to the "semiochemical diversity hypothesis" as a mechanism of associational resistance.

A model for peak and width of signaling windows: Ips duplicatus and Chilo partellus pheromone component proportions--does response have a wider window than production?

Pheromone communication systems have a reliable signal with a restricted window of amounts and ratios released and perceived. We propose a model based on a Gaussian response profile that allows a quantification of the response peak (location of optimum) and a measure of the peak width (response window). Interpreting the Gaussian curve, fitted by nonlinear regression (NLR), as a standard normal distribution, the peak location equals the mean (it) and the window width equals 2 x the standard deviation (2sigma). The NLR procedure can provide an objective measure for both peak location and width for a wide range of data sets. Four empirical data sets as well as 10 literature data sets were analyzed. The double-spined spruce engraver, Ips duplicatus, was field tested in four populations to find the optimum proportion for attraction to the two male aggregation pheromone components, ipsdienol (Id) and (E)-myrcenol(EM), ranging from 0 to 100% of Id. Tests in Norway and the Czech Republic confirmed the preference of western populations for a blend between 50 and 90% Id. A population in Inner Mongolia showed a preference for traps with the 10 and 50% Id baits. The NLR fitted values for response peak and width (mu; 2sigma) were: Norway 0.64, 0.73; Czech Republic 0.53, 0.73; NE China 0.77, 0.29; and Inner Mongolia 0.33, 0.50. The signal produced by Norwegian field-collected males had a narrower window width (2sigma = 0.12). Males of the maize stem borer, Chilo partellus, were tested in a flight tunnel for their response to variation in the two major female sex pheromone gland components, (Z)- l1-hexadecenal and the corresponding alcohol (OH). Variation of the alcohol in seven levels from 2 to 29% OH showed the highest male response for 17% OH. For all behavioral steps, the peak of male response was near mu = 0.14, while the window width fell from 2sigma = 0.5 to 0.2 for eight sequential behavioral steps from take-off to copulation. Female production had a similar peak location (mu = 0.13) but a narrower width, 2sigma = 0.14. Literature data from other moth species showed similar patterns, with a wider male response relative to the female production windows. Literature data on response to enantiomer ratios in a hymenopteran and to pheromone amounts in a dipteran were also described by our model. In a bark beetle population (Ips pini), with two hybridizing enantiomeric strains, the production peaks were narrower (0.1) than the response peaks (0.5). Thus, it in general, seems that in the pheromone systems analyzed, the width of the response window (2sigma = 0.1 to 0.8) is larger than that of the production window (2sigma = 0.03 to 0.14), irrespective of the sex of the sender.

Olfactory responses of Ips duplicatus from inner Mongolia, China to nonhost leaf and bark volatiles.

Leaf and bark volatiles from nonhost angiosperm trees were tested on Ips duplicatus by gas chromatographic-electroantennographic detection (GC-EAD) and by pheromone-baited traps in Sweden and Inner Mongolia, China, respectively. GC-EAD analysis of the headspace volatiles from fresh bark chips of Betula pubescens revealed trans-conophthorin, two green leaf volatiles (GLVs): 1-hexanol and (Z)-3-hexen-1-ol, and two C8 alcohols: 3-octanol and 1-octen-3-ol, that consistently elicited antennal responses by I. duplicatus. The identification of these EAD-active compounds was confirmed in further GC-EAD recordings with synthetic mixtures. Antennal responses were also found to synthetic (E)-2-hexen-1-ol and linalool, which have been identified from the leaves of nonhost birch and aspen species. No antennal responses of I. duplicatus were found to hexanal, (E)-2-hexenal, and (Z)-3-hexyl acetates. In field trapping experiments, blends of EAD-active green leaf alcohols or C8 alcohols, or transconophthorin alone resulted in significant reductions (27-60%) in the number of I. duplicatus captured compared with pheromone-baited traps. The unsuitable host compound, verbenone (Vn), also significantly reduced trap catches by up to 60% in both experiments. The strongest disruptive effect resulted from the addition of the combination of green leaf alcohols, C8 alcohols, and verbenone to the pheromone trap, which caused an 84% reduction in trap catch. The blend of two green leaf aldehydes plus the acetate increased the trap catches in 1998 and had no negative or positive effects in 1999. Our results suggest that these nonhost volatiles (NHVs) are important olfactory signals used by I. duplicatus in host selection. They may have great significance in developing semiochemical-based management programs for I. duplicatus by reducing or stopping attacks on suitable hosts.

Synthetic attractants for the bark beetle parasitoid Coeloides bostrichorum Giraud (Hymenoptera: Braconidae).

Coeloides bostrichorum Giraud parasitoids (Hymenoptera: Braconidae) attack late larval stages of various bark beetle species breeding in spruce. Volatile compounds collected from Norway spruce (Picea abies) infested by Ips typographus L. (Coleoptera: Scolytidae) were analysed by coupled gas chromatography-mass spectrometry (GC-MS) and GC-electroantennographic detection (GC-EAD). Monoterpene hydrocarbons are the predominant volatile compounds of fresh Norway spruce, while the presence of oxygenated monoterpenes indicates damaged trees. Between one and eight EAD-active oxygenated monoterpenes were used, in amounts reflecting their natural abundance in spruce trees containing bark beetle larvae, to prepare five synthetic baits which were tested in wind tunnel bioassays. Odour samples collected from spruce logs containing the preferred host stage were attractive, while similar samples from uninfested logs failed to elicit any flight activity. However, when a four- or an eight-component synthetic bait was added to volatiles collected from uninfested spruce logs, this combination was as attractive as volatiles collected from infested spruce logs.

Strategies of a bark beetle, Pityogenes bidentatus, in an olfactory landscape.

Volatiles from leaves or bark of nonhost birch (Betula pendula) and Norway spruce (Picea abies) dramatically reduced the attraction of the bark beetle, Pityogenes bidentatus (Coleoptera: Scolytidae), to their aggregation pheromone components (cis-verbenol and grandisol) in the field. In addition, odors from both the needles and bark of the host Scots pine (Pinus sylvestris) similarly inhibited attraction. Monoterpenes of pine and spruce (alpha-pinene, beta-pinene, terpinolene, and 3-carene) as well as ethanol, chalcogran and some nonhost green leaf alcohols [(Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, and 1-hexanol], also reduced catches. Collections of volatiles from the field-tested plant tissues indicated they released monoterpenes in amounts similar to the synthetics that inhibited responses. The various plant and insect sources of these inhibitory compounds indicate that P. bidentatus bark beetles have evolved several strategies to increase their fitness by avoiding nonhost and unsuitable host trees in a complex olfactory landscape.

Pheromones in white pine cone beetle,Conophthorus coniperda (schwarz) (Coleoptera: Scolytidae).

Female white pine cone beetles,Conophthorus coniperda, attacking second-year cones of eastern white pine,Pinus strobus L., produced a sex-specific pheromone that attracted conspecific males in laboratory bioassays and to field traps. Beetle response was enhanced by host monoterpenes. The female-produced compound was identified in volatiles collected on Porapak Q and in hindgut extracts as (+)-trans-pityol, (2R,5S)-(+)-2-(1-hydroxy-1-methylethyl)-5-methyltetrahydrofuran. Males and females produced and released the (E)-(-)-spiroacetal, (5S,7S)-(-)-7-methyl-1,6-dioxaspiro[4.5]decane, which was not an attractant for either sex, but acted as a repellent for males. Porapak Q-trapped volatiles from both sexes contained (+)-trans-pinocarveol and (-)-myrtenol. In addition, hindgut extracts of females containedtrans-verbenol, while males had pinocarvone and verbenone. Work in Georgia and Canada confirmed that the same isomers of pityol and spiroacetal are present in two distinct and widely separated populations ofC. coniperda.

Electroantennogram responses of the southern pine beetle parasitoidDinotiscus dendroctoni (Ashmead) (Hymenoptera: Pteromalidae) to potential semiochemicals.

In two studies using the electroantennogram (EAG) technique, bark beetle- and tree-produced semiochemicals were presented toDinotiscus dendroctoni (Ashmead), a larval parasitoid ofDendroctonus frontalis Zimm. In the first study, 20 test compounds and a standard mixture of oxygenated monoterpenes were presented individually at one concentration to the parasitoids. In the second study, the nine compounds that elicited the greatest EAGs in study 1 were then tested as serial dilutions of 10 to 0.0001µg/ul. The individual compounds did not elicit responses greater than the standard mixture of oxygenated monoterpenes. Males and females exhibited similar dose responses, although females showed lower thresholds of response than males to frontalin, terpinen-4-ol,E,Z-chalcogran, andexo-brevicomin. In both studies, pino-/isopinocamphone elicited the greatest responses at high concentrations. Tests of different ratios of the camphone mixture indicated that pinocamphone elicited the greatest response. Most of the test compounds elicited similar responses which suggests that several of the compounds may be used together byD. dendroctoni in habitat and/or host community location.

Isolation of pheromone synergists of bark beetle,Pityogenes chalcographus, from complex insect-plant odors by fractionation and subtractive-combination bioassay.

Capillary gas chromatography with columns of different polarity and two-dimensional fractionation of effluents were used with novel subtrac-tive-combination bioassays to rigorously isolate host- and insect-produced pheromone synergists of the bark beetlePityogenes chalcographus (Coleop-tera: Scolytidae). Methyl (E,Z)-2,4-decadienoate (E,Z-MD) and the previously identified chalcogran were found to be synergistically attractive to both sexes.E,Z-MD was produced sex-specifically in males, and only when they had fed on host-plant tissue. A Norway spruce monoterpene fraction (including α-pinene, ß-pinene, and camphene) increased the attractive response to the pheromone components. Dose-response curves forE,Z-MD and chalcogran in the laboratory bioassay indicated the two components are highly synergistic. The isolation methods are important for further progress in identifying certain semiochemical synergists found in trace amounts in complex chemical mixtures, such as when insects must feed in host plants in order to produce pheromone.

Volatiles released from individual spruce bark beetle entrance holes Quantitative variations during the first week of attack.

Volatiles released from individual entrance holes of eight spruce bark beetles (Ips typographus) were collected during the first week of attack on a resistant host tree. In order to quantify the release of the highly volatile 2-methyl-3-buten-2-ol (MB) from attacking males, a new method was developed with deuterated quantification standard released at the time of collection. The amounts of collected volatiles, as analyzed by GC and GC-MS, showed a large variation between individual holes and also between subsequent entrainments from the same hole. Most of the quantified compounds on the average have two maxima, with a pronounced intervening depression. The amounts of releasedcis-verbenol (cV) increased five times during the first two days, while the amounts of MB were consistently high. The attacked spruce tree was not taken by the beetles, and the average amounts of the two aggregation pheromone components, MB and cV, increased again after the first maxima. The first peak of oxygenated monoterpene, released in the beginning of the attack containing α-terpineol, terpinen-4-ol, bornyl acetate,trans-pinocarveol, and verbenone, was possibly due to spontaneous oxidation of monoterpene hydrocarbons from the tree. Microorganisms established in the gallery wall phloem probably participated in the production of oxygenated monoterpenes during the second increase.

Variation of enantiomeric composition of α-pinene in norway spruce,Picea abies, and its influence on production of verbenol isomers byIps typographus in the field.

The enantiomeric composition of α-pinene in individual Norway spruce trees [Picea abies (L.) Karst.] was determined on a chiral GC column after stereoselective hydroboration-oxidation followed by a reaction with isopropyl isocyanate to form the carbamate derivative. The enantiomeric composition varied considerably between trees of different genetic origin. There was a strong correlation between the chirality of α-pinene in host spruce trees and thecis/trans ratio of verbenols found in the hindguts of the bark beetleIps typographus (L.) infesting the trees.

Structure-activity studies on aggregation pheromone components ofPityogenes chalcographus (Coleoptera: Scolytidae) : All stereoisomers of chalcogran and methyl 2,4-decadienoate.

Syntheses of all four Stereoisomers (2S,5S; 2S,5R;2R,5R; and2R,5S) of chalcogran, a major component of the aggregation pheromone ofPityogenes chalcographus, and of all four isomers (2Z,4Z; 2Z,4E; 2E,4E; and 2E,4Z) of methyl 2,4-decadienoate (MD), the second major pheromone component, are briefly described. Attraction responses of walking beetles of both sexes were tested to mixtures of the synergistic pheromone components or analogs. These bioassays showed that theE,Z isomer of MD is the most active when tested with chalcogran. When tested with (E,Z)-MD, (2S,5R)-chalcogran was the most active stereoisomer, while 2R,5R and 2R,5S isomers had intermediate activities, and the 2S,5S isomer was inactive. There was no evidence that the relatively less active Stereoisomers of chalcogran inhibited or promoted attraction to (2S,5R)-chalcogran with (E,Z)-MD. Male beetles only produce the activeE,Z isomer of MD (inactive alone) and their hindguts contain the most active (2S,5R)- and least active (2S,5S)-chalcogran. A mixture of all MD isomers with racemic chalcogran was not significantly different in attractivity compared to (E,Z)-MD with racemic chalcogran, indicating no synergistic or inhibitory effects of the inactive isomers of MD.

Inhibition of attraction to aggregation pheromone by verbenone and ipsenol : Density regulation mechanisms in bark beetleIps typographus.

The semiochemicals verbenone (Vn), ipsenol (Ie), and ipsdienol (Id), present in late phases of host colonization, have been implicated as qualitative "shut-off" signals regulating attack density. Combinations of the three chemicals were released in pipe traps together with the aggregation pheromone components 2-methyl-3-buten-2-ol (MB) andcis-verbenol (cV) at different levels and in different ratios to MB + cV, and with two spacings of traps to test for possible effects on reducing catch at traps baited with aggregation pheromone. When they were released with the attractants Vn and Ie (alone or together) decreased the mean catch significantly at the higher release rates used (1 mg/day). Id alone or together with Vn at low release rates (0.1 mg/day), with the attractants, increased catch somewhat. A dose-response test of Vn, with the attractants held constant, showed a decline in catches, down to about < 10% of the control, at ratios of Vn to cV between 1∶1 and 150∶ 1. A larger spacing (25 m) of traps gave a stronger response to change in doses of Vn and MB + cV than a smaller (6 m) spacing. The sex ratio was more skewed towards females when two or three inhibitors were present and at higher doses of Vn. It is suggested that Vn could be the most important density-regulating signal in the natural system, as release of Vn from galleries is larger and starts earlier than that of Id and Ie.

Individual variation in aggregation pheromone content of the bark beetle,Ips typographus.

The total amounts of, and proportions among, components of the aggregation pheromone produced byIps typographus were found to vary considerably among individuals excised from attacks on standing spruce trees. Chemical analyses of 392 individual male beetles were made by GC-MS. Both unmated and mated males had log-normal frequency distributions in their content of the pheromone components 2-methyl-3-buten-2-ol (MB) andcis-verbenol (cV), since a large fraction of males had a low content. The amount of MB in male hindguts varied independently of cV and the other oxygenated monoterpenes, while the amount of cV covaried with the other pinene alcohols and showed a variation between beetles from different spruce trees. Mated males had, on average, lower amounts of MB than unmated, while the average content of cV in mated males varied with the resin content of their host trees. Ipsdienol and ipsenol were only found in mated males, but in less than 40% and 10%, respectively, of these mated males. Even-aged males exposed to α-pinene in the laboratory showed slightly less variation in the amounts of verbenols, and the variations in ratio between cV and tV were similar to those among males attacking the same spruce tree.

Field response of spruce bark beetle,Ips typographus, to aggregation pheromone candidates.

Six compounds previously identified from hindguts of unmated maleIps typographus (L.) during host colonization: 2-methyl-3-buten-2-ol (MB),cis-verbenol (cV),trans-verbenol (tV), myrtenol (Mt),trans-myrtanol (tM), and 2-phenylethanol (PE), were tested for their attractivity in the field with a subtractive method. The amounts of MB and cV released from a pipe trap were similar to those given off from the commercial bait Ipslure as well as that from a Norway spruce tree,Picea abies (L.) Karst., under mass attack. The blend of the compounds became nonattractive when either MB or cV was subtracted, while subtraction of any of the other four compounds had no effect. Addition of ipsdienol (Id) to the blend did not significantly increase the attraction. In a second comparative test, the addition of three compounds as a group (tV + Mt + PE) to MB + cV again had no effect on the attraction, but the addition of Id increased the catch somewhat. Addition of host logs to a bait releasing MB + cV at a rate lower than in previous experiments did not influence the attraction to pipe traps. Sticky traps containing natural pheromone sources (50 males in a log), which released 1-5 mg/day of MB as determined by aerations with deuterated MB as internal standard, were less attractive than a synthetic source releasing similar amounts of MB.

Quantitative variation of pheromone components in the spruce bark beetleIps typographus from different attack phases.

Ips typographus beetles were collected in the field, separated into eight attack phases (from beetles walking on the trunk of a tree under attack to those excavating gallery systems with a mother gallery longer than 4 cm), and immediately frozen in liquid nitrogen. 2-Methyl-3-buten-2-ol,cis- andtrans-verbenol, verbenone, myrtenol, trans-myrtanol, ipsenol, ipsdienol, and 2-phenylethanol were quantified from excised hindguts against an internal standard, heptyl acetate, in the extraction solvent. Methylbutenol, the pinene alcohols, and 2-phenylethanol showed the same pattern of variation between attack phases in males, with the largest amounts present before accepting females and then a fast decline. Ipsenol and ipsdienol were not detected in males before the females were accepted, and the amounts increased when the females start their egg laying. Verbenone occurred only in trace amounts. The beetles were sampled from five Norway spruce trees (Picea abies) of differing resin flow. The correlations between the nine pheromone components and five major host monoterpenes in the gut showed that the variation in the amount of methyl-butenol, ipsenol, and ipsdienol could not be explained by the variation in the amounts of host monoterpenes. In contrast over 80% of the quantitative variation ofcis-verbenol,trans-verbenol, and myrtenol was explained by the amount of α-pinene. The nine pheromone components from 36 individual males were also quantified. Both methylbutenol andcis-verbenol showed a large variation in both amounts and proportions. Females containedtrans-verbenol and traces of most other components found in males. When accepted by the male, they also contained a female-specific compound, ß-isophorone. Behavioral and biosynthetic implications of the results are discussed.