DOI: 10.5061/dryad.ns1rn8q2c
title: Data from: Neural and sensory basis of homing behavior in the invasive cane toad, Rhinella marina

description: The behavioral, sensory, and neural bases of vertebrate navigation are
 primarily described in mammals and birds. However, we know much less about
 the navigational abilities and mechanisms of vertebrates that move on
 smaller scales, such as amphibians. To address this knowledge gap, we
 conducted an extensive field study on navigation in the cane
 toad, Rhinella marina. First, we performed a translocation
 experiment to describe how invasive toads in Hawaiʻi navigate home
 following displacements of up to one kilometer. Next, we tested the effect
 of olfactory and magnetosensory manipulations on homing, as these senses
 are most commonly associated with amphibian navigation. We found that
 neither ablation alone prevents homing, suggesting that toad navigation is
 multimodal. Finally, we tested the hypothesis that the medial pallium, the
 amphibian homolog to the hippocampus, is involved in homing. By comparing
 neural activity across homing and non-homing toads, we found evidence
 supporting the involvement of the medial pallium, lateral pallium, and
 septum in navigation, suggesting the conservation of neural structures
 supporting navigation across vertebrates. Our study lays the foundation to
 understand the behavioral, sensory, and neural bases of navigation in
 amphibians and to further characterize the evolution of behavior and
 neural structures in vertebrates.
description: This manuscript includes datasets related to cane toad
 (<em>Rhinella marina</em>) spatial movements, the effects of
 sensory manipulations on toad spatial movements, and brain activity during
 toad navigation acitvities. The following types of data are
 presented: 1) Toad metadata: HI 2022 Toad List.csv,
 TransToadInfo.csv, and Toads.csv contain information about individuals in
 the experiment. This includes ID, sex, size (mass and snout-vent-length),
 tagging date and times, sensory ablation information (whether an ablation
 happened, what type, when it was performed, and information about how many
 coordinate points post-ablation fall into the area the toad occupied
 pre-ablation), the toad experimental group, whether the toad homed or not,
 the field site where the toad was monitored, the date the toad was
 translocated, and the day the toad was euthanized. HI 2022 Toad List.csv
 is used in analysis of baseline spatial data, TransToadInfo.csv is used in
 analysis of trajectories post translocation, and Toads.csv is used in
 analysis of brain activity related to homing.  2)
 Baseline spatial data (Baseline.csv): This dataset includes geographic
 coordinates and timestamps of toad positions collected in the wild on the
 island of O‘ahu in the state of Hawaiʻi. This data was gathered by using
 radio telemetry to track individual toads over several nights and record
 their positions with handheld GPS devices. Coordinates were uploaded to
 ArcGIS Online and ArcGIS Pro for visualization and to convert coordinates
 to UTM projection, and then processed in<strong> </strong>R
 Studio (v 2023.09.1+494, Posit Software, PBC, Sunnyvale, CA) running R (v
 4.3.1, R Foundation for Statistical Computing, Vienna, Austria).
 Trajectories were generated for each toad using the package ‘adehabitatLT’
 in R; "ltraj" objects generated in ‘adehabitatLT’ were used to
 calculate spatial and temporal attributes, including cumulative movement
 during baseline tracking, daily movement during this period, and total
 movement range. 3) Ablation related baseline spatial
 data (Ablation_Baseline.csv): This dataset includes the baseline
 coordinates and timestamps found in Baseline.csv, but only for toads
 involved in olfaction and magnetroeception manipulation experiments. There
 is a column that specifies whether the baseline point was recorded
 pre-ablation or post-ablation. Separate trajectories were made for pre-
 and post- periods using the package ‘adehabitatLT’ in R, and baseline
 metrics were compared to see if ablations or sham manipulations disrupted
 space baseline space use.  4) Coordinates from
 translocation experiment (Translocation_Coordinates.csv): This dataset
 includes coordinates and timestamps of tods during translocation
 experiments where toads were moved ~500 m or ~1000 m away from their
 outermost baseline coordinate point and tracked for three days to observe
 whether they would return home. Toads were tracked until they moved within
 200 meters of their nearest baseline point or 3 days had passed
 post-translocation. Trajectories were generated for each toad using the
 package ‘adehabitatLT’ in R; "ltraj" objects generated in
 ‘adehabitatLT’ were used to calculate cumulative movement, straightness
 index, homing duration/speed, and active homing duration/speed. The
 dataset includes a column "Grp" which specifies whether the
 toads were unablated (in which case "500" or "1000" is
 listed, corresponding to the translocation distance) or had an ablation or
 sham ablation (OAE for olfactory ablation, OAC for olfactory control/sham,
 MAE for magnetoreception ablation, and MAC for magnetoreception
 control/sham). 5) Angle correction for normalization of
 trajectories (Transect_Correction.csv): This dataset contains the
 coordinates of the translocation release site and the outermost  baseline
 reference point used to calculate translocation distance for each toad.
 The package ‘adehabitatLT’ in R was used to create trajectories from these
 points for each toad to extract the angle of travel needed to reach home,
 which was used to normalize trajectories for plotting. The timestamps are
 arbitrary time points picked to separate the two points for each toad:
 translocation release point is 2022-02-22 and baseline reference is
 2022-05-13 or 2022-05-14).  6) Metadata for analyzed
 brains (Brains.csv): This dataset contains information about brains
 extracted from toads, including the toad, aproximate trannslocation
 distane (0, 500, or 1000m), condition (either Homing, Lost, Baseline, or
 Homing* if brain collected at late time point after toad homed), ablation
 (ablation information if applicable), extraction (date that brain was
 collected), sodaz (0 if brain was not put in PBS with sodium azide, 1 if
 it was, depending on the time the brain was collected in relation to
 transport to the laboratory). This dataset was used to assign identifiers
 to count data described below. 7) Region specific pS6
 count data for analysis of brain activity differences between homing
 conditions (BranCounts_02.csv): This dataset inclusdes the count of
 pS6-positive cells in six different brain regions across 9 sections of
 brain in every toad considered in analysis. Count data was generated by
 completing pS6-immunohistochemistry on cryosectioned brain sections,
 imaging sections at 20x magnification using brightfield microscopy, and
 counting positive cells in each region manually with ImageJ. Count data
 was used to build models in R. We used generalized linear mixed models
 (‘glmmTMB’ package in R) to test for differences in brain activity and its
 relation with behavior.  Models used a negative binomial distribution and
 best model fit was confirmed using the ‘DHARma’ package in R . Homing
 condition, brain region, and their interaction were the main effects
 predicting the number of pS6-positive cells. Toad ID was included as a
 random factor and brain region area was included as an offset variable to
 account for brain area size. We modeled differences in brain activity due
 to ablations separately using the same model parameters as above except
 that ablation was also included as a main effect. Post hoc pairwise
 contrasts (between homing condition groups and ablation treatments) were
 calculated with estimated marginal means using the “emmeans” package in
 R. In addition, to test if brain activity was related
 to either straightness or duration of homing, we performed linear mixed
 effects models using the “lme4” package, where pS6-count was the response
 variable and the homing descriptor (straightness or duration), brain
 region, and their interaction were fixed effects. Toad ID was a random
 effect and brain region area was an offset variable in the model. Linear
 mixed effects models were also performed within count data specific to
 each brain region, in which the response variable was pS6 counts within a
 given region, the homing descriptor was the fixed effect, Toad ID was a
 random effect, and region area was an offset variable. 
 8) Count of pS6-positive cells in the olfactory bulbs of
 olfaction ablated and sham ablated toads (olf_ct.csv): This dataset
 includes pS6 count data from olfactory bulbs of toads in olfactory
 ablation experiment. Three sections of olfactory bulb were considered for
 each animal and pS6-postive cells were counted in a 90,000 micrometer
 squared box on each section, in order to test whether there was an effect
 of olfactory ablation on the number of pS6-positive cells in olfactory
 bulb. The comparison was made between sham and ablated animals using a
 Wilcoxon Rank Sum Test.  9) Weather data from
 translocation experiments (Weather.csv): This dataset includes daily
 weather data taken from NOAA for any day a translocation experiment was
 happening at a particular field site. This data includes precipitation
 amoutn, max temperature, and minmum temperature. These measurements were
 compared between field sites using Kruskal-wallis Tests and post hoc
 Dunn's Tests to identify differences in eather conditions between
 locations during tracking experiments.
description: # Neural and sensory basis of homing behavior in the invasive cane toad,
 *Rhinella marina* In this study, we characterize homing behavior in
 invasive cane toads in Hawaii, test the effects of sensory manipulations
 on toad navigation, and characterize brain activity differences between
 homing and non-homing toads. This project has both field and laboratory
 components, so many methods were employed. In the field, radio telemetry
 was used to track toad baseline movements and to track toads following
 translocations of up to 1 km from their home area to observe if they were
 able to return home. In addition, we manipulated olfaction and
 magnetoreception in a subset of toads to test the effect of these senses
 on homing behavior. Finally, in the lab, we used pS6-immunohistochemistry
 to model differences in brain activity between homing, non-homing, and
 non-translocated toads, in order to identify brain regions associated with
 navigation behavior in amphibians. The datasets generated by these
 experiments include toad metadata, coordinate points and timestamps of
 toad locations during baseline tracking and translocations, and pS6 count
 data for comparisons of neural activity between homing and non-homing
 toads.  With this data, we demonstrate that (a) toads show robust
 navigation ability following translocations, (b)  disruption of neither
 olfaction or magnetoreception alone prevents homing, suggesting that
 homing is multimodal, and (c) brain regions associated with navigation in
 other vertebrates, including the medial pallium and the septum, show
 greater levels of activity in homing toads, suggesting a conservation of
 neural structures associated with navigation across taxa. ## Description
 of the data and file structure There are 11 data-sheets associated with
 this manuscript. They are all used in R script that was written to perform
 the reported analysis, which is also provided. **Toad Metadata**: Toad
 metadata is provided in 3 data-sheets (HI Toad List 2022, TransToadInfo,
 and Toads) that are implemented in different parts of the R script. **HI
 Toad List 2022** contains the metadata for all toads tagged during the
 course of the three month field season in O‘ahu from February-May 2022.
 This data-sheet is implemented in Part 2 of the R script (*Analysis of
 Baseline Tracking Data*). Columns and data in these sheets include the
 following:  * Sex (M for Male, F for Female) * Mass (in grams) * SVL
 (snout-vent-length, in cm) * Tag Date (date of tagging) * Capture time
 (for tagging) * Release time (post-tagging) * Tag time (duration of
 tagging) * Ablation (either None, MAC/ MAE for magnetoreception
 control/experimental, or OAC/OAE for olfaction control/experimental) *
 PostPtsinPre (for ablated animals, a count of how many baseline coordinate
 locations post-ablation fall into the minimum convex polygon created from
 pre-ablation coordinates; NA for all animals without ablations) * PostPts
 (total number of coordinate points collected post-ablation; NA for all
 animals without ablations) * Condition (either Baseline, 500 or 1000 for
 translocations without ablations, Ablation code described above, or LOST
 TAG) * Homing (0 if non-homing, 1 if homing, NA if baseline or lost) and
 homing was not measured * Location (name of field site) * Ablation date
 (if applicable, NA if not ablated) * Translocation date (if applicable, NA
 if not translocated) * Sac Date (date of euthanasia, LOST if lost before
 euthanasia occurred) * Notes (additional observations) The sheet
 **TransToadInfo** contains same data as **HI 2022 Toad List**, except it
 only includes translocated toads (omits all animals only used for baseline
 tracking). The sheet also omits columns Ablation, PostPtsinPre, and
 PostPts. It is implemented in Part 1 of the R script (*Analysis of
 translocation related spatial activity*). The sheet **Toads** also
 contains the same data as **HI 2022 Toad List**, but was used in the
 analysis of brain activity (Part 4 of the R script: *Analysis of pS6 count
 data*). **Baseline Activity**: The sheets **Baseline and**
 **Ablation_Baseline** contain geographic coordinates and timestamps of
 toad locations collected during radio tracking prior to translocations.
 The coordinates in **Baseline** are used to generate "ltraj"
 objects in the 'adehabitatLT' package in R (script part 2:
 *Analysis of Baseline Tracking Data*) in order to calculate descriptors of
 baseline movements, including cumulative movement, daily movement, and
 movement range.  The following columns are included: * ID (unique
 identifier of each toad in experiment) * Condition (statement about
 activity at time of observation) * Notes (additional observations) *
 POINT_X (longitude in UTM of coordinate point of observation) * POINT_Y
 (latitude in UTM of coordinate point of observation) * Timestampe_E (Date
 and time of observation) The **Ablation_Baseline** sheet *contains the
 same data with the same columns, but only for toads that underwent sensory
 ablations or sham ablations, *and an additional column
 ,"PrePost," specifies whether the points were collected pre- (0)
 or post- (1) ablation. In Part 3 of the R script (*Script for looking at
 effects of ablations on pre-translocation movements*), this data was
 processed in the same way as the general baseline data, and used to see if
 there were any effects of the sensory manipulations on general space use
 prior to translocation. **Tracking during Translocations**: The sheet
 **Translocation_Coordinates** contains geographic coordinates and
 timestamps of toad locations collected during radio tracking of toads
 after they were displaces 500 m or 1000 m from their outermost baseline
 coordinate. The toads were tracked either until they moved within 200 m of
 their nearest baseline location or 3 days had elapsed without them
 returning. In Part 1 of the R script, the 'adehabitatLT' package
 in R was used to generate "ltraj" objects for each toad to
 calculate spatial and temporal properties of their movements, including
 cumulative movement, daily movement, straightness index, homing
 duration/speed, and active homing duration/speed. The following colums are
 included in the sheet: * ID (unique identifier of each toad in experiment)
 * Condition (Release, Sac, and other location/behavior information) *
 Notes (additional observations) * Timestamp_E (Date/time of observation) *
 POINT_X (longitude of observation in UTM) * POINT_Y (latitude of
 observation in UTM) * Grp (either 500 or 1000 if not ablated, MAC/MAE for
 magnetoreception control/experimental, or OAC/OAE for olfaction
 control/experimental). The sheet **Transect_Correction** contains the
 coordinates of the  translocation release site and the outermost baseline
 reference point used to calculate translocation distance for each toad.
 These points were used to create trajectories ("ltraj" objects)
 to extract the angle of travel needed to reach home, which was used to
 normalize plots (in script Part 5: *Normalize trajectories and plot with
 origin being translocation release site*). The timestamps are arbitrary
 time points used to separate the two points for each toad: translocation
 release point is 2022-02-22 and baseline reference is 2022-05-13 or
 2022-05-14). * ID (unique identifier of each toad in experiment) *
 Timestamp (arbitrary time points to separate translocation release and
 baseline reference points from each other: translocation release point is
 2022-02-22 and baseline reference is 2022-05-13 or 2022-05-14) * x
 (longitude of specific point in UTM) * y (latitude of specific point in
 UTM) **Weather:** The sheet **Weather** contains daily weather data taken
 from the National Oceanic and Atmospheric Administration. The data
 recorded includes precipitation, maximum temperature, and minimum
 temperature from weather stations near each field site for any day a
 translocation experiment was occurring at that field site. Each row
 represents one day of data for one field site. This information was used
 to compare weather conditions between locations to understand if these
 conditions were affecting homing performance. Weather data is analyzed at
 the end of Part 1 of the R script. The following columns are included: *
 loc (field site) * Date (date of weather data) * PRCP (daily
 precipitation, reported in inches) * TMAX (maximum temperature in degrees
 Fahrenheit) * TMIN (minimum temperature in degrees Fahrenheit) **Neural
 activity data:** The remaining sheets are associated with the analysis of
 pS6 immunohistochemistry used to compare brain activity between homing,
 non-homing, and translocated toads. Brains were cryosectioned into 20
 micrometer sections and mounted on slides, stained with our
 immunohistochemistry protocol, and imaged using brightfield microscopy.
 The sheet **Brains** includes metadata about each extracted brain. The
 following columns are included: * Brain (numerical identifier assigned to
 brain) * Toad (unique identifier of each toad in experiment) * Distance
 (approximate distance translocated in meters: 500, 1000, or 0) * Condition
 (either Homing, Lost, Baseline, or Homing* if brain collected at late time
 point after toad homed) * Ablation (ablation code or None) * Extraction
 (date that brain was collected) * sodaz (0 if brain was not put in PBS
 with sodium azide for storage, 1 if it was) The sheet **BrainCounts_02**
 contains the count information, generated by manually counting
 pS6-positive cells in ImageJ. Nine sections across the AP axis were
 counted for each animal. Each row represents one brain region in one brain
 section, and contains the number of pS6-positive cells and the area of the
 brain region in that section. The following columns are included: * Brain
 (numerical identifier of brain) * Toad (unique identifier of each toad in
 experiment) * Slice (section of brain numbered 1-9, moving posterior along
 the AP axis for each toad) * Image (number referring to TIF file exported
 from LAS X microscope software) * Brain Region (contains abbreviation of
 brain region count being reported; Dp, dorsal pallium; Lp, lateral
 pallium; Mp, medial pallium; Ms, medial septum; Ls, lateral septum; Str,
 striatum) * Count (number of pS6 positive cells counted in Image J) * Area
 (area of brain region in micrometers squared) Part 4 of the R script deals
 with *Analysis of pS6 count data*. We used generalized linear mixed models
 (‘glmmTMB’  package in R) to test for differences in brain activity and
 its relation with behavior.  Models used a negative binomial distribution
 and best model fit was confirmed using the ‘DHARma’ package in R . Homing
 condition, brain region, and their interaction were the main effects
 predicting the number of pS6-positive cells. Toad ID was included as a
 random factor and brain region area was included as an offset variable to
 account for brain area size. We modeled differences in brain activity due
 to ablations separately using the same model parameters as above except
 that ablation was also included as a main effect. Post hoc pairwise
 contrasts (between homing condition groups and ablation treatments) were
 calculated with estimated marginal means using the “emmeans” package in R.
 To test if brain activity was related to either straightness or duration
 of homing, we performed linear mixed effects models using the “lme4”
 package, where pS6 count was the response variable and the homing
 descriptor (straightness or duration), brain region, and their interaction
 were fixed effects. Toad ID was a random effect and brain region area was
 an offset variable in the model. Linear mixed effects models were also
 performed within count data specific to each brain region, in which the
 response variable was pS6 counts within a given region, the homing
 descriptor was the fixed effect, Toad ID was a random effect, and region
 area was an offset variable.   An additional sheet, **olf_ct**, contains
 counts of pS6-positive cells from the the olfactory bulbs of toads that
 received olfaction ablations or sham olfaction ablations. Three sections
 of olfactory bulb were counted from each toad. For each section, a 300
 micrometer by 300 micrometer box on the edge of the granule cell layer was
 sampled for counting. This data was used to test the effectiveness of the
 olfactory ablation treatment in disrupting olfaction related neural
 activity by comparing counts in ablated and sham animals with a Wilcoxon
 Rank Sum Test. The following columns are included: * id (unique identifier
 of each toad in experiment) * slice (section of brain, 1-3, moving from
 anterior to posterior in the olfactory bulb) * count (number of pS6
 positive cells counted in 90000 micrometer squared box) * density (cell
 count divided by sample area in mm squared) ## Sharing/Access information
 Data was derived from the following sources: * Manually curated GPS
 coordinates gathered in ArcGIS Field Maps from February to May 2022 * pS6
 Count Data generated by manually counting pS6-positive cells in stained
 brain tissue using ImageJ * Daily weather station record from NOAA
 National Centers for Environmental Information Climate Datasets Online
 (CDO) * Menne, Matthew J., Imke Durre, Bryant Korzeniewski, Shelley
 McNeill, Kristy Thomas, Xungang Yin, Steven Anthony, Ron Ray, Russell S.
 Vose, Byron E.Gleason, and Tamara G. Houston (2012): Global Historical
 Climatology Network - Daily (GHCN-Daily), Version 3. [ZIPs:
 96707,96731,96712]. NOAA National Climatic Data Center.
 doi:10.7289/V5D21VHZ [Accessed 31 October 2024] * Matthew J. Menne, Imke
 Durre, Russell S. Vose, Byron E. Gleason, and Tamara G. Houston, 2012: An
 Overview of the Global Historical Climatology Network-Daily Database. J.
 Atmos. Oceanic Technol., 29, 897-910. doi:10.1175/JTECH-D-11-00103.1. ##
 Code/Software We performed all analysis in R Studio (v 2024.12.0+467,
 Posit Software, PBC, Sunnyvale, CA) running R (v 4.4.2, R Foundation for
 Statistical Computing, Vienna, Austria**).  \**The attached R script
 contains all code for analysis in the manuscript. All necessary packages
 are listed in the beginning of the code. The script references the csv
 datasheets that have been described. The script is split into 7 sections,
 some of which have been mentioned above: PART 1: Analysis of translocation
 related spatial activity (lines 71-612) PART 2: Analysis of Baseline
 Tracking Data (lines 613-760) PART 3: Script for looking at effects of
 ablations on pre-translocation movements (lines 761-923) PART 4: Analysis
 of pS6 count data (lines 924-1212) PART 5: Normalize trajectories and plot
 with origin being translocation release site (lines 1213-1408) PART 6:
 Plot Temporal aspects of trajectories (lines 1409-1453) PART 7:
 Visualizations (lines 1454-1787) In order to run this code, you must
 download the provided data-files and change the working directories and
 file paths throughout the code to the location where you are storing the
 data.  Here are all packages loaded in the script with version numbers
 used :  devtools (v 2.4.5), tidyr (v 1.3.0), ggplot2 (v 3.4.4), DHARMa (v
 0.4.6), emmeans (v 1.8.9), psych (v 2.3.9), lme4 (v 1.1-34), nlme (v
 3.1-162), effects (v 4.2-2), cowplot (v 1.1.1), interactions (1.1.5),
 ggpubr (v 0.6.0), gtsummary (v 1.7.2), mblm (v 0.12.1), rcompanion (v
 2.4.35), coda (v 0.19-4), rjags (v 4-14), BayesFactor (v 0.9.12-4.5),
 Bolstad (v 0.2-41), dplyr (v 1.1.4), trajr (v 1.5.0), adehabitatLT (v
 0.3.27), adehabitatHR (v 0.4.21), raster (v 3.6-26), OneR (v 2.2), chron
 (v 2.3-61), datetime (v 0.1.4), knitr (v 1.44), spdep (v 1.2-8), lubridate
 (v 1.9.3), ggforce (v 0.4.1), sp (v 2.1-1), sf (v 1.0-14), geosphere (v
 1.5-18), glmmTMB (v 1.1.8), ggsignif (v 0.6.4), car (v 3.1-2), MASS (v
 7.3-60), broom (v 1.0.5), lmerTest (v 3.1-3), circular (v 0.5-0), FSA (v
 0.9.5)