DOI: 10.5061/dryad.n8pk0p369
title: Enzyme gene expression in house sparrow populations

description: Phenotypic plasticity is a major mechanism whereby organisms adjust their
 traits within generations to complement environmental
 conditions.  In the context of range expansions, plasticity is
 thought to be especially important, as plastic changes in traits can lead
 to rapid adaptation.  For these reasons, there has been growing
 interest in the roles of molecular epigenetic processes in the context of
 range expansions.  One epigenetic process in particular, DNA
 methylation, enables organisms to adjust gene expression contingent on the
 environment, which probably explains why it has played a role in some
 invasions.  Nevertheless, we still know very little about how
 methylation is regulated in wildlife, especially the expression of the
 enzymes responsible for altering methyl marks on the genome.  The
 most important forms of these enzymes in vertebrates are DNA
 methyltransferase 1, which largely maintains existing methyl marks, DNA
 methyltransferase 3, which creates most de novo methyl marks, and TET2,
 which is a major demethylator of CpG motifs, genomic regions where most
 methyl marks occur.  In this study, we compared the expression of
 these genes in three different tissues (i.e., gut, liver, and spleen) of
 house sparrows (Passer domesticus) from 9 locations. 
 Some populations derived from the native range of the species (i.e.,
 Israel, the Netherlands, Norway, Spain, and Vietnam), whereas others came
 from areas where birds were introduced &lt;150 years ago (i.e.,
 Australia, Canada, New Zealand, and Senegal).  We hypothesized
 that non-native birds and/or birds from sites with comparatively
 unpredictable climates would express the highest levels of all three
 genes.  We found, however, that DNMT expression differences,
 while extensive, were reversed from predictions: Sparrows from the native
 range and from areas with more stable temperatures expressed more of all
 three genes.  Surprisingly, too, expression of all three genes
 was strongly correlated among countries and within individuals. 
 Our results reveal no simple role for these enzymes in range expansions,
 but they do indicate appreciable among and within-population variation,
 which we hope motivates more detailed investigations of these enzymes in
 other wildlife.
description: <strong>Bird capture, husbandry, and tissue
 collection:</strong> We captured adult house
 sparrows using mist nets from sunrise until ~1100 at each location (Table
 1). Upon capture, we measured wing chord (to the nearest 1 mm), tarsus
 length (to the nearest 1 mm), and body mass (to the nearest 0.1 g). We
 also collected approximately 50 µl of blood from the brachial vein of each
 bird, which was stored in 300 µl of DNA/RNA Shield (Zymo R1100-50).
 Immediately after, each bird was injected subcutaneously with 100 µl of 1
 mg ml⁻¹ LPS (from <em>E. coli</em> 055; Fisher L4005) in
 sterile saline over the breast muscle. Birds were housed individually in
 wire songbird cages (approx. 35.6 x 40.6 x 44.5 cm) with food and water
 provided <em>ad libitum</em>, while maintaining visual and
 vocal contact. Forty-eight hours post-injection, between 0700 and 1000,
 birds were euthanized via isoflurane overdose followed by rapid
 decapitation. We collected liver, spleen, and gut samples in 500 µl of
 DNA/RNA Shield, and all samples were stored at -80°C until further
 analysis. We chose these tissues in particular because the goal of the
 larger project for which we collected sparrows involves epigenetic
 regulation of immune gene expression; these tissues are among the most
 active lymphoid tissues in the body.  All animal procedures complied with
 local ethical guidelines, approved by the USF IACUC (IS00011653) and
 relevant authorities in the countries of capture. Export and import of
 animal tissues followed all relevant U.S. regulations, including
 USDA-APHIS permits. <strong>RNA extraction and
 gene expression analyses:</strong> We extracted
 RNA from liver, gut, and spleen tissue samples of each sparrow using a
 standard phenol: chloroform protocol (Sambrook and Russell, 2012). Reverse
 transcription was carried out using the iScript cDNA Synthesis kit
 (Bio-Rad 1708891) according to the manufacturer’s instructions. We then
 quantified the absolute copy numbers of DNMT1, DNMT3, and  TET2 using
 droplet digital PCR (ddPCR). Each ddPCR reaction contained 5 µl ddPCR
 Multiplex Supermix (12005909, Bio-Rad), 2.25 µl of forward and reverse
 primers (10 µM), 0.63 µl of probe FAM, 0.63 µl of probe HEX, and 0.63 µl
 of FAM + HEX probe mixture (for 50% FAM + HEX, 0.31 µl of each), and 1 µl
 of cDNA sample (3500 ng/µl). The reactions were run on a C1000 Touch™
 Thermal Cycler with a 96–Deep Well Reaction Module (1851197, Bio-Rad).
 After amplification, droplets were separated and analyzed as positive
 (containing the target sequence) or negative (without the target sequence)
 using the QXDx Droplet Reader (12008020, Bio-Rad). Expression data were
 analyzed using QuantaSoft™ Analysis Pro software (version 1.05).
description: # Enzyme gene expression in house sparrow populations
 [https://doi.org/10.5061/dryad.n8pk0p369](https://doi.org/10.5061/dryad.n8pk0p369) ## Description of the data and file structure ### Files and variables #### File: DNMTdataforDryad.xlsx For R code used on these data, see R_Code__enzyme_gene_expression_in_house_sparrow_populations.txt **Description:**  ##### Variables * Band = individual identifier for a bird * Country = location of capture * pop.type = category of introduction history (native or non-native) * latitude = location on globe in degrees and minutes * genetic group = ancestry based on assignments implicated by Ravinet et al., 2018 * altitudem = in meters * urbanization = percent of habitat experiencing urbanization within 10km of location where a bird was caught * tempP = Colwell's index for temperature predictability * prep = Colwell's index for precipitation predictability * Gex = gene expression * LogGex = log10-transformed gene expression * bodymass0 = body mass of a bird at the time of capture in grams * wing chord = wing chord of a bird at capture in mm * sex = sex of an individual bird R_Code__enzyme_gene_expression_in_house_sparrow_populations.txt: This code analyzes factors affecting gene expression across tissues, genes, and populations using mixed-effects models, estimating fixed effects and random variance components (band and country), and exploring multivariate correlations across three genes. It also visualizes model results and correlation structures using `ggplot2` and `patchwork`