Yeast Volatomes Differentially Affect Larval Feeding in an Insect Herbivore.

Yeasts form mutualistic interactions with insects. Hallmarks of this interaction include provision of essential nutrients, while insects facilitate yeast dispersal and growth on plant substrates. A phylogenetically ancient chemical dialogue coordinates this interaction, where the vocabulary, the volatile chemicals that mediate the insect response, remains largely unknown. Here, we used gas chromatography-mass spectrometry, followed by hierarchical cluster and orthogonal partial least-squares discriminant analyses, to profile the volatomes of six Metschnikowia spp., Cryptococcus nemorosus, and brewer's yeast (Saccharomyces cerevisiae). The yeasts, which are all found in association with insects feeding on foliage or fruit, emit characteristic, species-specific volatile blends that reflect the phylogenetic context. Species specificity of these volatome profiles aligned with differential feeding of cotton leafworm (Spodoptera littoralis) larvae on these yeasts. Bioactivity correlates with yeast ecology; phylloplane species elicited a stronger response than fruit yeasts, and larval discrimination may provide a mechanism for establishment of insect-yeast associations. The yeast volatomes contained a suite of insect attractants known from plant and especially floral headspace, including (Z)-hexenyl acetate, ethyl (2E,4Z)-deca-2,4-dienoate (pear ester), (3E)-4,8-dimethylnona-1,3,7-triene (DMNT), linalool, α-terpineol, ß-myrcene, or (E,E)-α-farnesene. A wide overlap of yeast and plant volatiles, notably floral scents, further emphasizes the prominent role of yeasts in plant-microbe-insect relationships, including pollination. The knowledge of insect-yeast interactions can be readily brought to practical application, as live yeasts or yeast metabolites mediating insect attraction provide an ample toolbox for the development of sustainable insect management.IMPORTANCE Yeasts interface insect herbivores with their food plants. Communication depends on volatile metabolites, and decoding this chemical dialogue is key to understanding the ecology of insect-yeast interactions. This study explores the volatomes of eight yeast species which have been isolated from foliage, from flowers or fruit, and from plant-feeding insects. These yeasts each release a rich bouquet of volatile metabolites, including a suite of known insect attractants from plant and floral scent. This overlap underlines the phylogenetic dimension of insect-yeast associations, which according to the fossil record long predate the appearance of flowering plants. Volatome composition is characteristic for each species, aligns with yeast taxonomy, and is further reflected by a differential behavioral response of cotton leafworm larvae, which naturally feed on foliage of a wide spectrum of broad-leaved plants. Larval discrimination may establish and maintain associations with yeasts and is also a substrate for designing sustainable insect management techniques.

Common Cerambycid Pheromone Components as Attractants for Longhorn Beetles (Cerambycidae) Breeding in Ephemeral Oak Substrates in Northern Europe.

Longhorn beetles are ecologically important insects in forest ecosystems as decomposers of woody substrates, microhabitat engineers, and as components of forest food webs. These species can be greatly affected both positively and negatively by modern forestry management practices, and should be monitored accordingly. Through headspace sampling, coupled gas chromatography-electroantennography, gas chromatography-mass spectrometry, and field bioassays, we identified two compounds, 2-methyl-1-butanol and 3-hydroxy-2-hexanone, that constitute aggregation-sex pheromone attractants of three cerambycid species which breed primarily in different types of fresh, recently dead oak wood in Northern Europe: Pyrrhidium sanguineum (L.), Phymatodes alni ssp. alni (L.), and Phymatodes testaceus (L.) (Cerambycinae: Callidiini). Analyses of headspace volatiles collected from live insects indicated that the male-produced aggregation-sex pheromone of P. sanguineum is a 1-15:100 blend of (R)-2-methyl-1-butanol and (R)-3-hydroxy-2-hexanone, whereas the corresponding ratios for P. alni were 70-110:100. In field bioassays, adult P. sanguineum and P. alni were significantly attracted to multiple blends with varying ratios of the two compounds. When tested individually, the compounds were minimally attractive. In contrast, adult P. testaceus exhibited nonspecific attraction to both of the individual compounds and to different blends, despite the hydroxyketone not being part of its pheromone, which consists of (R)-2-methyl-1-butanol alone. Overall, our results suggest that a blend of 50:100 of racemic 2-methyl-1-butanol and 3-hydroxy-2-hexanone is appropriate for parallel, cost-efficient pheromone-based monitoring of all three species. In particular, these species could serve as useful indicators of how modern forestry practices affect a whole guild of saproxylic insects that require ephemeral deadwood substrates for successful breeding.

"This is not an apple"-yeast mutualism in codling moth.

The larva of codling moth Cydia pomonella (Tortricidae, Lepidoptera) is known as the worm in the apple, mining the fruit for food. We here show that codling moth larvae are closely associated with yeasts of the genus Metschnikowia. Yeast is an essential part of the larval diet and further promotes larval survival by reducing the incidence of fungal infestations in the apple. Larval feeding, on the other hand, enables yeast proliferation on unripe fruit. Chemical, physiological and behavioral analyses demonstrate that codling moth senses and responds to yeast aroma. Female moths are attracted to fermenting yeast and lay more eggs on yeast-inoculated than on yeast-free apples. An olfactory response to yeast volatiles strongly suggests a contributing role of yeast in host finding, in addition to plant volatiles. Codling moth is a widely studied insect of worldwide economic importance, and it is noteworthy that its association with yeasts has gone unnoticed. Tripartite relationships between moths, plants, and microorganisms may, accordingly, be more widespread than previously thought. It, therefore, is important to study the impact of microorganisms on host plant ecology and their contribution to the signals that mediate host plant finding and recognition. A better comprehension of host volatile signatures also will facilitate further development of semiochemicals for sustainable insect control.

High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.

BACKGROUND: Field-grown leafy vegetables can be damaged by biotic and abiotic factors, or mechanically damaged by farming practices. Available methods to evaluate leaf tissue damage mainly rely on colour differentiation between healthy and damaged tissues. Alternatively, sophisticated equipment such as microscopy and hyperspectral cameras can be employed. Depending on the causal factor, colour change in the wounded area is not always induced and, by the time symptoms become visible, a plant can already be severely affected. To accurately detect and quantify damage on leaf scale, including microlesions, reliable differentiation between healthy and damaged tissue is essential. We stained whole leaves with trypan blue dye, which traverses compromised cell membranes but is not absorbed in viable cells, followed by automated quantification of damage on leaf scale. RESULTS: We present a robust, fast and sensitive method for leaf-scale visualisation, accurate automated extraction and measurement of damaged area on leaves of leafy vegetables. The image analysis pipeline we developed automatically identifies leaf area and individual stained (lesion) areas down to cell level. As proof of principle, we tested the methodology for damage detection and quantification on two field-grown leafy vegetable species, spinach and Swiss chard. CONCLUSIONS: Our novel lesion quantification method can be used for detection of large (macro) or single-cell (micro) lesions on leaf scale, enabling quantification of lesions at any stage and without requiring symptoms to be in the visible spectrum. Quantifying the wounded area on leaf scale is necessary for generating prediction models for economic losses and produce shelf-life. In addition, risk assessments are based on accurate prediction of the relationship between leaf damage and infection rates by opportunistic pathogens and our method helps determine the severity of leaf damage at fine resolution.