DOI: 10.5061/dryad.3n5tb2rd0
title: Hormonal and neural correlates of care in active versus observing poison frog parents

description: The occasional reversal of sex-typical behavior suggests that many of the
 neural circuits underlying behavior are conserved between males and
 females and can be activated in response to the appropriate social
 condition or stimulus. Most poison frog species (Family Dendrobatidae)
 exhibit male uniparental care, but flexible compensation has been observed
 in some species, where females will take over parental care duties when
 males disappear. We investigated hormonal and neural correlates of
 sex-typical and sex-reversed parental care in a typically male uniparental
 species, the Dyeing Poison Frog (Dendrobates tinctorius). We first
 characterized hormone levels and whole brain gene expression across
 parental care stages during sex-typical care. Surprisingly, hormonal
 changes and brain gene expression differences associated with active
 parental behavior in males were mirrored in their non-caregiving female
 partners. To further explore the disconnect between neuroendocrine
 patterns and behavior, we characterized hormone levels and neural activity
 patterns in females performing sex-reversed parental care. In contrast to
 hormone and gene expression patterns, we found that patterns of neural
 activity were linked to the active performance of parental behavior, with
 sex-reversed tadpole transporting females exhibiting neural activity
 patterns more similar to those of transporting males than non-caregiving
 females. We suggest that parallels in hormones and brain gene expression
 in active and observing parents are related to females’ ability to
 flexibly take over parental care in the absence of their male partners.The
 occasional reversal of sex-typical behavior suggests that many of the
 neural circuits underlying behavior are conserved between males and
 females and can be activated in response to the appropriate social
 condition or stimulus. Most poison frog species (Family Dendrobatidae)
 exhibit male uniparental care, but flexible compensation has been observed
 in some species, where females will take over parental care duties when
 males disappear. We investigated hormonal and neural correlates of
 sex-typical and sex-reversed parental care in a typically male uniparental
 species, the Dyeing Poison Frog (Dendrobates tinctorius). We first
 characterized hormone levels and whole brain gene expression across
 parental care stages during sex-typical care. Surprisingly, hormonal
 changes and brain gene expression differences associated with active
 parental behavior in males were mirrored in their non-caregiving female
 partners. To further explore the disconnect between neuroendocrine
 patterns and behavior, we characterized hormone levels and neural activity
 patterns in females performing sex-reversed parental care. In contrast to
 hormone and gene expression patterns, we found that patterns of neural
 activity were linked to the active performance of parental behavior, with
 sex-reversed tadpole transporting females exhibiting neural activity
 patterns more similar to those of transporting males than non-caregiving
 females. We suggest that parallels in hormones and brain gene expression
 in active and observing parents are related to females’ ability to
 flexibly take over parental care in the absence of their male partners.
description: <i>Tissue collection</i>
 <em>D. tinctorius</em> poison frogs were housed in
 breeding pairs to ensure parental/partner identity. To control for
 potential effects of parental experience, we allowed all pairs to
 successfully rear at least one clutch from egg-laying through tadpole
 transport prior to the study. For the ‘no care’ group, we collected frog
 pairs between parental bouts when they were not caring for eggs or
 tadpoles (N=8 pairs for hormones, N=5 pairs for RNAseq). For the ‘egg
 care’ group, we collected frogs one week after they laid a clutch of eggs
 (N=7-10 pairs for hormones, N=5 pairs for RNAseq). We collected only frogs
 that were caring for eggs, as evidenced by the healthy development of
 embryos. For the ‘tadpole transport’ group, we checked daily for transport
 behavior and collected tissue when we found frogs actively transporting
 (N=7-8 pairs for hormones, N=5 pairs for RNAseq). For all behavioral
 groups we simultaneously collected tissue from parental males and their
 non-caregiving female partners. <i>Library
 construction and sequencing</i> Flash frozen
 brains were placed in Trizol (Life Technologies, Grand Island, NY) and RNA
 was extracted according to manufacturer instructions. RNA quality was
 confirmed on an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) where all
 samples had a RIN (RNA Integrity Number) score &gt;7; one sample (a
 male transporter) had a RIN score below 5 and was excluded from the
 further tissue processing and gene expression analysis. Poly-adenylated
 RNA was isolated from each sample using the NEXTflex PolyA Bead kit (Bioo
 Scientific, Austin, TX, USA) following manufacturer guidelines. Lack of
 contaminating ribosomal RNA was confirmed using an Agilent 2100
 Bioanalyzer. Strand specific libraries for each sample were prepared using
 the dUTP NEXTflex RNAseq kit (Bioo Scientific), which includes a magnetic
 bead-based size selection. 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),
 both according to manufacturer instructions. Libraries were sequenced on
 an Illumina HiSeq 2500 at the Bauer Sequencing Core at Harvard
 University. <i>Transcriptome construction and
 annotation</i> We constructed <i>de
 novo</i> transcriptomes using a pipeline established in our
 laboratory (Caty
 et al., 2019). We amended sequencing errors using
 Rcorrector (Song and Florea, 2015) and trimmed raw reads using Trim
 Galore! (Babraham Bioinformatics, Babraham Institute) to remove Illumina
 adapters and restrict all reads to high-quality sequence. Following
 developer recommendations, we used a quality score of 33, a stringency of
 5, and a minimum read length of 36 bp. Corrected, trimmed reads from all
 individuals were pooled prior to <i>de novo</i> transcriptome
 construction with Trinity (Grabherr et al, 2011;  Haas et al, 2013). Our
 initial assembly contained 2,655,485 isoforms representing 2,272,431
 presumptive genes. We filtered the raw assembly using
 several approaches. We clustered overlapping contigs using CD-HIT-EST
 (http://weizhongli-lab.org/cd-hit/) and removed contigs
 that were smaller than 250 bp following clustering, leaving 1,479,791
 total contigs. Preliminary annotations of this and other assemblies from
 closely related poison frog species revealed a high percentage of
 contaminant (i.e. non-vertebrate) sequences. To remove non-vertebrate
 (i.e. bacterial, fungal and other pathogen/parasite) contaminants, we
 annotated sequences using blastx queries with default parameters and an
 e-value cutoff of 10<sup>-10</sup> against the SwissProt
 database (www.uniprot.org) and retained only those contigs with
 annotations to known vertebrate genes. Our final assembly contained 68,399
 contigs representing 35,058 genes. Additional assembly annotation was done
 using Trinotate (Bryant et al, 2017) and assembly completeness was
 assessed using BUSCO (Simão et al, 2015). Final assembly completeness was
 estimated at 87% (37% duplicated; 3.5% fragmented, 9.0% missing). All
 high-powered computing for transcriptome assembly and filtering was
 performed on the Odyssey computing cluster supported by the FAS Science
 Division Research Computing Group at Harvard University.