Identification of key yeast species and microbe-microbe interactions impacting larval growth of Drosophila in the wild.

Microbiota consisting of various fungi and bacteria have a significant impact on the physiological functions of the host. However, it is unclear which species are essential to this impact and how they affect the host. This study analyzed and isolated microbes from natural food sources of Drosophila larvae, and investigated their functions. Hanseniaspora uvarum is the predominant yeast responsible for larval growth in the earlier stage of fermentation. As fermentation progresses, Acetobacter orientalis emerges as the key bacterium responsible for larval growth, although yeasts and lactic acid bacteria must coexist along with the bacterium to stabilize this host-bacterial association. By providing nutrients to the larvae in an accessible form, the microbiota contributes to the upregulation of various genes that function in larval cell growth and metabolism. Thus, this study elucidates the key microbial species that support animal growth under microbial transition.

The relative abundances of yeasts attractive to Drosophila suzukii differ between fruit types and are greatest on raspberries.

Fungal metabolic volatiles attract Drosophila suzukii which oviposits in ripening fruits, but there are few data describing the fungal microbiomes of commercial fruits susceptible to this insect pest. We tested the hypothesis that fruit type and ripening stage have a significant effect on fruit surface fungal communities using DNA metabarcoding approaches and found strong support for differences in all three fungal community biodiversity metrics analysed (numbers, types, and abundances of taxa). There was an average fivefold greater difference in fungal communities between sites with different fruit types (strawberry, cherry, raspberry, and blueberry) than across fruit developmental stages, demonstrating site and/or fruit type is the greater factor defining fungal community assemblage. The addition of a fungal internal standard (Plectosphaerella cucumerina) showed cherry had relatively static fungal populations across ripening. Raspberry had a greater prevalence of Saccharomycetales yeasts attractive to D. suzukii, including Hanseniaspora uvarum, which aligns with reports that raspberry is among the fruits with greatest susceptibility and attraction to D. suzukii. Greater knowledge of how yeast communities change during fruit maturation and between species or sites may be valuable for developing methods to manipulate fruit microbiomes for use in integrated pest management strategies to control D. suzukii.

Hanseniaspora uvarum Attracts Drosophila suzukii (Diptera: Drosophilidae) With High Specificity.

Since the early phase of the intercontinental dispersal of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), fermentation baits have been used for monitoring. Self-made lures and commercial products are often based on wine and vinegar. From an ecological perspective, the formulation of these baits is expected to target especially vinegar flies associated with overripe fruit, such as Drosophila melanogaster (Meigen) (Diptera: Drosophilidae). Hanseniaspora uvarum (Niehaus) (Ascomycota: Saccharomyceta) is a yeast closely associated with D. suzukii and fruit, and furthermore attractive to the flies. Based on this relation, H. uvarum might represent a suitable substrate for the development of lures that are more specific than vinegar and wine. In the field, we therefore, compared H. uvarum to a commercial bait that was based on vinegar and wine with respect to the number of trapped D. suzukii relative to other drosophilids and arthropods. Trap captures were higher with the commercial bait but specificity for D. suzukii was greater with H. uvarum. Moreover, H. uvarum headspace extracts, as well as a synthetic blend of H. uvarum volatiles, were assayed for attraction of D suzukii in a wind tunnel and in the field. Headspace extracts and the synthetic blend induced strong upwind flight in the wind tunnel and confirmed attraction to H. uvarum volatiles. Furthermore, baited with H. uvarum headspace extract and a drowning solution of aqueous acetic acid and ethanol, 74% of field captured arthropods were D. suzukii. Our findings suggest that synthetic yeast headspace formulations might advance the development of more selective monitoring traps with reduced by-catch.

Field and greenhouse application of an attract-and-kill formulation based on the yeast Hanseniaspora uvarum and the insecticide spinosad to control Drosophila suzukii in grapes.

BACKGROUND: The invasive insect Drosophila suzukii (Matsumura) is an important pest of several red grape varieties. The yeast Hanseniaspora uvarum (Niehaus), which is associated with D. suzukii, strongly attracts flies and stimulates them to feed on yeast-laden food. In the present study, a formulation based on H. uvarum culture with spinosad insecticide was applied to the foliage of vineyards and control of D. suzukii was compared to applying spinosad to the whole plant. After successful H. uvarum and insecticide application in the vineyard, we tested additional H. uvarum-based formulations with spinosad in a greenhouse to determine their capacity to control D. suzukii. RESULTS: Application of the H. uvarum-spinosad formulation at 36.4 g of spinosad per hectare reduced the D. suzukii field infestation at the same rate as applying 120 g of spinosad per hectare and prevented spinosad residues on grapes. Leaves treated with H. uvarum and spinosad in the field and transferred to a laboratory assay caused high mortality to flies and reduced the number of eggs laid on fruits. Formulations with spinosad applied in the greenhouse showed that both H. uvarum culture and the yeast cell-free supernatant of a centrifuged culture increased fly mortality and reduced the number of eggs laid compared to the unsprayed control. CONCLUSION: In comparison to typical spinosad spray applications, the use of H. uvarum in combination with spinosad as an attract-and-kill formulation against D. suzukii reduces pesticide residues on the fruits by targeting the treatment to the canopy and decreasing the amount of insecticide per hectare without compromising control efficacy.

Behavioral manipulation of Drosophila suzukii for pest control: high attraction to yeast enhances insecticide efficacy when applied on leaves.

BACKGROUND: The invasive pest, Drosophila suzukii attacks fresh soft-skinned fruit. Broad-spectrum insecticides are implemented for control but there is a need to reduce environmental risks and insecticide residues on fruits. Hanseniaspora uvarum is a yeast frequently found on ripe fruits and associated with D. suzukii. We aim to exploit the ecological association and attraction of D. suzukii to H. uvarum by developing an attract-and-kill strategy, with spray-application on canopy but not fruit. We therefore investigated D. suzukii attraction, egg-laying and mortality when exposed to insecticidal yeast-based formulations. RESULTS: Hanseniaspora uvarum strongly attracted D. suzukii when applied on leaves of grapevine, Vitis vinifera. Notably, this attractiveness was competitive to ripe grape berries that were susceptible to D. suzukii infestation. Moreover, adding H. uvarum enhanced the efficacy of insecticidal formulations against D. suzukii. Flies exposed to leaves treated with yeast-insecticide formulations showed higher mortality and laid a lower number of eggs compared to flies exposed to insecticide alone. In a wind tunnel, all treatments containing H. uvarum alone or in combination with insecticides, caused similar upwind flight and landing at the odor source, which provides evidence that the addition of insecticide did not reduce D. suzukii attraction to yeast. CONCLUSION: Hanseniaspora uvarum can be used to manipulate the behavior of D. suzukii by attracting flies to insecticide formulations. Yeast attraction is competitive to grape berries and improves insecticide effectiveness, suggesting that sprays covering canopy only, could reduce residues on fruit without compromising management efficacy.

Impact of Background Fruit Odors on Attraction of Drosophila suzukii (Diptera: Drosophilidae) to Its Symbiotic Yeast.

Background odors produced by plants in the environment can interfere with the response of insects to a point-releasing attractant, especially when their compositions overlap. In this study, a series of binary choice tests was conducted in a wind tunnel to investigate whether background odors emitted from cherry, blueberry, blackberry, or raspberry fruits would affect the level of Drosophila suzukii (Matsumura) attraction to its symbiotic yeast, Hanseniaspora uvarum (Niehaus) (Saccharomycetales: Saccharomycetaceae). Whether an increase in the intensity of background odors would affect the attractiveness of H. uvarum to D. suzukii was also investigated, either by increasing the number of cherry or raspberry fruit per cup or by increasing the number of fruit cups surrounding the cup baited with the yeast. In wind tunnel assays, background fruit odors interfering with D. suzukii attraction to the yeast varied among fruit types. Raspberry odor inhibited the attractiveness of H. uvarum to the fly the most, followed by blackberry odor, whereas cherry and blueberry odors had no significant impact on the attraction. An increase in the intensity of odors by adding more cherry or raspberry fruit per cup did not increase the impact of fruit odor on the attraction; however, adding more raspberry cups around H. uvarum linearly decreased its attractiveness, suggesting that background host fruit abundance and likely increase in host odor may influence D. suzukii attraction to yeast odor depending on host species.

Exploring the Efficacy and Mechanisms of a Crop Sterilant for Reducing Infestation by Spotted-Wing Drosophila (Diptera: Drosophilidae).

Vinegar flies (Diptera: Drosophilidae) are well known to be associated with yeasts, which provide important nutrients and emit attractive semiochemicals. Drosophila suzukii (Matsumura) has become a major pest of berries and cherries around the world, requiring intensive management to maintain fruit quality. Although insecticides remain a dominant control approach, disruption of fly-yeast-host interactions remains a promising avenue for reducing the economic impact of this pest. We conducted field and laboratory experiments to explore whether a crop sterilant (peroxyacetic acid and hydrogen peroxide) developed for disease control can affect D. suzukii. In 2 yr of field tests in highbush blueberries, we found significantly lower infestation by D. suzukii in plots treated with the crop sterilant, both alone and in a rotation program with zeta-cypermethrin. When shoots from treated plots were tested in no-choice bioassays, crop sterilant treatments did not affect adult mortality or oviposition, but they reduced infestation. To explore the mechanisms in the laboratory, we found that the crop sterilant did not affect adult mortality, nor oviposition on treated fruit under no-choice settings, but adult flies settled and oviposited less on treated fruit in choice settings. When the crop sterilant was applied to colonies of Hanseniaspora uvarum (Niehaus) (Saccharomycetales: Saccharomycodaceae) and Issatchenkia terricola (Van der Walt) (Saccharomycetales: Saccharomycetacea) yeasts that are attractive and provide nutrition to D. suzukii, there was a dose-dependent inhibition of their growth. We highlight the potential for microbial management as a component of integrated pest management programs and prioritize research needs to incorporate this approach into control programs.

Differential Impacts of Yeasts on Feeding Behavior and Development in Larval Drosophila suzukii (Diptera:Drosophilidae).

Larval Drosophila encounter and feed on a diverse microbial community within fruit. In particular, free-living yeast microbes provide a source of dietary protein critical for development. However, successional changes to the fruit microbial community may alter host quality through impacts on relative protein content or yeast community composition. For many species of Drosophila, fitness benefits from yeast feeding vary between individual yeast species, indicating differences in yeast nutritional quality. To better understand these associations, we evaluated how five species of yeast impacted feeding preference and development in larval Drosophila suzukii. Larvae exhibited a strong attraction to the yeast Hanseniaspora uvarum in pairwise yeast feeding assays. However, larvae also performed most poorly on diets containing H. uvarum, a mismatch in preference and performance that suggests differences in yeast nutritional quality are not the primary factor driving larval feeding behavior. Together, these results demonstrate that yeast plays a critical role in D. suzukii's ecology and that larvae may have developed specific yeast associations. Further inquiry, including systematic comparisons of Drosophila larval yeast associations more broadly, will be necessary to understand patterns of microbial resource use in larvae of D. suzukii and other frugivorous species.

Laboratory and Field Evaluation of Host-Related Foraging Odor-Cue Combinations to Attract Drosophila suzukii (Diptera: Drosophilidae).

The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura), is a major pest of soft-skinned fruits. Since its introduction into North America and Europe, significant progress has been made in understanding the volatile cues used by this fly during food, oviposition site, and mate finding. Despite this progress, commercially available lures are non-selective. Here, we tested two Hanseniaspora uvarum (Niehaus) yeast compounds (isoamyl acetate and isobutyl acetate) and a leaf compound ß-cyclocitral alone and in combination with a blend of four fermentation compounds ('Fermentation lure': acetic acid, ethanol, methionol, and acetoin) to improve D. suzukii attraction and selectivity. In laboratory assays, males and females were attracted to all seven individual compounds, although in electrophysiological assays, their antennae exhibited a dose-dependent response to only four of these compounds. In two-choice cage studies, the Fermentation lure was more attractive to D. suzukii than water controls, whereas ß-cyclocitral and the mixture of isoamyl acetate and isobutyl acetate were not attractive in this larger-cage study. Moreover, adding the two-component H. uvarum compound blend to the Fermentation lure reduced D. suzukii attraction to the Fermentation blend. When these experiments were repeated in blueberry, raspberry, blackberry, and cherry orchards across several states in the United States over 2 yr, similar outcomes were observed: ß-cyclocitral or the mixture of the H. uvarum blend did not improve the attractiveness of the Fermentation lure or its selectivity. This study demonstrates that cues from different sources may interfere with each other and reduce D. suzukii attraction to otherwise attractive odor combinations.

Associations Between Drosophila suzukii (Diptera: Drosophilidae) and Fungi in Raspberries.

The invasive vinegar fly, Drosophila suzukii Matsumura, has emerged as one of the most serious arthropod pests of primocane red raspberries (Rubus ideaus L.) in the United States. In raspberries, D. suzukii encounter a diverse community of microbes, including fruit rot pathogens such as Botrytis cinerea Pers and Cladosporium cladosporioides de Vries. In this study, our primary objectives were to evaluate D. suzukii-fungal associations and determine D. suzukii's influence on fungal communities in raspberry fruit. Through culture-based surveys of larval gut microbes, we isolated several yeast fungi (primarily Hanseniaspora spp.), as well as Cladosporium, Botrytis, and several other non-yeast fungi from larval frass, suggesting that D. suzukii larvae encounter and feed on these fungi. Subsequent field surveys confirmed that D. suzukii larvae occurred in berries affected by Botrytis fruit rot and Cladosporium fruit rot. Under laboratory conditions, D. suzukii may facilitate C. cladosporioides infections, likely through the introduction of epiphytic propagules on the fruit surface. We could not detect impacts on B. cinerea infections or establish a clear vectoring relationship for either fruit rot. These studies provide evidence for an association between D. suzukii and fungal fruit rot pathogens. Understanding interactions between raspberry fruit, D. suzukii, and fungal microbes-especially whether D. suzukii facilitates the development of fruit rots or conversely, if fruit rots influence D. suzukii infestation patterns-may improve pest and pathogen management programs.

Drosophila-associated yeast species in vineyard ecosystems.

Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem.

The thalidomide-binding domain of cereblon defines the CULT domain family and is a new member of the ß-tent fold.

Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded ß-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A ß-hairpin inserted into the first ß-meander extends across the bottom of the structure towards the C-terminal edge of the second ß-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the ß-tent fold for the striking arrangement of its constituent ß-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.

Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries.

A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii.