Behavioural and electrophysiological responses of the malaria mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations

Behavioural and electrophysiological responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations collected on glass beads were studied using a dual-port olfactometer and electroantannography. Glass beads to which skin emanations from human hands had been transferred elicited a level of attraction similar to a human hand. The attractiveness of these handled glass beads faded away 4 h after transfer onto the beads. Storage at -20 degrees C for up to 8 weeks showed a decreased but still attractive effect of the beads. In a choice test between one individual and four others, the emanations from the reference individual were significantly more attractive in three out of four cases. The headspace of handled glass beads elicited a dose-dependent EAG response. The substances causing EAG activity could be removed partially by dichloromethane, ethanol and pentane-ether. Glass beads provide a suitable neutral substrate for the transfer of human odour to enable chemical analysis of the human skin emanations for identification of kairomones of anthropophilic mosquitoes.     

Genetic Divergence in the <Emphasis Type="Italic">cacophony</Emphasis> IVS6 Intron Among Five BrazilianPopulations of <Emphasis Type="Italic">Lutzomyia longipalpis</Emphasis>

Genes involved in the reproductive isolation are particularly useful as molecular markers in speciation studies. Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), a putative species complex, is a vector of visceral leishmaniasis in Latin America. We isolated from this species a fragment homologous to cacophony, a Drosophila gene that encodes features of the lovesong, an acoustic signal that is important in the sexual isolation of closely related species and known to vary considerably among L. longipalpis putative siblings species. Using an intron of the sandfly cacophony as a marker, we analyzed the molecular variation and sequence divergence among five populations of L. longipalpis from Brazil, three allopatric (Jacobina, Lapinha and Natal) and two putative sympatric sibling species from the locality of Sobral. A high level of polymorphism was found and analysis of the data indicates that very little gene flow is occurring among the populations of Jacobina, Lapinha, and Natal. A high level of differentiation was also observed between the two putative sympatric species of Sobral, one of which seems to be the same sibling species found in Natal, while the other is somewhat more related to Jacobina and Lapinha. However, the amount of estimated gene flow among the Sobral siblings is about seven times higher than the previously estimated for period, another lovesong gene, perhaps indicating that introgression might be affecting cacophony more than period. The results suggest that L. longipalpis is not a single species in Brazil, but it is yet not clear whether the different populations studied deserve species status rather than representing an incipient speciation process.     

Response of female Lutzomyia longipalpis to host odour kairomones from human skin

Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) is the vector of Leishmania chagasi, the aetiologic agent of visceral leishmaniasis in the New World. In the present study, the response of female sandflies from Jacobina, Brazil, to human odours from six different volunteers was investigated. Glass Petri dishes were handled by different volunteers and then exposed to female sandflies. There was a significant difference between subjects in that some individuals were more attractive or less repellent to sandflies. Response of flies to handled Petri dishes was higher during the first minutes of observation, suggesting the presence of volatile compounds in hand odours. Extracts of glass Petri dishes that had been handled by the volunteers were made with organic solvents such as acetone, methanol, pentane and ether. These were then concentrated and tested for sandfly response. Only extracts carried out with non-polar solvents such as pentane and ether were able to transfer odours from handled glass Petri dishes onto clean dishes. The attractivity of male and female human subjects was monitored for 80 days, and minor fluctuations in attractiveness were observed.     

Differential behaviour of Anopheles gambiae sensu stricto (Diptera: Culicidae) to human and cow odours in the laboratory

A dual port olfactometer was used to study the response of Anopheles gambiae Giles sensu stricto to odours of human and animal origin. Human odour consisted of human skin emanations collected on a nylon stocking, which was worn for 24 h. This was tested alone or together with 4.5% carbon dioxide, the concentration in human and cattle breath. Cattle odours consisted of cow skin emanations and/or carbon dioxide. Cow skin emanations were collected by tying a nylon stocking ('cow sock') around the hind leg of a cow for 12 h. Anopheles gambiae s.s. was consistently highly attracted by human odour, which is consistent with the high degree of anthropophily in this mosquito. Anopheles gambiae s.s. was not attracted by human or cattle equivalent volumes of carbon dioxide and this gas did not enhance the effect of human skin residues. Furthermore, A. gambiae s.s. showed a high degree of aversion to cow odour. When human odour and cow odour were tested together in the same port, mosquitoes were still highly attracted, indicating that whilst cattle odour may deter A. gambiae s.s., these mosquitoes can detect human odour in the presence of cattle odour. It was concluded that carbon dioxide plays a minor role in the host seeking behaviour of A. gambiae s.s., whilst host specific cues such as human skin residues play a major role and very effectively demonstrated anthropophilic behaviour in the laboratory.     

Interindividual variation in the attractiveness of human odours to the malaria mosquito Anopheles gambiae s. s

Differences between human individuals in their attractiveness to female mosquitoes have been reported repeatedly, but the underlying mechanisms are not well understood. Skin emanations from 27 human individuals, collected on glass marbles, were tested against ammonia in a dual-choice olfactometer to establish their degrees of attractiveness to anthropophilic Anopheles gambiae s.s. Giles (Diptera: Culicidae) mosquitoes. Ammonia was used as a standard odour source because of its proven attractiveness to An. gambiae s.s. Skin emanations from most volunteers attracted significantly more mosquitoes than ammonia. There were clear differences in the attractiveness of skin emanations from different volunteers relative to that of ammonia, as well as in the strength of the trap entry response. Consistent differences were observed when emanations from the three most and the three least attractive volunteers were tested pairwise. No gender or age effect was found for relative attractiveness or trap entry response. Emanations from volunteers with higher behavioural attractiveness elicited higher electroantennogram response amplitudes in two pairs, but in a third pair a higher electroantennogram response was found for the less attractive volunteer. These results confirm that odour contributes to the differences in attractiveness of humans to mosquitoes.     

Life cycle differences among Brazilian sandflies of the Lutzomyia longipalpis sibling species complex

The developmental cycles of five Brazilian populations of the Lutzomyia longipalpis Lutz & Neiva species complex (Diptera: Psychodidae) were compared under laboratory conditions. Three of the populations were derived from insects collected in allopatric sites at Natal (Rio Grande do Norte State), Jacobina (Bahia State) and Lapinha Cave (Minas Gerais State). The other two originated from Sobral (Ceará State), where the males of two sympatric species can be distinguished by the presence of one (1S) or two (2S) pairs of abdominal spots. The results of the present study clearly show that all three populations whose males produce C₁₆ pheromones and use pulse-type copulation songs (Jacobina, Lapinha Cave and Sobral 1S) are more easily adapted to the colonization conditions used in our laboratory, producing larger egg batches, with higher survival and an overall faster developmental cycle. This contrasts with populations producing C₂₀ male pheromones and using burst-type copulation songs (Natal and Sobral 2S) that produce smaller egg batches, have higher oviposition mortality and a slower rate of development under identical laboratory conditions. In conclusion, these phenological differences are a further indication of the differentiation of the siblings within the Lu. longipalpis species complex.     

Metaphase karyotypes and G-banding in sandflies of the Lutzomyia longipalpis complex

Mitotic metaphase chromosomes (2n = 8) from brain cells of fourth instar sandfly larvae of four geographical strains of the Lutzomyia longipaplis complex were examined microscopically, with bright-field illumination, after staining by a new G-banding technique involving exposure of air-dried chromosome preparations to quinacrine and ultraviolet light. Differences of G-banding and/or position of the centromere on chromosome 4 (the smallest chromosome pair) distinguished four putative sibling species from Costa Rica, Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves). The karyotype of the population from Jacobina, Brazil, showed an apparently plesiomorphic pattern of G-banding. On the basis of their recognizably different mitotic karyotypes, cytogenetic identification of separate taxa in the L. longipalpis complex should be useful for specific female vector competence and ecology studies.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Phlebotomine sandfly responses to carbon dioxide and human odour in the field

Responses of Lutzomyia sandflies (Diptera: Psychodidae: Phlebotominae) to carbon dioxide (CO2) and human odour were investigated by field experiments in Parana State, southern Brazil. Catches of two predominant species: Lu. intermedia (Antunes & Coutinho) and Lu. whitmani Lutz & Neiva, were compared between traps baited with a human adult or with CO2 emitted at the human-equivalent rate. When the baits were only 40 cm apart, no difference of attractiveness was detected. When baits were separated by 20 m, however, significantly fewer sandflies (44% Lu. intermedia, 46% Lu. whitmani) were trapped with CO2 compared with human bait. This is the first field evidence that anthropophilic sandflies are attracted by human kairomones in addition to CO2. For both species [Lutzomyia intermedia and Lu. whitmani] [corrected], the proportion of human attractiveness attributable to CO2 was significantly more [corrected] for males than females; for Lu. intermedia males human bait was no more attractive than CO2 alone. Gender differences in sandfly olfactory sensitivity are likely to be associated with behavioural differences on the host, where females feed on blood and males find mates. With traps 20 m apart, both Lutzomyia spp. showed roughly linear increased responses (log-log scale) to 0.08-0.55% CO2 equivalent to 0.5-4 humans. This would explain why host size is generally proportional to attractiveness, as observed for other species of phlebotomine sandflies.     

The study of fingerprint characteristics of the emanations from human arm skin using the original sampling system by SPME-GC/MS

An efficient and noninvasive method consisting of an original sampling device, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was developed to analyze volatile organic emanations from the skin of human arms. The emanations were sampled by SPME connected with the active sampling device for 30 min and transferred into GC-MS immediately for the consequent analysis. The sampling projects for 15 candidates were scheduled in both winter and spring with the same optimized conditions. Thirty-five compounds were finally identified according to various degrees of certainty. Different emission behaviors specified with principal component analysis (PCA) and similar fingerprint characteristics were observed clearly by comparisons of chromatograms of different seasons. Top ten emanations contributing to characteristics in different seasons were attempted to be described using comparisons based on common model strategy. The large amounts of experimental data were all handled by the corresponding chemometrics strategies with the homemade chromatographic data processing system. The results suggest that the analysis based on fingerprint characteristics of human skin emanations could provide useful and important clues to reveal biomarkers among the mixture of human skin emanations.     

Effect of human odours and positioning of CO2 release point on trap catches of the malaria mosquito Anopheles gambiae sensu stricto in an olfactometer

Abstract.  The anthropophilic malaria mosquito Anopheles gambiae sensu stricto responds to CO₂ and human skin emanations. How these odorants affect the behaviour of this mosquito species is studied in an olfactometer. Carbon dioxide is released either as an homogeneous plume or in a turbulent fashion at two different positions from the trap entrance. Anopheles gambiae is deterred from entering a trap with CO₂ as the only kairomone, when presented as an homogeneous or turbulent plume. This effect is completely overcome by the addition of skin emanations to the CO₂ plume, with a high proportion of mosquitoes found in the trap with skin emanations. Rearrangement of the position of the turbulent CO₂ source so that it is 5 cm downwind of the trap entrance overcomes the deterrent effect of CO₂. Carbon dioxide alone, however, does not elicit higher proportions caught compared with clean air. Further studies with the CO₂ source positioned 5 cm downwind of the trap entrance show that skin emanations alone result in fewer mosquitoes entering the trap than CO₂+ skin emanations. Skin emanations induce more mosquitoes to fly into a trap than a synthetic blend of NH₃+ l-lactic acid when both are combined with CO₂. It is concluded that CO₂ is a poor kairomone when offered alone and that its presence in the plume at the trap entrance deters mosquitoes from entering. By contrast, when positioned just downwind of the trap entrance, CO₂ appears to guide mosquitoes to the vicinity of the trap, where skin emanations then become the principle attractant, causing the mosquito trap entry response. The results of the study have implications for the design of odour-baited traps for this mosquito species.     

Sugar meal sources for the phlebotomine sandfly Lutzomyia longipalpis in Ceará State, Brazil

Abstract. In the Brazilian village of BoqueiraTo do Renato Parente, Ceará State, where visceral leishmaniasis is endemic, significant variation in the abundance of phlebotomine sandflies between different types of vegetation was demonstrated by castor oil sticky trap collections. Population densities of Lutzomyia longipalpis (Lutz & Neiva) sandflies were higher in beans and maize crops than in the surrounding open and tree scrub. Significant relationships were detected between the abundances of sandflies and aphids, suggesting aphid honey dew as a potential food source. Sugar meal analysis, using high-performance anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) and the cold anthrone test, was used to demonstrate that significantly more sandflies fed on bean plants contaminated with aphids and honeydew than on uncontaminated beans. Furthermore, higher concentrations of sugars were detected in flies which fed on aphid/honeydew-contaminated beans, suggesting that sugar was more easily available and/or that honeydew/aphids act as phagostimulants for sandflies. Amongst wild sandflies collected from a bean field, significantly more female sandflies were 'sugar-positive' than males, though the sugar concentrations in positive sandflies were similar for both sexes. The concentrations of di- and trisaccharides in sandfly homogenates, honeydew extracts and phloem exudates were very low.     

Behavioural and electrophysiological responses of the phlebotomine sandfly Lutzomyia longipalpis (Diptera: Psychodidae) when exposed to canid host odour kairomones

Compounds from the odour-producing glands of the fox Vulpes vulpes were collected. This complex mixture of compounds was used to stimulate the ‘ascoid’ olfactory organs of female sandflies in single sensillum and gas chromatography-linked single sensillum recordings. Sixteen of these compounds were identified using gas chromatography-linked mass spectrometry and amounts present were determined. The compounds fell into four organic classes: ketones, carboxylic acids, alcohols and aldehydes. Specific neurones present in the ascoid sensillum that responded to each of these classes of compound were characterized. A bioassay chamber was developed that gave female sandflies the choice of two odour sources. Female sandflies were attracted upwind by fox odour and were trapped in closer proximity to the fox odour port than the control port. Synthetic compounds were recombined in appropriate quantities to mimic the fox odour. In this bioassay, the synthetic blend attracted sandflies upwind, and again they were caught closer to the test port than the control port. Furthermore, the synthetic fox odour induced an electrophysiological response from neurones in the ascoid sensillum that was very similar to that induced by natural fox odour. No synthetic compound alone induced the same behavioural response from sandflies as did whole fox odour. However, benzaldehyde, 4-hydroxy-4-methyl-2-pentanone and 4-methyl-2-pentanone alone did cause sandflies to fly upwind and to be caught closer to the test port than the control.     

Schistosoma mansoni: Adult worm chemoattraction,with and without barriers

Heterosexual and homosexual chemoattraction studies were done with 8- to 12-week-old Schistosoma mansoni adults in linear chambers containing Earle's saline, in the absence and presence of both perforated and unperforated dialysis sac chimney barriers. In the absence of barriers attraction was seen in all heterosexual and homosexual combinations. After 90 min of observation at 37 C, organisms maintained at 4 or 37 C for at least 1 hr prior to the assay showed heterosexual attraction similar to those used immediately after perfusion. There was significantly more heterosexual attraction between worms from same than different pairs. In the presence of perforated barriers the greatest attraction was heterosexual, with males moving toward females; heterosexual attraction with females moving toward males was approximately equal to homosexual female attraction, and male homosexual attraction was not seen. In the presence of unperforated barriers the greatest attraction of females toward males occurred during the first 0.5 hr; within 3 to 4 hr females were no longer significantly attracted to males. However, after 0.5 hr males were more significantly attracted to females in unperforated than perforated chimneys. These studies demonstrate that adult schistosomes attract each other in vitro, and that there is a chemical basis for this attraction.     

Genetic inherence of the response to human kairomones by two allopatric members of the Lutzomyia longipalpis complex

Abstract.  The sandfly Lutzomyia longipalpis (Lutz & Neiva) is the main vector of Leishmania infantum in the New World. Several studies show that Lu. longipalpis is a species complex of at least three members. The feeding habits among the members of the complex vary from one geographical location to another. These differences in feeding habits may be related to differences between different members of the complex. The present study investigates differences in the response to human kairomones by two members of the complex, as well as the possibility that differences in the response have a genetic basis. One of the members used in this study is from Jacobina Bahia State, Brazil. Males from this population are known to produce a sex pheromone characterized by a C16 molecule identified as 3-methyl-α-himachelene. The other member is from a population originating in Marajó Island, Pará State, Brazil. Males from this location secrete a sex pheromone characterized by a C20 molecule whose structure remains to be elucidated, but is known to be a diterpene type. Our findings indicate that Jacobina females are significantly more responsive to human odours than Marajo females. When Jacobina and Marajó populations of Lu. longipalpis complex are cross-mated, the response of F1 females to the human odours is found to be genetically controlled.     

The relationship between CDC light-trap and human-bait catches of endophagic sandflies (Diptera: Psychodidae) in the Peruvian Andes

Abstract. A study was carried out in the Peruvian Andes to test the suitability of CDC light traps for monitoring changes in the human-landing rate of endophagic phlebotomine sandflies, following house-spraying with pyrethroid insecticide. On four pairs of consecutive nights, sandflies were caught inside eight sprayed and eight unsprayed houses, either by human bait or by CDC light traps. The sandflies collected were Lutzomyia verrucarum (97%) and Lu. peruensis (3%), both probable vectors of Leishmania peruviana , and the species composition was unaffected by house-spraying. A non-linear relationship was detected between light-trap and human-bait catches, but the relationship did not diverge significantly from linearity within the range of sandfly abundance found in most houses in the endemic area (i.e. between 3 and 200 sandflies/house-night), and did not differ significantly between sprayed and unsprayed houses. However, light trap catches had a significantly lower proportion of blood-fed females in sprayed than in unsprayed houses, probably due to an insecticidal effect on post-blood-feeding behaviour. The proportion of Lu. verrucarum was significantly higher in light trap than in human bait catches, indicating that Lu. peruensis is either more anthropophilic or less phototropic than Lu. verrucarum.     

Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), S?o José do Ribamar (MA), Canindé (CE), Natal (RN) and Gruta da Lapinha, Lagoa Santa (MG). No morphological differences were observed that could distinguish between these populations. Homogeneity tests showed that the allopatric populations display a certain heterogeneity and that the sympatric populations, with different patterns of spots, are homogeneous. The Student-Newman-Keuls test, represented by Euler-Venn diagrams, showed a disjunction between the populations from the north/northeast and the one from Gruta da Lapinha. Genetic distances between the four populations (excluding the Canindé population) were within the range of intrapopulational differences. The Gruta da Lapinha population displayed a heterozygotic deficiency that could be a consequence of high levels of inbreeding due to cryptic habits of living in a small cave. These results do not favor the hypothesis of a L. longipalpis species complex in Brazil, and the species should be considered high polymorphic.     

Sandfly distribution and abundance in a tropical rain forest

1. The distribution patterns of sandflies (Diptera: Psychodidae) upon tree buttresses were studied in tropical rain forest at Finca la Selva in the Caribbean lowlands of Costa Rica. 2. Four species of sandfly, Lutzomyia shannoni Dyar, L. trapidoi F. & H., L. ylephiletor F. & H. and L. vespertilionis F. & H. comprising 97% of those caught, used tree buttresses as diurnal resting sites. Their distribution on the buttresses was aggregated. 3. During the dry season tree species had no significant effect upon the distribution of the sandflies. However, during the wet season the distributions of two of the species, L. trapidoi and L. ylephiletor, were significantly affected by the species of tree; it is suggested that some species of tree may provide greater protection from rainfall than others. 4. L. vespertilionis was restricted to a single buttress on each positive tree. Distribution of this species is evidently determined by the distribution of its host animal, the bat (Emballonuridae). Female flies feed upon the bat's blood and male flies may be attracted to the bat as it provides a source of female sandflies. It is suggested, therefore, that tree buttresses serve as sandfly swarming sites. 5. Within a large buttress the sandflies are not randomly distributed but are aggregated in particular areas. Within these aggregations, the sandflies are vertically zoned upon the buttress with a shift in species composition with height. Two hypothesis were suggested to account for this distribution pattern: a response to an environmental gradient or an interaction between the four species of fly.     

Wind tunnel behavioural response and field trapping of the blowfly Calliphora vicina

The attraction of the blowfly Calliphora vicina Robineau‐Desvoidy, 1830 (Diptera: Calliphoridae) to single synthetic compounds, blends and authentic odours was investigated in a wind tunnel. A total of 1850 C. vicina (1750 females and 100 males) were tested. A comparison of male and female responses showed significant differences in attraction between the sexes. Females were more attracted than males to liver odour. The attraction of females lay in the ranges of 0–22% for single compounds, 26–64% for synthetic blends and 58–88% for authentic odours. Dimethyl trisulphide was the most attractive single compound. Significant improvement in attraction was achieved with blends and a three‐component lure, consisting of dimethyl trisulphide, mercaptoethanol and o‐cresol, was found to be the best solution for field trapping of C. vicina. Authentic odours from dead fish and mice were significantly more attractive than liver and the three‐component blend, and the blend and liver were similarly effective as attractants. Field tests support the results of the wind tunnel study and a high number of C. vicina were caught in funnel traps. Overall, 99.1% of the specimens caught were females.     

Parasitoids and dipteran predators exploit volatiles from microbial symbionts to locate bark beetles

Host location by parasitoids and dipteran predators of bark beetles is poorly understood. Unlike coleopteran predators that locate prey by orienting to prey pheromones, wasps and flies often attack life stages not present until after pheromone production ceases. Bark beetles have important microbial symbionts, which could provide sources of cues. We tested host trees, trees colonized by beetles and symbionts, and trees colonized by symbionts alone for attractiveness to hymenopteran parasitoids and dipteran predators. Field studies were conducted with Ips pini in Montana. Three pteromalid wasps were predominant. All were associated with the second and third instars of I. pini. Heydenia unica was more attracted to logs colonized by either I. pini or the fungus Ophiostoma ips than logs alone or blank controls (screen with no log). Rhopalicus pulchripennis was more attracted to logs colonized by I. pini than logs alone or blank controls. Dibrachys cavus was attracted to logs but did not distinguish whether or not they were colonized. Two dolichopodid predators were predominant. A Medetera species was more attracted to colonized than uncolonized logs and more attracted to logs than blank controls. It was also more attracted to logs colonized with the yeast Pichia scolyti than uncolonized logs, but attraction was less consistent. An unidentified dolichopodid was more attracted to logs colonized with I. pini, O. ips, and the bacteria Burkholderia sp., than to uncolonized logs. It was also attracted to uncolonized logs. Its responses were less consistent and pronounced than H. unica. These results suggest some parasitoids and dipteran predators exploit microbial symbionts of bark beetles to locate hosts. Overall, specialists showed strong attraction to fungal cues, whereas generalists were more attracted by plant volatiles. These results also show how microbial symbionts can have conflicting effects on host fitness.