DOI: 10.5061/dryad.n2z34tnb4
title: Heliconiini butterflies display flight behaviours reminiscent of orientation flights when using new floral sources

description: Despite their small brains, many insects form long-term memories of the
 spatial distribution of resources. To support this, some species display
 ‘orientation’ flights to increase capture of landscape cues around novel
 resources. The role of orientation behaviour in spatial learning has been
 broadly explored in Hymenoptera, which are well known to navigate between
 their nests and floral resources. Here, we describe exaggerated flight
 behaviours expressed in the foraging context in Heliconiini butterflies,
 resembling orientation behaviours in Hymenoptera. Heliconiini butterflies
 are of particular interest as they include the genus Heliconius, which is
 reported to have a greater capacity for spatial memory in association with
 a novel dietary strategy of pollen feeding, including formation of stable
 foraging routes. We hypothesised that these flight behaviours we observe
 may provide a strategy to memorize the location of floral resources. We
 compared Heliconius and non-Heliconius butterflies to evaluate this
 ability in pollen and non-pollen-feeding Heliconiini species. We
 characterized three behavioural patterns that are directed towards a new
 floral resource: circle flights, hovering, and feeding bouts. Although all
 Heliconiini studied displayed these patterns, Heliconius spent more time
 performing these flight patterns, in particular hovering before landing.
 We suggest that these behaviours could provide the context in which
 individuals acquired sensory information to guide future foraging events,
 and our data indicate greater emphasis on this process during memory
 formation. Our findings of behaviours reminiscent of orientation flights
 in Heliconiini butterflies provide new avenues of research on how spatial
 learning and memory have converged between Lepidoptera and Hymenoptera.
description: Behavioural experiments were performed in an experimental cage
 with a group of 6 butterflies of a single species each time. We used two
 species of <em>Heliconius</em>, <em>H. erato</em>
 and <em>H. melpomene</em>, and, for comparison purposes, two
 closely related species that do not feed on pollen, <em>Dryas
 iulia</em> and <em>Dryadula phaetusa</em>. Behavioural
 video footage was analysed using BORIS software (Behavioural Observation
 Research Interactive Software, Friard and Gamba 2016). <em>Experimental
 procedure</em> Experiments were performed in an
 experimental cage (3 m x 3 m x 2 m, Figure S1) with a group of 6
 butterflies of a single species each time. Individuals were marked and
 sexed to allow individual identification. The food resource available in
 the cage consisted of three pots of flowering plants:
 <em>Stachytarpheta mutabilis</em> and/or <em>Pentas
 lanceolata</em>, sparsely positioned in the cage (Figure S1). These
 plant species are well known to attract butterflies, are regularly used to
 feed Heliconiini butterflies, and provide both nectar and pollen (Estrada
 and Jiggins, 2002). The butterflies had two days to become habituated in
 the new cage and could freely feed from the flowers.
 After the cage habituation period, a new floral resource was
 placed in the centre of the cage each morning (~ 9:00 AM), when all
 species are motivated to feed (Dell’Aglio et al., 2024). The experimental
 floral resource consisted of inflorescences of <em>Lantana
 camara</em> freshly collected and put in a 50ml Falcon tube with
 water. Two action cameras (GoPro©) were positioned to film approaches to
 the flowers from the side and from below (Figure S1). To capture the first
 interactions between the butterflies and the resource, we recorded the
 first 40 minutes after this resource was introduced. Subsequently, the
 cameras and the experimental floral resource were removed from the cage.
 This procedure was repeated over three consecutive days.
 Behavioural video footage was analysed using BORIS software
 (Behavioural Observation Research Interactive Software, Friard and Gamba
 2016). The behaviours recorded and described in the manuscript were
 defined during previous observations in the wild. Using the videos
 recorded from the side of the floral resource, we recorded: i) time until
 first feeding (time to find the new resource), ii) duration and number of
 feeding attempts, and iii) duration and number of ‘hovering’ and ‘circular
 flights’ behaviours around the flowers (see Table 1 for full description).
 To calculate the average duration for which a behaviour was expressed, we
 used the total duration of the behaviour (seconds) divided by the number
 of occurrences of the behaviour. To calculate the number of hovering
 flights per feeding attempt, we used the total number of hovering
 occurrences divided by the total number of feeding occurrences. Feeding
 bouts were annotated using tabular events data from BORIS, in which
 periods of consecutive feeding occurrences (the maximum time between
 feeding events to be included in the same bout was stipulated to be 3
 minutes) were grouped per individual. Using the videos captured from below
 the floral resource, we recorded the diameter (maximum observable distance
 from the individual take off) and duration of the circular flights. We
 note that the field of view of the camera was less than the total diameter
 of the cage, and as a result, the diameters of the largest circular
 flights could not be quantified, which may impact our later analyses of
 how this trait changes with time. To explore the
 importance of floral odours to these behaviours, we added a final trial
 for each species with naïve butterflies, repeating the same methodology,
 but using an artificial flower (orange foam sheet with a 1 ml Eppendorf
 tube for solution attached in the centre) instead of
 <em>Lantana</em> flowers. In this experiment, all real flowers
 were removed from the cage and only the new artificial flower was
 positioned in the centre of the cage. On the 1<sup>st</sup>
 and 2<sup>nd</sup> day, the artificial flower contained a
 sugar-water solution (20% sugar and 80% water), and on the
 3<sup>rd</sup> day, a clean, previously unused artificial
 flower was empty. During this trial, we recorded the same behaviours as
 listed above. Naïve butterflies are not strongly attracted to artificial
 flowers, and typically must be trained to use them. Therefore, we expected
 feeding attempts from this experiment to be low in number, as we focused
 on innate behaviours of untrained individuals. As such, quantitative data
 from this experiment are limited. However, it allowed us to assess whether
 visual cues associated with the feeder would stimulate flight behaviours
 around the potential food source, or if olfactory cues were essential to
 initiate them. <em>Statistical
 analysis</em> We used linear mixed-effect models (GLMM)
 implemented with the ‘lme4’ package in R (Bates et al., 2015),
 transforming data to a Log-normal distribution to fit the normality
 assumption. We included day and species as fixed factors, and individuals
 and group (butterflies tested on the same day) as random
 factors. Significance of fixed factors was determined through analysis of
 deviance test (Type II Wald Χ<sup>2</sup> test) and followed
 by Tukey's post hoc tests in R (R Core Team, 2020). Count data was
 analysed using generalized linear model (GLM) with the Poisson family,
 followed by analysis of deviance tests. To test for differences between
 pollen-feeding and non-pollen-feeding Heliconiini, some analyses were
 calculated grouping the species in “<em>Heliconius”</em>
 (<em>H. erato</em> and <em>H. melpomene</em>) and
 “Non-<em>Heliconius</em>” (<em>D. iulia</em> and
 <em>D. phaetusa</em>). 28 individuals who did not interact
 with the flowers were removed from the data set. Therefore, results are
 based on a final dataset of 14 <em>H. erato</em> (8 females, 6
 males), 19 <em>H. melpomene</em> (6 females, 13 males), 15
 <em>D. iulia</em> (6 females, 9 males) and 15 <em>D.
 phaetusa</em> (5 females, 10 males), all of which interacted with
 the flowers on all three days of the experiment (a total of 63 out of 91
 individuals). None of the behaviours observed were influenced by sex (all
 <em>p</em> &gt; 0.05), so this factor was removed from the
 analysis.
description: # Heliconiini butterflies display flight behaviours reminiscent of
 orientation flights when using new floral sources Dataset DOI:
 [10.5061/dryad.n2z34tnb4](https://doi.org/10.5061/dryad.n2z34tnb4) ##
 Description of the data and file structure ### Files and variables ####
 File: artificial_flower_07march2025.txt **Description:** Artificial
 feeders data processed with Boris. ##### Variables * "species"
 (name of the observed species), "sex" (male or female),
 "ind_id" (individual identification), "day" (day of
 the experiment), "week" (group of individuals tested in the same
 week), "date" (date of the video recording), "sugar"
 (with sugar water or empty), "behavior" (behaviors described
 above), "Total_occur" (number of events),
 "Total_duration" (total duration of the event in seconds),
 "Duration_mean" (mean of duration in seconds). #### File:
 circular_27sep2024.txt **Description:** The behaviour of circular flight
 was analysed using the below-view videos with Boris. ##### Variables *
 "species" (name of the observed species), "Hgroup"
 (Heliconius or Non-Heliconius), "sex" (male or female),
 "ind_id" (individual identification), "day" (day of
 the experiment), "week" (group of individuals tested in the same
 week), "date" (date of the video recording),
 "behavior" (behaviors described
 above), "behavior_type" (STOP, START or POINT), "time"
 (time in the video in seconds), "num_cf" (cumulative number of
 circle flights), "turn" (left or right), "diam_cm"
 (diameter of the circle flight in cm). #### File: fb_flower_27FEB2025.txt
 **Description:** Feeding bouts data were analysed using tabular events
 data, and periods of consecutive feeding events were grouped per
 individual. ##### Variables * "Hgroup" (Heliconius or
 Non-Heliconius), "species" (name of the observed species),
 "sex" (male or female), "ind_id" (individual
 identification), "day" (day of the experiment), "week"
 (group of individuals tested in the same week), fb_n (feeding bout
 number), n_feed (number of feeding events), total_time_seg (time in
 seconds), interval_seg (interval time in seconds). #### File:
 Flower_flight_RScript_DDD.R **Description:** R script file. This file
 contains all the packages, statistical models, and graphs used in the
 manuscript. It is divided into analyses in the same order as the published
 article. #### File: time_summ_21Feb2025.txt **Description:** Summary of
 the tabular events, by individual and behavioural category. #####
 Variables * "species" (name of the observed species),
 "sex" (male or female), "ind_id" (individual
 identification), "day" (day of the experiment), "week"
 (group of individuals tested in the same week), "date" (date of
 the video recording), "behavior" (behaviors described above),
 "Total_occur" (number of events), "Total_duration"
 (total duration of the event in seconds), "Duration_mean" (mean
 of duration in seconds), "per_total_length" (percentage of the
 behaviour by total time), "Hgroup" (Heliconius or
 Non-Heliconius). #### File: tabular_events_20Feb2025.txt
 **Description:** Raw data. Behaviour analysis processed with Boris. #####
 Variables * "species" (name of the observed species),
 "sex" (male or female), "ind_id" (individual
 identification), "day" (day of the experiment), "week"
 (group of individuals tested in the same week), "date" (date of
 the video recording), "behavior" (behaviors described above),
 "behavior_type" (STOP, START or POINT), "time" (time
 in the video in seconds), "Hgroup" (Heliconinii group:
 Heliconius or Non-Heliconius). ## Code/software Software used: R version
 4.5.0. Script: ***Flower_flight_RScript_DDD.R*** This file contains all
 the packages, statistical models, and graphs used in the manuscript. It is
 divided per analysis in the same order as the published article. To use
 the script, you need to add the .txt files attached.