Butterfly-mediated microbial dispersal experiment (Nymphalidae)

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Method synopsis

Controlled experiment using marker bacterium to track microbial transfer from inoculated donor flowers to recipient flowers via butterfly foraging behavior.

Synthesized from method descriptions across 3 papers using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — The dispersal of microbes among and within flowers by butterflies (2023), Ecological Entomology. The protocol was originally introduced by No evidence that gut microbiota impose a net cost on their butterfly host (2019), Molecular Ecology. Implementations in other papers (listed below) may differ.

1. Set up dispersal experiment arena
   Used 3 × 6 m room with large south-facing window. Placed Pyrrocoma crocea inflorescences near window in large floral pick with water to prevent wilting. Caught 26 S. mormonia adults and kept them in autoclave glass envelopes to starve overnight.
   Quantities: 26 S. mormonia butterflies, 3 × 6 m experimental arenaDuration: Overnight starvationConditions: Large south-facing window for natural lighting
   Equipment: floral picks, autoclave glass envelopes
2. Inoculate donor florets
   Dispensed about 1 μL of solution containing 8.5% NaCl and bacterial cells using a micropipette into each floret of donor inflorescence. Allowed inflorescence to dry, then placed in trial arena and allowed butterfly to feed.
   Quantities: 1 μL bacterial solution per floret, 8.5% NaCl concentrationDuration: Time to dry before butterfly feedingConditions: Allowed to dry before butterfly access
   Equipment: micropipette, 8.5% NaCl solution
3. Test microbial transfer to recipient florets
   After butterfly fed on donor inflorescence, replaced with recipient inflorescence (formerly training inflorescence) and allowed butterfly to feed. Removed and plated samples from stigmas, anthers, and nectaries using sterile needle-tip forceps. Made corolla tube slice to expose nectaries for sampling.

Typical Equipment

• floral picks
• micropipette
• sterile needle-tip forceps
• permanent markers

Output Measurements

• bacterial colony counts on floral organs

Papers Using This Protocol (3)

Species Studied With This Protocol

Places Where Used