DOI: 10.5061/dryad.h9w0vt4s2
title: Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor

description: The ways in which animals sense the world changes throughout development.
 For example, young of many species have limited visual capabilities, but
 still make social decisions, likely based on information gathered through
 other sensory modalities. Poison frog tadpoles display complex social
 behaviors that have been suggested to rely on vision despite a century of
 research indicating tadpoles have poorly-developed visual systems relative
 to adults. Alternatively, other sensory modalities, such as the lateral
 line system, are functional at hatching in frogs and may guide social
 decisions while other sensory systems mature. Here, we examined
 development of the mechanosensory lateral line and visual systems in
 tadpoles of the mimic poison frog (Ranitomeya imitator) that use
 vibrational begging displays to stimulate egg feeding from their
 mothers. We found that tadpoles hatch with a fully developed
 lateral line system. While begging behavior increases with development,
 ablating the lateral line system inhibited begging in pre-metamorphic
 tadpoles, but not in metamorphic tadpoles. We also found that the increase
 in begging and decrease in reliance on the lateral line co-occurs with
 increased retinal neural activity and gene expression associated with eye
 development. Using the neural tracer neurobiotin, we found that axonal
 innervations from the eye to the brain proliferate during metamorphosis,
 with little retinotectal connections in recently-hatched tadpoles. We then
 tested visual function in a phototaxis assay and found tadpoles prefer
 darker environments. The strength of this preference increased with
 developmental stage, but eyes were not required for this behavior,
 possibly indicating a role for the pineal gland. Together, these data
 suggest that tadpoles rely on different sensory modalities for social
 interactions across development and that the development of sensory
 systems in socially complex poison frog tadpoles is similar to that of
 other frog species.
description: <strong><em>Ranitomeya imitator</em> tadpole
 transcriptome:</strong> Thirty <em>R. imitator</em>
 tadpoles in an unrelated study were anesthetized with benzocaine and
 euthanized by cervical transection. Eyes, brains, and intestines were
 placed in RNAlater. RNA was extracted from each tissue using Trizol
 (ThermoFisher, Waltham, MA, USA) according to manufacturer instructions.
 Poly-adenylated RNA was isolated from each sample using the NEXTflex PolyA
 Bead kit (Bioo Scientific, Austin, TX, USA) according to manufacturer
 instructions. Strand-specific libraries for each sample were prepared
 using the dUTP NEXTflex RNAseq kit (Bioo Scientific). Libraries were
 pooled in equimolar amounts after library quantification using both
 quantitative PCR with the KAPA Library Quantification Kit (KAPA
 Biosystems, Wilmington, MA, USA) and the fluorometric Qubit dsDNA high
 sensitivity assay kit (Life Technologies, Carlsbad, CA, USA), both
 according to manufacturer instructions. Libraries were sequenced on an
 Illumina HiSeq 2500 over a full flow cell to obtain 1,286,107,683 paired
 end 250 bp reads.  We used Trinity to de novo assemble the <em>R.
 imitator</em> tadpole transcriptome (parameters: --seqType fq
 --SS_lib_type RF --normalize_reads --trimmomatic --full_cleanup). We used
 blastx to compare contigs in the assembly to proteins in the Uniprot
 Swiss-Prot database (e-value threshold of 1e-5) and retained contigs with
 a match to a protein in this database. As we were specifically interested
 in frog transcripts, we removed contigs that had homology to
 non-vertebrate proteins in the Uniprot Swiss-Prot database, including
 microbes, nematodes, and arthropods. Our final <em>R.
 imitator</em> tadpole draft assembly contained 129,863 contigs
 (N50=499 bp). We then assessed the completeness of this filtered assembly
 by examining vertebrate ortholog representation using BUSCO v.4.1.4
 (Benchmarking Universal Single-Copy Orthologs). BUSCO estimated our
 assembly’s completeness to be at 87% with 824 complete single-copy BUSCOS,
 2092 complete and duplicated BUSCOs, 271 fragmented BUSCOs, and 167
 missing BUSCOs out of 3354 total BUSCO groups searched. We annotated the
 transcriptome using Trinotate. 
 <strong>PhosphoTRAP: </strong>RNAseq is performed on
 the total (TOT) input RNA sample and the immunoprecipitated (IP) sample
 that. RNA samples were purified using a SMART-Seq v4 Ultra Low Input RNA
 Kit for Sequencing (Takara, Mountain View, CA, USA), followed by library
 preparation using the Nextera XT DNA Library Prep kit (Illumina, San
 Diego, CA, USA), both according to manufactures’ protocols. Pooled
 equimolar library samples were run on an Illumina Hi-Seq 2500. The R
 script for phosphoTRAP analysis is included. Illumina reads were aligned
 to a <em>R. imitator</em> transcriptome. Count data were
 generated using DESeq2 and analyzed in R using paired t-tests on each
 transcript between the TOT and IP samples. Fold changes were also
 calculated for each transcript as log2 (IP count / TOT count).
 Differentially expressed genes were defined as having a p-value under 0.05
 and a fold change greater than 1.5 in either direction. We chose to use
 paired t-tests of transcripts within each group because this better
 reflects changes in expression associated with begging and reduces the
 variation present due to intrinsic variables (developmental stage, hunger,
 etc) that affect total count data.  <strong>Video
 of light preference behavior trial:</strong> Example video is
 included of a light preference behavior trial. Tadpole were allowed to
 acclimate in the chamber for ∼10 min. The small petri dish (tadpole arena)
 was filled with 40 ml of frog water. Tadpoles were transferred to the
 middle of the arena and behaviors were recorded for 3 minutes. We then
 flipped the light arena and recorded for an additional 3 minutes. Behavior
 was recorded from above with a GoPro Hero 6 camera.
description: # Tadpoles rely on mechanosensory stimuli for communication when visual
 capabilities are poor
 [https://doi.org/10.5061/dryad.h9w0vt4s2](https://doi.org/10.5061/dryad.h9w0vt4s2) ## Description of the data and file structure Supplemental Excel File contains output from the phosphoTRAP analysis, including the count matrix output from DESeq2, a list of significantly enriched or depleted transcripts in begging and non-begging tadpoles, and a GO enrichment analysis on transcripts depleted in begging tadpoles. This file can be viewed in Excel or Google Sheets. An *R. imitator* transcriptome is included. This was de novo assembled from Illumina reads obtained from sequencing RNA from eyes, brain, and intestines of thirty R. imitator tadpoles. This can be viewed with a text editor. The included R script contains the R code used in the phosphoTRAP analysis. This can be viewed or used in R. Required packages include glmmTMB, dplyr, stringr, Biostrings, and ggplot2. The video includes an example of a tadpole light preference behavior trial. This MP4 files can be viewed from common video software such as QuickTime Player. ## Sharing/Access information Links to other publicly accessible locations of the data: * Sequencing data is available on NCBI (Accession No. PRJNA1124168) * [https://www.protocols.io/view/a-simple-phototaxis-assay-for-aquatic-larvae-x54v9p294g3e/v1](https://www.protocols.io/view/a-simple-phototaxis-assay-for-aquatic-larvae-x54v9p294g3e/v1) * [https://doi.org/10.1016/j.ydbio.2024.05.006](https://doi.org/10.1016/j.ydbio.2024.05.006 "Persistent link using digital object identifier") ## Code/Software An R script file is included that was used to analyze phosphoTRAP data. This script was run in R (version 4.0.2).