Six-arm Olfactometer Research Articles

Behavioral responses of Rhyzopertha dominica (F.) to volatiles of different stored grains

The aim of this study was to explore the behavioral responses of the lesser grain borer, Rhyzopertha dominica (F.) to volatiles of different stored grains. The olfactory responses of R. dominica to the volatiles of five grains, i.e. wheat, rice, maize, sorghum, and soybean, were studied in Y-tube and six-arm olfactometer experiments. The volatile components of these grain were analyzed by gas chromatography-mass spectrometry (GC-MS). Y-tube olfactometer bioassays showed that both adults and larvae of R. dominica showed significant preferences for volatiles from the five grains, compared with clean air. In six-arm olfactometer bioassays, adults of R. dominica showed the strongest olfactory preference for wheat, followed by rice, maize, sorghum, and soybean, and larve showed similar preferences for the volatiles of these stored grains. The GC-MS analysis identified 31, 24, 17, 11, and 10 components in the volatile profiles of wheat, soybean, rice, maize, and sorghum, respectively. 1-hexanol was the most abundant component of the volatiles of wheat (27.43%) and soybean (48.28%), whereas nonanal was the most abundant component of the volatiles of rice (21.75%), maize (31.22%), and sorghum (40.78%). Therefore, the different olfactory responses of R. dominica are reasonably related to the differences in the types and contents of volatile components among these five grains. Noteworthy, the volatile components identified in grains may be useful as attractants in traps combined with insect phermones for monitoring or even control R. dominica.

Rugose spiralling whitefly, Aleurodicus rugioperculatus Martin infested host plant volatiles elicit a host locating behavior of aphelinid parasitoid, Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae)

Parasitoids find their hosts by using cues released by herbivores and their host plants. In crop fields, these semiochemicals could be used for insect pest control by attracting and retaining natural enemies. The hymenopteran aphelinidae endoparasitoid, Encarsia guadeloupae (E. guadeloupae), selectively parasitizes rugose spiralling whitefly (RSW), Aleurodicus rugioperculatus Martin, an invasive pest of coconut in India, thus offering an effective way to improve RSW control under natural field conditions. However, the chemical interactions that occur in between the RSW infected host plants and E. guadeloupae were unknown. Hence, using olfactometer assays and chemical analyses, we investigated the volatile organic compounds (VOCs) mediating attraction of the parasitoid to RSW infested host plants. To investigate the attraction of the E. guadeloupae towards the healthy and RSW infected leaves of coconut (Cocos nucifera L), banana (Musa paradisiaca L) and custard apple (Annona squmosa L), we employed a six-arm olfactometer. The E. guadeloupae preferred the volatile compounds from RSW-infested host plants rather than healthy plants. A higher percent response rate of E. guadeloupae was observed in RSW-infected banana (35.83 %), followed by healthy banana (24.33 %), RSW-infested custard apple (16.33 %), and RSW-infested coconut (9.00 %) leaf volatiles, respectively. The volatile compounds emitted from RSW-infested and healthy plants have been studied using GC–MS with thermal desorption (TD). The results revealed more than 20 volatile compounds belonging to seven chemical classes were identified and characterized from infested plants by RSW. Of these, α-Ocimene, α-Pinene, 3-Carene, trans-α-Ocimene, and (E)-hex-3-ene-2, 5-diol were emitted at higher levels than other compounds its proven through PCA and Heat map analysis. These five compounds elicited the greatest attraction of E. guadeloupae towards RSW damaged plants. The synthetic blend of the above volatiles could be used as attractants to enhance the population of E. guadeloupae to manage RSW under field conditions.

Exogenous Calcium Suppresses the Oviposition Choices of Frankliniella occidentalis (Thysanoptera: Thripidae) and Promotes the Attraction of Orius similis (Hemiptera: Anthocoridae) by Altering Volatile Blend Emissions in Kidney Bean Plants.

Frankliniella occidentalis is a destructive pest of horticultural plants, while Orius similis is a natural enemy of thrips. It has been demonstrated that exogenous calcium could induce plant defenses against herbivore attack. We examined whether CaCl2 supplementation altered the volatile emissions of kidney bean plants, which influence the oviposition preference of F. occidentalis. We also assessed the influence of volatile cues on O. similis. Using Y-tube olfactometer tests, we found that exogenous CaCl2 treatment inhibited the selectivity of F. occidentalis but attracted O. similis. In addition, CaCl2 treatment reduced the oviposition preference of F. occidentalis. Gas chromatography-mass spectrometry analyses revealed that CaCl2 treatment altered the number and relative abundance of the volatile compounds in kidney bean plants and that (E)-2-hexen-1-ol, 1-octen-3-ol, β-lonone, and (E,E)-2,4-hexadienal might be potential olfactory cues. Furthermore, the results of the six-arm olfactometer test indicated that 1-octen-3-ol (10-2 μL/μL), β-lonone (10-2 μL/μL), and (E,E)-2,4-hexadienal (10-3 μL/μL) repelled F. occidentalis but attracted O. similis. Overall, our results suggested that exogenous CaCl2 treatment induced defense responses in kidney bean plants, suggesting that CaCl2 supplementation may be a promising strategy to enhance the biological control of F. occidentalis.

Host preference of Tribolium castaneum (Herbst) on six kinds of flour

The red flour beetle, Tribolium castaneum (Herbts) is a major pest of flour from various grains. The infested flour is discoloured and will emit a disagreeable odour due to the secretion of benzoquinone from the insect’s abdominal glands. Considering the economic losses effected by T. castaneum, new alternative control measures are needed for this pest. This study sought to determine the host preferences of T. castaneum from amongst six kinds of flour to assess their vulnerability to infestation. The research was conducted in the Plant Pest Laboratory, Department of Plant Pests and Diseases, Faculty of Agriculture, University of Brawijaya. The host preferences of T. castaneum were examined using a six-arm olfactometer. Feeding preference tests were performed for a duration of 12 hours at 200 ml/minute airflow in each chamber-arm, followed by oviposition preference observations one week later. Feeding preference was calculated for the total pool of adults observed, segregated by sex, whereas oviposition preference was calculated as the number of eggs laid. Flour nutrition (proximate composition, phenolic content, and riboflavin content) were analyzed. Our results showed a feeding preference by T. castaneum for bran, soy and tapioca flour was greater over wheat, corn, and white gelatinous rice flour, while for oviposition, bran flour was the most preferred.

Behavioral Responses of Thrips hawaiiensis (Thysanoptera: Thripidae) to Volatile Compounds Identified from Gardenia jasminoides Ellis (Gentianales: Rubiaceae)

Thrips hawaiiensis is a common thrips pest of various plant flowers with host preference. Plant volatiles provide important information for host-searching in insects. We examined the behavioral responses of T. hawaiiensis adults to the floral volatiles of Gardenia jasminoides Ellis, Gerbera jamesonii Bolus, Paeonia lactiflora Pallas, and Rosa chinensis Jacq. in a Y-tube olfactometer. T. hawaiiensis adults showed significantly different preferences to these four-flower plants, with the ranking of G. jasminoides > G. jamesonii > P. lactiflora ≥ R. chinensis. Further, 29 components were identified in the volatile profiles of G. jasminoides, and (Z)-3-hexenyl tiglate (14.38 %), linalool (27.45 %), and (E3,E7)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (24.67 %) were the most abundant. Six-arm olfactometer bioassays showed that T. hawaiiensis had significant positive responses to (Z)-3-hexenyl tiglate, linalool, and (E3,E7)-4,8,12-trimethyltrideca-1,3,7,11-tetraene tested at various concentrations, with the most attractive ones being 10−3 μL/μL, 10−2 μL/μL and 100 μg/μL for each compound, respectively. In pairing of these three compounds at their optimal concentrations, T. hawaiiensis showed the preference ranking of (Z)-3-hexenyl tiglate > linalool > (E3,E7)-4,8,12-trimethyltrideca-1,3,7,11-tetraene. Large numbers of T. hawaiiensis have been observed on G. jasminoides flowers in the field, which might be caused by the high attraction of this pest to G. jasminoides floral volatiles shown in the present study. Our findings shed light on the olfactory cues routing host plant searching behavior in T. hawaiiensis, providing important information on how T. hawaiiensis targets particular host plants. The high attractiveness of the main compounds (e.g., linalool, (E3,E7)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, particular (Z)-3-hexenyl tiglate) identified from volatiles of G. jasminoides flowers may be exploited further to develop novel monitoring and control tools (e.g., lure and kill strategies) against this flower-inhabiting thrips pest.

Differential Response of a Local Population of Entomopathogenic Nematodes to Non-Native Herbivore Induced Plant Volatiles (HIPV) in the Laboratory and Field.

Recent work has shown the potential for enhanced efficacy of entomopathogenic nematodes (EPN) through their attraction to herbivore induced plant volatiles. However, there has been little investigation into the utilization of these attractants in systems other than in those in which they were identified. We compared (E)-β-caryophyllene and pregeijerene in the highbush blueberry (Vaccinium corymbosum) agroecosystem in their ability to enhance the attraction of EPN to and efficacy against the system's herbivore, oriental beetle (Anomala orientalis). The relative attractiveness of (E)-β-caryophyllene and pregeijerene to a local isolate of the EPN species Steinernema glaseri was tested in a six-arm olfactometer in the laboratory to gather baseline values of attraction to the chemicals alone in sand substrate before field tests. A similar arrangement was used in a V. corymbosum field by placing six cages with assigned treatments and insect larvae with and without compound into the soil around the base of 10 plants. The cages were removed after 72h, and insect baits were retrieved and assessed for EPN infection. The lab results indicate that in sand alone (E)-β-caryophyllene is significantly more attractive than pregeijerene to the local S. glaseri isolate Conversely, there was no difference in attractiveness in the field study, but rather, native S. glaseri were more attracted to cages with G. mellonella larvae, no larvae, and cages with the blank control and G. mellonella larvae.

Attraction of entomopathogenic nematodes to sugarcane root volatiles under herbivory by a sap-sucking insect

Few systems have been described in which herbivore-induced root volatiles mediate attraction of entomopathogenic nematodes (EPNs), and they only concern root damage inflicted by chewing insects. EPNs, especially Heterorhabditis indica and Steinernema carpocapsae, are potential biological control agents of sugarcane spittlebug (Mahanarva fimbriolata) populations. Here, we investigated the response of these two species of EPNs to sugarcane root volatiles damaged by M. fimbriolata nymphs in a belowground six-arm olfactometer. We also examined changes on root volatile profile in response to herbivory of sugarcane spittlebug nymphs. Results showed that both EPN species did not discriminate between odors of undamaged sugarcane and moistened sand (blank). However, when EPNs were exposed to odors of spittlebug-damaged and undamaged sugarcane roots, both species significantly preferred odors of spittlebug-damaged roots. Headspace collection followed by GC–MS analyses showed no qualitative difference (total of 11 compounds) between volatile profiles of spittlebug-damaged and undamaged sugarcane roots. In contrast to the previous studies involving feeding by root chewing insects, our root volatile analysis did not reveal any up-regulation resulting from sugarcane spittlebug damage, but the down-regulation of the terpenes dihydromyrcenol and β-isomethyl ionone when compared with the profile of undamaged sugarcane roots. Here, we propose alternative explanations for the EPN attraction to spittlebug-damaged roots as it is unlikely that reduced concentrations of the volatiles play a role in this interaction. Further studies are necessary to determine the key compounds of the root volatile emission to enhance biological control efficacy with EPNs against M. fimbriolata in sugarcane.

Comparing the attraction of two parasitoids to herbivore-induced volatiles of maize and its wild ancestors, the teosintes

Artificial selection of crop plants for desired traits such as increased yield and improved seed or fruit quality has been hypothesized to have had a cost for other potentially useful traits, including resistance to herbivores. Besides direct defences, such as the production of toxins, plants may also indirectly protect themselves by emitting volatile organic compounds (VOCs) that attract the natural enemies of herbivores. Parasitoid wasps are known to use these VOCs to localize hosts for their offspring. However, domestication and selective breeding of crop plants have reportedly led to the loss of such signals. The aim of this study was to identify possible differences in the attraction of parasitoid wasps by modern maize and its wild ancestors, the teosintes. In a six-arm olfactometer, we compared the capacity of teosintes and maize to attract the parasitoid wasps Cotesia marginiventris (Hymenoptera: Braconidae) and Campoletis sonorensis (Hymenoptera: Ichneumonidae). We studied the attractiveness of plants in which VOC emission was induced by the application of artificial damage and caterpillar regurgitant, as well as the attractiveness of extracts of volatiles that we collected from plants exposed to herbivory. C. sonorensis did not distinguish between the odours of maize and teosintes, whereas C. marginiventris showed a significant preference for the odours of teosintes over the odours of maize. The fact that we obtained very similar results with extracts of collected volatiles implies that we could use these extracts to identify the key compounds that are responsible for wasp attraction. Restoring and/or enhancing such key parasitoid attractants in cultivated plants could be an effective way to increase natural pest control.

A six-arm olfactometer for analysing olfactory responses of Goniozus legneri Gordh (Hymenoptera: Bethylidae), the larval ectoparasitoid of carob moth

The behavioural responses of <em>Goniozus</em> <em>legneri</em> were investigated in a six-arm olfactometer. Among the different odours examined, carob moth (<em>Ectomyelois</em> <em>ceratoniae</em> Zeller) frass elicited the highest olfactory responses, while <em>Ephestisa</em> larvae, which were less suitable hosts, elicited the lowest response. The different preferences to various odours suggest that <em>Goniozus</em> <em>legneri</em> can discriminate among suitable and less suitable insect hosts.

Effects of Rewarding and Unrewarding Experiences on the Response to Host-induced Plant Odors of the Generalist Parasitoid Cotesia marginiventris (Hymenoptera: Braconidae)

Associative learning is known to modify foraging behavior in numerous parasitic wasps. This is in agreement with optimal foraging theory, which predicts that the wasps will adapt their responses to specific cues in accordance with the rewards they receive while perceiving these cues. Indeed, the generalist parasitoid Cotesia marginiventris shows increased attraction to a specific plant odor after perceiving this odor during contact with hosts. This positive associative learning is common among many parasitoids, but little is known about the effects of unrewarding host searching events on the attractiveness of odors. To study this, preferences of female C. marginiventris for herbivore-induced odors of three plant species were tested in a six-arm olfactometer after the wasps perceived one of these odors either i) without contacting any caterpillars, ii) while contacting the host caterpillar Spodoptera littoralis, or iii) while contacting the non-host caterpillar Pieris rapae. The results confirm the effects of positive associative learning, but showed no changes in innate responses to the host-induced odors after “negative” experiences. Hence, a positive association is made during an encounter with hosts, but unsuccessful host-foraging experiences do not necessarily lead to avoidance learning in this generalist parasitoid.

Subterranean Herbivore-induced Volatiles Released by Citrus Roots upon Feeding by Diaprepes abbreviatus Recruit Entomopathogenic Nematodes

Herbivore-induced volatile emissions benefit plant hosts by recruiting natural enemies of herbivorous insects. Such tritrophic interactions have been examined thoroughly in the above-ground terrestrial environment. Recently, similar signals have also been described in the subterranean environment, which may be of equal importance for indirect plant defense. The larvae of the root weevil, Diaprepes abbreviates, are a serious pest of citrus. Infestations can be controlled by the use of entomopathogenic nematodes, yet the interactions between the plant, insect and nematode are poorly understood and remain unpredictable. In bioassays that used a root zone six-arm olfactometer, citrus roots ('Swingle citrumelo' rootstock) recruited significantly more entomopathogenic nematodes (Steinernema diaprepesi) when infested with root weevil larvae than non-infested roots. Infested plants were more attractive to nematodes than larvae alone. Roots damaged by weevil larvae attracted more nematodes than mechanically damaged roots and sand controls. By dynamic in situ collection and GC-MS analysis of volatiles from soil, we determined that four major terpene compounds were produced by infested plant roots that were not found in samples from non-infested roots or soil that contained only larvae. Solvent extracts of weevil-infested roots attracted more nematodes than extracts of non-infested roots in a two choice sand-column bioassay. These findings suggest that Swingle citrus roots release induced volatiles as an indirect defense in response to herbivore feeding, and that some of these induced volatiles function as attractants for entomopathogenic nematodes.

Fungal Infection Reduces Herbivore-Induced Plant Volatiles of Maize but does not Affect Naïve Parasitoids

Plants attacked by insects release volatile compounds that attract the herbivores' natural enemies. This so-called indirect defense is plastic and may be affected by an array of biotic and abiotic factors. We investigated the effect of fungal infection as a biotic stress agent on the emission of herbivore-induced volatiles and the possible consequences for the attraction of two parasitoid species. Maize seedlings that were simultaneously attacked by the fungus Setosphaeria turcica and larvae of Spodoptera littoralis emitted a blend of volatiles that was qualitatively similar to the blend emitted by maize that was damaged by only the herbivore, but there was a clear quantitative difference. When simultaneously challenged by fungus and herbivore, the maize plants emitted in total 47% less of the volatiles. Emissions of green leaf volatiles were unaffected. In a six-arm olfactometer, the parasitoids Cotesia marginiventris and Microplitis rufiventris responded equally well to odors of herbivore-damaged and fungus- and herbivore-damaged maize plants. Healthy and fungus-infected plants were not attractive. An additional experiment showed that the performance of S. littoralis caterpillars was not affected by the presence of the pathogen, nor was there an effect on larvae of M. rufiventris developing inside the caterpillars. Our results confirm previous indications that naïve wasps may respond primarily to the green leaf volatiles.

A comparison of naïve and conditioned responses of three generalist endoparasitoids of lepidopteran larvae to host-induced plant odours

Abstract Many parasitic wasps that exploit herbivores as their hosts make use of herbivoreinduced plant odours to locate their victims and these wasps often exhibit an ability to learn to associate specific plant-produced odours with the presence of hosts. This associative learning is expected to allow generalist parasitoids to focus on cues that are most reliably associated with current host presence, but evidence supporting this hypothesis is ambiguous. Using a six-arm olfactometer we compared the responses of three generalist larval endoparasitoids, Cotesia marginiventris (Hymenoptera: Braconidae), Microplitis rufiventris (Hymenoptera: Braconidae) and Campoletis sonorensis (Hymenoptera: Ichneumonidae), to the induced odours of three plant species: maize (Zea mays), cowpea (Vigna unguiculata), and cotton (Gossypium hirsutum). We tested the responses of naïve females as well as of females that were first conditioned by parasitising host larvae feeding on one of the plant species. Despite similarities in biology and host range the three wasp species responded entirely differently. Naïve C. marginiventris and C. sonorensis chose equally among the induced odours of the three plants, whereas naïve M. rufiventris, which may have a somewhat more restricted host range, tended to prefer the odour of maize. After conditioning, most C. marginiventris females chose the odour of the plant species that they had experienced, but conditioned M. rufiventris showed an even stronger preference for maize odours, independently of the plant they had experienced. Cotesia sonorensis did not show any change in its preference after conditioning. We speculate that its extremely broad host range allows C. sonorensis females to use fixed responses to cues commonly associated with plants damaged by Lepidoptera. These results imply that different generalist parasitoids may employ different foraging strategies and that associative learning is not necessarily part of it.

Evaluating the Induced-Odour Emission of a Bt Maize and its Attractiveness to Parasitic Wasps

The current discussion on the safety of transgenic crops includes their effects on beneficial insects, such as parasitoids and predators of pest insects. One important plant trait to consider in this context is the emission of volatiles in response to herbivory. Natural enemies use the odours that result from these emissions as cues to locate their herbivorous prey and any significant change in these plant-provided signals may disrupt their search efficiency. There is a need for practical and reliable methods to evaluate transgenic crops for this and other important plant traits. Moreover, it is imperative that such evaluations are done in the context of variability for these traits among conventional genotypes of a crop. For maize and the induction of volatile emissions by caterpillar feeding this variability is known and realistic comparisons can therefore be made. Here we used a six-arm olfactometer that permits the simultaneous collection of volatiles emitted by multiple plants and testing of their attractiveness to insects. With this apparatus we measured the induced odour emissions of Bt maize (Bt11, N4640Bt) and its near-isogenic line (N4640) and the attractiveness of these odours to Cotesia marginiventris and Microplitis rufiventris, two important larval parasitoids of common lepidopteran pests. Both parasitoid species were strongly attracted to induced maize odour and neither wasp distinguished between the odours of the transgenic and the isogenic line. Also wasps that had previously experienced one of the odours during a successful oviposition divided their choices equally between the two odours. However, chemical analyses of collected odours revealed significant quantitative differences. The same 11 compounds dominated the blends of both genotypes, but the isogenic line released a larger amount of most of these. These differences may be due to altered resource allocation in the transgenic line, but it had no measurable effect on the wasps' behaviour. All compounds identified here had been previously reported for maize and the differential quantities in which they were released fall well within the range of variability observed for other maize genotypes.

The Role of Fresh versus Old Leaf Damage in the Attraction of Parasitic Wasps to Herbivore-Induced Maize Volatiles

The odor produced by a plant under herbivore attack is often used by parasitic wasps to locate hosts. Any type of surface damage commonly causes plant leaves to release so-called green leaf volatiles, whereas blends of inducible compounds are more specific for herbivore attack and can vary considerably among plant genotypes. We compared the responses of naïve and experienced parasitoids of the species Cotesia marginiventris and Microplitis rufiventris to volatiles from maize leaves with fresh damage (mainly green leaf volatiles) vs. old damage (mainly terpenoids) in a six-arm olfactometer. These braconid wasps are both solitary endoparasitoids of lepidopteran larvae, but differ in geographical origin and host range. In choice experiments with odor blends from maize plants with fresh damage vs. blends from plants with old damage, inexperienced C. marginiventris showed a preference for the volatiles from freshly damaged leaves. No such preference was observed for inexperienced M. rufiventris. After an oviposition experience in hosts feeding on maize plants, C. marginiventris females were more attracted by a mixture of volatiles from fresh and old damage. Apparently, C. marginiventris has an innate preference for the odor of freshly damaged leaves, and this preference shifts in favor of a blend containing a mixture of green leaf volatiles plus terpenoids, after experiencing the latter blend in association with hosts. M. rufiventris responded poorly after experience and preferred fresh damage odors. Possibly, after associative learning, this species uses cues that are more directly related with the host presence, such as volatiles from host feces, which were not present in the odor sources offered in the olfactometer. The results demonstrate the complexity of the use of plant volatiles by parasitoids and show that different parasitoid species have evolved different strategies to exploit these signals.