Volatiles produced by symbiotic yeasts improve trap catches of Carpophilus davidsoni (Coleoptera: Nitidulidae): an important pest of stone fruits in Australia.

Carpophilus davidsoni (Dobson) is an important pest of Australian stone fruit. Current management practices for this beetle include the use of a trap that contains an attractant lure comprised of aggregation pheromones and a 'co-attractant' mixture of volatiles from fruit juice fermented using Baker's yeast, Saccharomyces cerevisiae (Hansen). We explored whether volatiles from yeasts Pichia kluyveri (Bedford) and Hanseniaspora guilliermondii (Pijper), which are closely associated with C. davidsoni in nature, might improve the effectiveness of the co-attractant. Field trials using live yeast cultures revealed that P. kluyveri trapped higher numbers of C. davidsoni compared to H. guilliermondii, and comparative GC-MS of volatile emissions of the two yeasts led to the selection of isoamyl acetate and 2-phenylethyl acetate for further investigation. In subsequent field trials, trap catches of C. davidsoni were significantly increased when 2-phenylethyl acetate was added to the co-attractant, compared to when isoamyl acetate was added, or both isoamyl acetate and 2-phenylethyl acetate. We also tested different concentrations of ethyl acetate in the co-attractant (the only ester in the original lure) and found contrasting results in cage bioassays and field trails. Our study demonstrates how exploring volatile emissions from microbes that are ecologically associated with insect pests can result in more potent lures for use in integrated pest management strategies. Results from laboratory bioassays screening volatile compounds should be treated with caution when making inferences regarding attraction under field conditions.

A Synthetic Blend of Fruit and Live Yeast Odours Shows Promise for Trapping Mated Female Queensland Fruit Fly, Bactrocera tryoni, in the Field.

Tephritid fruit flies are major horticultural pests of fruit and vegetable crops. Developing an odour lure that can attract mated female flies under field conditions has presented a major challenge to chemical ecologists around the globe. We have designed a new lure to attract female Queensland fruit fly, Bactrocera tryoni, based on the knowledge that this pest tephritid is attracted not only to odours from ripening fruits, but also to odours of symbiotic (gut-associated) yeasts on which the larvae feed. Initial field trials screened four volatile mixtures, each containing a base mix of three, short-chain "fruit ripening" esters and differing in the addition of long-chain "fruity" esters and / or alcohols produced by live yeasts. Results demonstrated that the lure formulated from short-chain esters and live yeast alcohols attracted the most female flies, with significantly higher catches than an odourless control trap, and a protein-baited trap. As electrophysiological studies exploring peripheral responses in tephritids to alcohols associated with fermentation have not been extensively investigated, we conducted this work on both the antennae and maxillary palps, and discovered that three yeast alcohols were detected by both sensory organs. Performance trials conducted in pome fruit and stone fruit orchards evaluated the six-component blend of short-chain ester and live yeast alcohol lure against a commercially available synthetic fruit-odour lure and a protein-based lure. In the apple orchard, the new lure caught significantly more female flies, and in particular mated females, compared to the commercially available lures. In the stone fruit orchard, while the new lure again caught the most mated female flies compared to the other lures, differences were not significant. The visual stimulus (trap design) used had a significant effect on capture, with a Ladd trap (red sphere on a yellow panel) being considerably more effective than the other traps. We discuss the implications of this study, including why the degree of effectiveness of the new lure might have differed among the test orchards (citrus, apple, stonefruit), and why yeast odours might increase attractiveness to "ripening fruit" volatile blends under field conditions.

Divergence in floral scent and morphology, but not thermogenic traits, associated with pollinator shift in two brood-site-mimicking Typhonium (Araceae) species.

BACKGROUND: Flowers which imitate insect oviposition sites probably represent the most widespread form of floral mimicry, exhibit the most diverse floral signals and are visited by two of the most speciose and advanced taxa of insect - beetles and flies. Detailed comparative studies on brood-site mimics pollinated exclusively by each of these insect orders are lacking, limiting our understanding of floral trait adaptation to different pollinator groups in these deceptive systems. METHODS: Two closely related and apparent brood-site mimics, Typhonium angustilobum and T. wilbertii (Araceae) observed to trap these distinct beetle and fly pollinator groups were used to investigate potential divergence in floral signals and traits most likely to occur under pollinator-mediated selection. Trapped pollinators were identified and their relative abundances enumerated, and thermogenic, visual and chemical signals and morphological traits were examined using thermocouples and quantitative reverse transcription-PCR, reflectance, gas chromatography-mass spectrometry, floral measurements and microscopy. KEY RESULTS: Typhonium angustilobum and T. wilbertii were functionally specialized to trap saprophagous Coleoptera and Diptera, respectively. Both species shared similar colour and thermogenic traits, and contained two highly homologous AOX genes (AOX1a and AOX1b) most expressed in the thermogenic tissue and stage (unlike pUCP). Scent during the pistillate stage differed markedly - T. angustilobum emitted a complex blend of sesquiterpenes, and T. wilbertii, a dung mimic, emitted high relative amounts of skatole, p-cresol and irregular terpenes. The species differed significantly in floral morphology related to trapping mechanisms. CONCLUSIONS: Functional specialization and pollinator divergence were not associated with differences in anthesis rhythm and floral thermogenic or visual signals between species, but with significant differences in floral scent and morphological features, suggesting that these floral traits are critical for the attraction and filtering of beetle or fly pollinators in these two brood-site mimics.

Yeasts Influence Host Selection and Larval Fitness in Two Frugivorous Carpophilus Beetle Species.

We explored how gut-associated yeasts influence olfactory behaviour and resource use in two pest species of Carpophilus beetle that co-exist in Australian stone fruits. Molecular analysis of yeasts isolated from the gut of C. davidsoni (prefers ripe fruits) and C. hemipterus (prefers overripe and rotting fruits) revealed that the predominant species were Pichia kluyveri and Hanseniaspora guilliermondii. In olfactory attraction and oviposition trials, adult beetles preferred H. guilliermondii over P. kluyveri, and follow up GC-MS analysis revealed unambiguous differences between the odour profiles of these yeasts. In contrast to behavioural trials, larval feeding assays showed that fruit substrates inoculated with P. kluyveri yielded significantly faster development times, higher pupal mass, and a greater number of adult beetles, compared to H. guilliermondii - in other words, the lesser preferred yeast (by foraging adults) was more suitable for larval survival. Moreover, whilst larvae of both species survived to adulthood when fed solely on P. kluyveri (i.e. without a fruit substrate), only larvae of C. davidsoni could develop on H. guilliermondii; and only C. davidsoni reached adulthood feeding on a yeast-free fruit substrate. We discuss how these findings may relate to adaptations towards early colonising of fruits by C. davidsoni, enabling differences in resource use and potentially resource partitioning in the two beetles. More broadly, consideration of microbial interactions might help develop host selection theory. Our results could pave the way to more powerful attractants to mass-trap and monitor Carpophilus pests in fruit orchards.

Not Led by the Nose: Volatiles from Undamaged Eucalyptus Hosts Do Not Influence Psyllid Orientation.

Psyllids (Hemiptera: Psylloidea) are small sucking insects with high host plant specificity. Despite the primitive olfactory system of psyllids, some species have been suggested to rely on host plant volatiles (HPVs) for seasonal migration between summer deciduous hosts and winter coniferous hosts. Similarly, enhanced attraction of psyllid vectors has been observed as a result of the manipulation of host odors by plant pathogens. As yet, there are no studies of olfaction in psyllids that utilize evergreen eucalypt hosts. We investigated the behavioral responses of adults of four Eucalyptus-feeding psyllids-Ctenarytaina eucalypti, C. bipartita, Anoeconeossa bundoorensis and Glycaspis brimblecombei-to their respective HPVs in Y-tube olfactometer bioassays. We also used existing physiological data for C. eucalypti to investigate potential olfactory tuning that may modulate the preference for morphologically juvenile leaves over morphologically adult leaves. Although adult C. eucalypti were consistently repelled by HPVs from damaged host leaves, none of the species exhibited positive chemotaxis to HPVs from undamaged leaves. Surprisingly, G. brimblecombei was repelled by HPVs from undamaged host leaves. Our findings provide little support for a significant role of olfaction in host location by Eucalyptus-feeding psyllids. We propose a number of ecological hypotheses to explain these unexpected findings.

Behavioral and Physiological Evidence for Palp Detection of the Male-Specific Attractant Cuelure in the Queensland Fruit Fly (Bactrocera tryoni).

The Queensland fruit fly, Bactrocera tryoni, is considered one of the worst horticultural pests in Australia attacking a large variety of fruit crops. To defeat pest insects, olfactory attractants have been developed and widely used in lure and kill strategies. Male B. tryoni are strongly attracted to the compound raspberry ketone and its synthetic analog, cuelure. Despite the strong behavioral response, a recent study failed to show any activation of antennal receptors to cuelure. Therefore, we hypothesized that cuelure may be detected by an accessory olfactory organ, the maxillary palp. Combining behavioral and physiological experiments we clearly demonstrate that male flies, but not female flies, primarily use the maxillary palps and not the antennae to detect and respond to cuelure. Furthermore, regardless of satiety status, male flies always preferred cuelure over a sugar rich source, unless the maxillary palps were excised.

Does foliage metal accumulation influence plant-insect interactions? A field study of two sympatric tree metallophytes.

Gossia (Myrtaceae) is a highly restricted tree genus most speciose in New Caledonia and eastern Australia. The latter group accumulates above-normal foliar manganese (Mn) concentrations, with some individuals exhibiting the rare Mn-hyperaccumulative trait. Whether foliar metals contribute to chemical defence has been addressed via numerous feeding experiments and very few field studies. This investigation exploited specifically different insect activities on the foliage of sympatric Gossia grayi (N.Snow & Guymer) and Gossia shepherdii (F.Muell.) N.Snow & Guymer, endemic to north-eastern Australia, to test for direct and indirect effects of foliar Mn enrichment on plant-insect interactions. Leaf organic and inorganic chemistries, specific weight, surface damage, gall infestation and occupancy were quantified. Discovery that both species are Mn hyperaccumulators augments the world listing by 5-7%. Highly elevated gall-Mn concentrations coupled with negligible gall parasitisation suggested chemical fortification and adaptation by the host insect - a Cecidomyiidae fly. Linear mixed modelling (LMM) showed differences in leaf Mn, phenolics, toughness and surface damage across tree species and leaf age. There was no direct relationship between leaf Mn and insect impact. However, LMM did resolve indirect effects, i.e. between insect impact and certain foliar elements, consistent with nutritional dynamics in a physiologically novel plant system where Mn is vastly overaccumulated.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival.

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the 'deterrent odour' yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the 'attractive odour' yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new "attract and kill" lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

The Long and the Short of Mate Attraction in a Psylloid: do Semiochemicals Mediate Mating in Aacanthocnema dobsoni Froggatt?

Mating is preceded by a series of interdependent events that can be broadly categorized into searching and courtship. Long-range signals convey species- and sex-specific information during searching, while short-range signals provide information specific to individuals during courtship. Studies have shown that cuticular hydrocarbons (CHCs) can be used for mate recognition in addition to protecting insects from desiccation. In Psylloidea, four species rely on semiochemicals for long-range mate attraction. Psyllid mating research has focused on long-range mate attraction and has largely ignored the potential use of cuticular hydrocarbons (CHCs) as mate recognition cues. This study investigated whether CHCs of Aacanthocnema dobsoni have semiochemical activity for long- and short-range communication prior to mating. Using a solid sampler for solvent-less injection of whole psyllids into coupled gas chromatography/mass spectrometry, we found quantitative, sex- and age-related differences in CHC profiles. Males had higher proportions of 2-MeC28, 11,15-diMeC29, and n-C33 alkanes, while females had higher proportions of 5-MeC27, 3-MeC27, 5,15-diMeC27, n-C29 and n-C30 alkanes. In males and females, 84 and 68 % of CHCs varied with age, respectively. Y-tube olfactometer bioassays provided no evidence that males or females responded to odors emanating from groups of conspecifics of the opposite sex. Tests of male and female psyllids for attraction to branchlets previously occupied by conspecifics showed no evidence of attraction to possible semiochemical residues. Our short-range chemoreception bioassay showed that males were as indifferent to freshly killed individuals of either sex with intact CHC profiles as to those treated with hexane (to remove CHCs). Aacanthocnema dobsoni utilizes substrate-borne vibrations (SBVs) for communication. Therefore, our results indicate that SBVs are probably more important than semiochemicals for long-range mate attraction. Furthermore, CHCs are unlikely to mediate short-range mate recognition or provide mate assessment cues.

Visual acuity trade-offs and microhabitat-driven adaptation of searching behaviour in psyllids (Hemiptera: Psylloidea: Aphalaridae).

Insects have evolved morphological and physiological adaptations in response to selection pressures inherent to their ecology. Consequently, visual performance and acuity often significantly vary between different insect species. Whilst psychophysics has allowed for the accurate determination of visual acuity for some Lepidoptera and Hymenoptera, very little is known about other insect taxa that cannot be trained to positively respond to a given stimulus. In this study, we demonstrate that prior knowledge of insect colour preferences can be used to facilitate acuity testing. We focused on four psyllid species (Hemiptera: Psylloidea: Aphalaridae), namely Ctenarytaina eucalypti, Ctenarytaina bipartita, Anoeconeossa bundoorensis and Glycaspis brimblecombei, that differ in their colour preferences and utilization of different host-plant modules (e.g. apical buds, stems, leaf lamellae) and tested their visual acuity in a modified Y-maze adapted to suit psyllid searching behaviour. Our study revealed that psyllids have visual acuity ranging from 6.3 to 8.7 deg. Morphological measurements for different species showed a close match between inter-ommatidial angles and behaviourally determined visual angles (between 5.5 and 6.6 deg) suggesting detection of colour stimuli at the single ommatidium level. Whilst our data support isometric scaling of psyllids' eyes for C. eucalypti, C. bipartita and G. brimblecombei, a morphological trade-off between light sensitivity and spatial resolution was found in A. bundoorensis. Overall, species whose microhabitat preferences require more movement between modules appear to possess superior visual acuity. The psyllid searching behaviours that we describe with the help of tracking software depict species-specific strategies that presumably evolved to optimize searching for food and oviposition sites.

Specificity and sensitivity of plant odor-detecting olfactory sensory neurons in Ctenarytaina eucalypti (Sternorrhyncha: Psyllidae).

The blue gum psyllid, Ctenarytaina eucalypti (Sternorrhyncha: Psyllidae), is an economic threat to Eucalyptus subgenus Symphyomyrtus plantations worldwide. To date, no generally applicable control method is available and the potential for semiochemical-based monitoring or control methods has not yet been investigated. Hence, we conducted the first study on the olfactory sense of C. eucalypti, investigating the specificity and sensitivity of its olfactory sensory neurons (OSNs) to host plant volatiles using single sensillum recordings (SSR). Synthetic compounds were selected from published identifications of Eucalyptus volatiles and after analysis of headspace collections from Eucalyptus cordata. The antenna of C. eucalypti carries four cavities containing olfactory sensilla (S1-S4). Our recordings revealed that each of these sensilla houses three OSNs that could be distinguished electrophysiologically based on spike amplitude differences (A, B, and C neuron with large, intermediate, and small amplitude, respectively). The A neuron in sensillum S1 responded primarily to ß-caryophyllene and weaker to ß-ocimene, whereas the accompanying B-neuron responded strongly and very specifically to linalool. Furthermore, the B-neuron in both S2 and S3 responded strongly to 1-hexanol, Z3-hexenol, and Z3-hexenyl acetate. OSNs in S4 responded only weakly to a few of the synthetic compounds. Response thresholds in strongly responding OSNs to putative key compounds were close to the 1ng dose on the filter paper and responses exhibited a phasic-tonic profile irrespective of compound dose. C. eucalypti may use the physiologically active compounds for long-range host finding. Future laboratory and field experiments will reveal whether plant volatiles can be used in the management and monitoring of C. eucalypti.

Ctenarytaina bipartita sp. n. (Hemiptera, Psylloidea), a new eucalypt psyllid from Southeast Australia.

Ctenarytaina bipartita sp.n., associated with Eucalyptus kitsoniana and E. viminalis, is described from the Australian Capital Territory, New South Wales, Tasmania and Victoria. It differs from other described Ctenarytaina species in the paramere which bears a small posterior lobe. Taxonomically relevant morphological details are illustrated and the species is diagnosed from other eucalypt inhabiting congeners. Information on the biology is also given. C. bipartita has the potential to become an exported pest species to countries with significant eucalypt plantations.

Anoeconeossa bundoorensis sp. n., a new psyllid (Hemiptera: Psylloidea) from Eucalyptus camaldulensis (Myrtaceae) from Southeast Australia.

Anoeconeossa bundoorensis sp. n. is described from Eucalyptus camaldulensis (Myrtaceae) from southern Victoria in Southeast Australia. It is placed in the A. communis Taylor species-group as the paramere lacks combs of black rods. It differs from other members of the species-group, A. communis and A. bullata Taylor as it lacks an anterobasal expansion on the paramere and from A. unicornuta Taylor as the inner horn-shaped process of the paramere is reduced to a short spine and the apical expansion is more elongate, with a corresponding greater length of equidistant setae. Taxonomically relevant morphological details are illustrated and the species is diagnosed from other eucalypt inhabiting congeners. In-formation on the biology is presented.

Novel bioassay demonstrates attraction of the white potato cyst nematode Globodera pallida (Stone) to non-volatile and volatile host plant cues.

Potato cyst nematodes (PCNs) are a major pest of solanaceous crops such as potatoes, tomatoes, and eggplants and have been widely studied over the last 30 years, with the majority of earlier studies focusing on the identification of natural hatching factors. As a novel approach, we focused instead on chemicals involved in nematode orientation towards its host plant. A new dual choice sand bioassay was designed to study nematode responses to potato root exudates (PRE). This bioassay, conducted together with a traditional hatching bioassay, showed that biologically active compounds that induce both hatching and attraction of PCNs can be collected by water extraction of incised potato roots. Furthermore, our results demonstrated that PCN also were attracted by potato root volatiles. Further work is needed to fully understand how PCNs use host plant chemical cues to orientate towards hosts. Nevertheless, the simple attraction assay used in this study provides an important tool for the identification of host-emitted attractants.