Data from: Neural and sensory basis of homing behavior in the invasive cane toad, Rhinella marina
Data files
Feb 03, 2025 version files 439.09 KB
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Ablation_Baseline.csv
72.54 KB
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Baseline.csv
122.59 KB
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BrainCounts_02.csv
126.36 KB
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Brains.csv
2.40 KB
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HI_2022_Toad_List.csv
11.54 KB
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olf_ct.csv
1.23 KB
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README.md
15.77 KB
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Toads.csv
10.92 KB
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Transect_Correction.csv
5.25 KB
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Translocation_Coordinates.csv
60.22 KB
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TransToadInfo.csv
8.12 KB
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Weather.csv
2.15 KB
Abstract
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.
README: 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)
Methods
This manuscript includes datasets related to cane toad (Rhinella marina) 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 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.