DOI: 10.5061/dryad.xpnvx0kp5
title: Olfactory learning in Pieris brassicae butterflies is dependent on the intensity of a plant-derived oviposition cue

description: Butterflies, like many insects, use gustatory and olfactory cues innately
 to assess the suitability of an oviposition site and are able to associate
 colours and leaf shapes with an oviposition reward. Studies on other
 insects have demonstrated that the quality of the reward is a crucial
 factor in forming associative memory. We set out to investigate whether
 the large cabbage white Pieris brassicae (Linnaeus) has the ability to
 associate an oviposition experience with a neutral olfactory cue. In
 addition, we tested whether the strength of this association is dependent
 on the gustatory response to the glucosinolate sinigrin, which is a known
 oviposition stimulus for P. brassicae. Female butterflies were able to
 associate a neutral odour with an oviposition experience after a single
 oviposition experience, both in a greenhouse and in a semi-natural outdoor
 setting. Moreover, butterflies performed best when trained with
 concentrations of sinigrin that showed the strongest response by specific
 gustatory neurons on the forelegs. Our study provides novel insight into
 the role of both gustatory and olfactory cues during oviposition learning
 in lepidopterans and contributes to a better understanding of how these
 insects might be able to adapt to a rapidly changing environment.
description: Butterflies were conditioned by spraying 1 ml sinigrin solution
 on a green paper disc (9.5 cm in diameter) as an unconditioned stimulus,
 coupled with 20 microliters of vanilla scent as a conditioned stimulus.
 Three different concentrations of sinigrin solution were used. 3 different
 conditioning treatments plus one group of non-conditioned naive animals
 --&gt; four different treatments. Tests consisted of releasing one
 animal in a testing environment provided with four green paper
 discs, identical to the ones used during training, but without sinigrin.
 Two of these were scented with vanilla scent, while the other two were
 scented with 30% alcohol-water solution. Each test lasted 30
 minutes. Parameters recorded for each test are listed and explained below.
 Please note that not all of the recorded parameters have been used for
 analysis.
 <strong>Electrophysiology</strong>
 Response of the trichoid taste sensilla to sinigrin was assessed
 with single sensillum recordings. Sinigrin was dissolved in 1 mM KCl, 4
 different concentrations were used (1, 10, 20, 30 mM), while 1 mM KCl was
 used as blank control. The foretarsi of female Pieris brassicae present
 two different sensilla classes, namely medial and lateral, with two
 clusters for each class. One random sensillum for each class was screened
 for responses to sinigrin.
description: # Olfactory learning in *Pieris brassicae* butterflies is dependent on the
 intensity of a plant-derived oviposition cue ## Folder structure and
 contents: **These files are related to the behavioural observations in
 both indoor and outdoor settings:** * GHdata.xlsx * OUTdata.xlsx *
 GH_Sinigrin_Behaviour.R * OUT_Sinigrin_Behaviour.R *
 Peftuloglu_et_al_Electronic_Supplementary_Material.pdf **These files are
 related to the electrophysiological taste recordings performed:** *
 pierislegtaste.csv * Pierislegtaste script.R **Pieris_ovi.mp4**: Mp4 video
 of oviposition instance by a female Pieris brassicae on green paper disc
 impregnated with sinigrin.
 **Peftuloglu_et_al_Electronic_Supplementary_Material.pdf**: This file
 contains a picture of the indoor set-up, as well as graphs showing the
 proportion of time spent on vanilla-scented vs. unscented discs for
 control butterflies as well as butterflies trained with different
 concentrations of sinigrin in the indoor trails (figure S1). The file also
 contains images of the outdoor set-up and graphs showing the proportion of
 time spent on vanilla-scented vs. unscented discs for control butterflies
 as well as butterflies trained with different concentrations of sinigrin
 in the outdoor trails (figure S2). Finally, the file provides additional
 statistical results for the time spent and the number of visitations to
 scented and unscented discs in the indoor and outdoor set-up by both
 trained and untrained butterflies (table 1-4).   ##### MEASUREMENTS IN
 "GHdata" and "OUTdata": * Consolidation: time
 (expressed in minutes) used to consolidate memory formation after
 conditioning. * Testing time: length (expressed in minutes) of the
 behavioural observation. * Flight: binary expression (1 or 0) to express
 whether flight (i.e., behavioural response) occurred. * Training: used to
 express whether the tested animal was conditioned or naive (i.e. control)
 * ConcentrationTraining: milli molar concentration value of the
 unconditioned stimulus used during training. * EggsTraining: number of
 eggs laid during the training event by the conditioned animal. *
 TimeScentedDiscs: time spent on vanilla-scented discs during testing,
 expressed in seconds. * TimeControlDiscs: time spent on control discs
 during testing, expressed in seconds. * Total time: sum of the time spent
 on both disc classes, expressed in seconds. * PercentScent: percentage of
 the time spent on vanilla-scented discs in Total time. * PercentControl:
 percentage of the time spent on control scented discs on Total time. *
 PrefIndexTime: preference index for time spent on vanilla scented discs,
 calculated as: (TimeScentedDiscs)-(TimeControlDiscs)/(TotalTime) *
 TotalTrial: time spent on vanilla scented discs during the total trial,
 calculated as TimeScentedDiscs/1800 * TotalTrialC: time spent on control
 discs during the total trial, calculated as TimeControlDiscs/1800 *
 VisitsScent: total number of visits (i.e. approaches) to vanilla-scented
 discs during testing. * VisitsControl: total number of visits (i.e.
 approaches) to control discs during testing. * TotalVisits: sum of
 VisitsScent and VisitsControl. * EggsScentedDiscs: number of eggs laid on
 vanilla-scented discs during testing if oviposition occurred. *
 EggsScented: binary value (1 or 0) that expresses whether oviposition on
 vanilla-scented discs occurred during testing. * EggsControlDiscs: number
 of eggs laid on control discs during testing if oviposition occurred. *
 EggsControl: binary value (1 or 0) that expresses whether oviposition on
 control discs occurred during testing. * T: temperature measured in
 Celsius degrees at the beginning of each test. * rH: percent relative
 humidity (for outdoor observations only). * Wind: windspeed in m/s (for
 outdoor observations only). * Pressure: pressure value measured in
 millibars at the beginning of each test (hPa). * Lux: light intensity (lx)
 measured at the beginning of each test. * Notes: additional remarks. *
 Empty cells indicate missing values. ##### MEASUREMENTS IN
 "Pierislegtaste": * ID: numerical value used to identify an
 animal used for a complete electrophysiological recording round. * Animal:
 numerical value used to identify recordings within a single day (not used
 in analysis). * Sensillum: identifies the sensillum type implied with the
 recording, namely "medial" or "lateral". *
 Spikes_large: number of spikes with amplitude greater than 1 mV during the
 1st second after stimulus onset. * Spikes_medium: number of spikes with
 amplitude between 0.5 and 1 mV during the 1st second after stimulus onset.
 * Spikes_small: - Spikes_large: number of spikes with amplitude smaller
 than 0.5 mV during the 1st second after stimulus onset. Not used in
 analysis since not easily distinguishable from recording artifacts. *
 Empty cells indicate missing values. ### Version changes:
 **26-July-2024:** A new version of
 "Peftuloglu_et_al_Electronic_Supplementary_Material.pdf" was
 added including updated supplementary statistical results tables. ##
 Software Required: * R 4.3.1 * Rstudio 2023.12.1 Contact:
 [dimitri.peftuloglu@wur.nl](mailto:dimitri.peftuloglu@wur.nl),
 [alexander.haverkamp@wur.nl](mailto:alexander.haverkamp@wur.nl)