A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Abstract

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

Learning about natural variation of odor mixtures enhances categorization in early olfactory processing

“…Behavioral conditioning of the proboscis extension reflex (PER) followed standard protocols (Smith and Burden, 2014). Two groups 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 0 2 4 6 47 45 48 55 52 25 43 62 19 38 60 37 42 49 23 27 29 36 18 33 35 17 28 24 Glomerulus no.…”

“…We used PER conditioning (Smith and Burden, 2014) to evaluate whether honey bees can discriminate variation in A. majus. PER conditioning has been used to show that variation in molecular structures (Guerrieri et al, 2005;Smith and Menzel, 1989b), concentration (Wright et al, 2009) and blends (Fernandez et al, 2009;Wright et al, 2005b) define perceptual dimensions in the honey bee olfactory system.…”

SwarmSight: Real-Time Tracking of Insect Antenna Movements and Proboscis Extension Reflex using a Common Preparation and Conventional Hardware

“…Video Analysis 4.1. Download and install the Antenna Tracking module of SwarmSight by following the steps provided online 58 .…”

“…The raw x and y coordinate values produced by the software are post-processed with a 3-frame rolling median filter 57 (see Discussion). The instructions to download the full source code can be found online 58 .…”

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (<em>Bombus terrestris</em>)

“…The proboscis extension reflex (PER) experiments involve restraining individual bees in a harness and then stimulating the bee's antennal sensilla to produce the feeding reflex [14][15][16][17] . Bees can be restrained in individual harnesses and then stimulated to produce the feeding reflex as an assay of their ability to taste compounds 18,19 .…”