Fungal volatiles mediate the orientation response of eusocial wasps Thomas “Seth” Davis, Kyria Boundy-Mills, Pete Landolt Davis et al. (2012) Volatile emissions from an epiphytic fungus are semiochemicals for eusocial wasps. In press, Microbial Ecology. DOI: 10.1007/s00248-012-0074-2

Talk overview Observation/Discovery stage Refining stage Experimental stage

Codling moth larvae (Cydia pomonella) Observation stage Our journey begins with a yeast-like fungus found in the frass (fecal material [poop]) of a frugivorous orchard pest… Yeast?! Frass Codling moth larvae (Cydia pomonella)

Observation stage Microbial symbionts can be attractive to their host arthropod (Hulcr et al 2011, Becher et al 2012) These symbioses can potentially be exploited to identify bioactive natural products (Poulsen et al 2011) So we tried to use yeasts to trap moths!

Not a single target moth with yeast-baits, but plenty of yellowjackets Observation stage From this… To this! Not a single target moth with yeast-baits, but plenty of yellowjackets

Observation stage Question 1 Hypothesis 1 Q1: Is there evidence for semiochemical attraction of yellowjacket wasps to yeast? Hypothesis 1 H1: Cell cultures (raw material) of the attractive fungus can attract wasps

Cell cultures were used to bait dome traps Traps selective for insect orientation: insects do not passively enter traps—they have to want in (directed flight) Treatments: Unbaited control, media with no yeast, and media with yeast growth. Replication: n = 10 for each treatment Traps placed in a random configuration in experimental orchard (USDA)

Observation stage Sure enough, traps baited with yeast cultures were more attractive than controls in the field!

Refining stage Objective 1 Identify epiphytic fungi associated with apples

Refining stage (1) Collected apples and performed serial dilutions of apple washes in saline buffer (2) Sorted cultures by morphotype and identified representative cultures by sequencing D1/D2 domain of 26 S (large ribosomal RNA gene) (3) DNA sequences were compared against the NCBI database using BLAST: identification assumed at ≥ 99% sequence matches

Refining stage Community represented by two species—a black yeast and a pink yeast All cultures listed are stored as vouchers in the Phaff Yeast Culture Collection (UC Davis)

Refining stage Objective 2 Identify and quantify volatile compounds produced by the attractive yeast species

Used a 2-port collection system to characterize compounds in A Used a 2-port collection system to characterize compounds in A. pullulans headspace Yeast samples compared against system blanks and growth media alone Compounds eluted from a polymer adsorbent and analyzed using GC/MS

Refining stage Yeast volatile profiles were simple: only three compounds present Quantified emissions per unit mass (g) yeast over time (h)

Experiment stage Question 2 Hypothesis 2 Q2: What components of yeast headspace do wasps orient to? Hypothesis 2 H2: Synthetic compounds isolated from yeast headspace are attractive to wasps

(1) 2-methyl-1-butanol (2) 3-methyl-1-butanol (3) 2-phenylethanol (4) 51:39:10 % blend

Experiment stage Wasp attraction highest in response to 2-methyl-1-butanol Additional compounds appeared to negatively affect the orientation response

Experiment stage Question 3 Hypothesis 3 Q2: Do wasps carry the organisms that they are attracted to? Hypothesis 3 H3: Wasps vector and deposit A. pullulans

All wasps we sampled were found to externally vector A All wasps we sampled were found to externally vector A. pullulans, and often several putative yeast species. This frequency (100%) was significantly greater (χ2 = 13.2, P<0.01) than expected value of 72%, which was previously reported for a bumblebee-yeast association.

Conclusions H3: Wasps vector and deposit A. pullulans H2: Synthetic compounds isolated from yeast headspace are attractive to wasps H1: Cell cultures (raw material) of the attractive yeast can attract wasps Supported! Wasps were significantly attracted to cultures in the field and displayed directed flight to traps baited with cultures. Supported! Wasps oriented to all compounds, but more wasps on average were attracted to 2-methyl-1-butanol, the most abundant compound. Supported! All sampled wasps carried A. pullulans externally, as well as several other putative yeast species.

Conclusions Certain eusocial wasps appear to be strongly attracted to microbial volatiles Arthropod attraction to microbes may be more widespread than currently recognized…how many insect species orient to microbial volatiles? Ongoing biodiversity studies to evaluate the greater community of insects that are associated with volatiles from the black yeast A. pullulans

Davis, TS; Boundy-Mills, KB; Landolt, PJ Volatile emissions from an epiphytic fungus are semiochemicals for eusocial wasps. In press, Microbial Ecology. DOI: 10.1007/s00248-012-0074-2