Guidelines for conducting, analyzing, and interpreting electrical penetration graph (EPG) experiments on herbivorous piercing–sucking insects

Abstract

The electrical penetration graph (EPG) allows researchers to obtain information on the feeding behavior of herbivorous piercing–sucking insects, mostly Hemiptera. Guidelines are proposed for designing and conducting EPG experiments on insect–plant interactions, and for analyzing and interpreting the data. Several statistical issues common in EPG experiments are discussed. Pre-recording insect handling, wiring, and being tethered can affect feeding behavior and recommendations are made to minimize such stresses. Recording duration can have a large effect on some variables and interpretation of the data. The pros and cons of long vs. short recording duration are discussed, and suggestions are made for reconciling these conflicting concerns. Replicates lacking a specific waveform pose problems for analyzing variables associated with that waveform. Proper ways for handling these replicates when analyzing data are discussed. The waveform in progress when the recording ends will be terminated before it would naturally end; consequently, its duration will be artificially short (truncated). This creates a problem for calculating mean or median duration of waveform periods (a waveform period is a single occurrence of the waveform), especially for waveforms that tend to occur relatively few times during a recording and in long periods, such as waveforms E2 and G (phloem and xylem sap ingestion for aphids) or Xi (xylem sap ingestion for xylem feeders). Based on a detailed analysis (see the supporting information), guidance is given on how to calculate mean and median duration of waveform periods when the last period is truncated. A modified experimental design is suggested to avoid the problem of truncated waveform periods. Most EPG variables are interdependent, and their values can affect one another. This interdependence should be considered when drawing conclusions from the data. In studies on plant resistance against vascular sap-feeders, variables that are associated with resistance encountered during the penetration route to the vascular tissue and those encountered in the vascular tissue are discussed.