3.3.1 Biology of Plants and Pollinators

As recognized in the first step of Figure 3.1, an understanding of the basic ecology of the plant and pollinator is fundamental for effective chemical ecology investigations. Furthermore, knowledge of the distribution, abundance and seasonality of the plant and pollinators is vital for planning an effective bioassay-guided approach. Knowledge of the source of active compounds is also crucial, with prior studies on related species potentially offering strong clues. If unknown, pollinator choice experiments using dissected flower parts should be performed.

3.3.2 Treatment of Biological Material for Chemical Analysis and Bioassays

Careful treatment of samples is critical for reliable outcomes of bioassays and chemical analysis. The safest approach is to process samples as soon as possible after their collection in the field. Solvent extractions can be conducted in the field, with a kit of solvents, syringes and vials, an insulated storage box with ice or a portable refrigerator. If processing in the field is not possible, it is important to keep the plant or insect material fresh by storing below 10°C and packed in such a way as to minimize contamination risk.

Irrespective of where the sampling is conducted, utmost care needs to be taken if re-using syringes or other means of sample transfer while handling solvent extracts. Multiple rinses with two or more solvents of contrasting polarity is recommended to avoid cross-contamination between samples. Using containers of glass or other inert material with appropriate lids will also minimize the background of extractable degradation products which can originate from plastic containers and rubber seals. Chemical analysis should be conducted as soon as possible, as volatile compounds easily evaporate or may degrade over time, with subsequent chemical profile changes as samples age. If this is not possible, storage in suitable vials at -80°C is recommended.

Once the active tissue is identified, it is crucial to design the isolation protocol so that it fits the purpose of the study. Chapter 1 provides a comprehensive review of the methods available for volatile sampling and analysis. Here we highlight the importance of following a bioassay-guided approach, particularly when the target compounds are unknown. Implementing bioassays to test which isolation method(s) retain maximum activity, are recommended (Figure 3.1). To minimize variability and reduce the number of bioassays, we suggest, where possible, to work with bulk multi-flower/multi-insect extracts.

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