DOI: 10.5061/dryad.bk3j9kdjr
title: The reproductive microbiome and maternal transmission via eggs in Sceloporus virgatus

description: Maternal transmission of microbes occurs across the animal kingdom and is
 vital for the  development and long-term health of offspring. The
 mechanisms of this transfer are most well studied in humans and other
 mammals, but are less well understood in egg-laying animals, especially in
 those with no parental care. Here we investigate the transfer of maternal
 microbes in Sceloporus virgatus, an oviparous spiny lizard. We compared
 three maternal tissue microbiomes – oviduct, cloaca, and intestine – to
 three offspring sample types: egg contents and eggshells on the day of
 oviposition, and hatchling intestinal tissue on the day of hatching. We
 found that dam ID is an important factor in hatchling microbiome
 composition, indicating that maternal transmission is occurring. The
 maternal cloacal and oviductal communities contribute to offspring
 microbiomes in all three sample types, but there was minimal influence of
 maternal intestinal microbes. This indicates that the maternal
 reproductive microbiome is more important for microbial inheritance than
 the gut microbiome, and that the tissue-level variation of the adult S.
 virgatus microbiome must develop as the hatchling matures. Despite
 differences between adult and hatchling communities, the offspring
 microbiome was still dominated by Enterobacteriaceae and Yersiniaceae,
 consistent with past studies of adult S. virgatus microbiomes.
description: Sequences of the 16s V4 regions were generated at IIDS Genomics
 and Bioinformatics Resources Core at the University of Idahoon the
 Illumina MiSeq platform. Detailed methodology can be found in the attached
 README file.
description: # The reproductive microbiome and maternal transmission via eggs in
 Sceloporus virgatus M. E. Bunker and S. L. Weiss University of Puget
 Sound, 1500 N. Warner Street, Tacoma, WA, USA 98416-1088 ## **Sample and
 Data collection** Ten gravid female *Sceloporus virgatu*s were collected
 by lasso near the Southwestern Research Station in Cochise County,
 Arizona, on 26-27 June 2022. Animals were kept in 23 x 15 cm cages
 sterilized with 70% ethanol and had sterile water available ad libitum
 until 29 June, when they were induced to oviposit with a 0.1mL injection
 of oxytocin. Eggs were collected as they were laid and placed into
 individual cups of sterile vermiculite. For each female, the first and
 last egg was punctured with sterile scissors and the contents and shells
 were stored in separate 1.5ml microcentrifuge tubes (total of n = 20 per
 sample type). The remaining eggs (n = 78) were incubated until hatching at
 30°C, either in sterile vermiculite or vermiculite treated with a single
 species of fungal spores (as part of a different study). On 30 June,
 females were euthanized with two injections of buffered tricaine
 methanesulfonate, followed by decapitation. Tissue samples were taken with
 heat-sterilized instruments from the cloaca, oviduct, and intestine.
 Oviduct and intestine were each sampled in two locations which were
 sequenced separately. Hatchlings were sacrificed within 24 hours of
 hatching by decapitation. The entire intestinal tract, from just below the
 stomach to the cloaca, was sampled together due to the small size of the
 intestine (the average mass of hatchling tissue samples was 3.5 mg).
 Sample were kept at -80C until they could be extracted and sequenced. 
 FastQC and MultiQC were utilized to determine a length cutoff of 260 bp
 (forward reads) and 190 bp (reverse reads). We processed the raw data
 using the DADA2 pipeline
 (). The Decontam
 package was used to remove contaminants, with a threshold of 0.1. 
 Further, any ASVs that had &lt; 27 reads total were removed from the
 datasets, as was any sample with &lt;500 reads after processing (9 samples
 were removed from the dataset for low reads, out of  193 samples total). 
 Read numbers were log transformed to account for differences in read
 depth. Phylogenetic trees were made using Phangorn and DECIPHER, based on
 this protocol: . All
 parameters in processing were chosen based on analysis of a mock
 community, which was processed in the same manner.  All processing and
 analyses were performed in R.  ## **Included Files:** MT_analysis.html:
 File generated from Rmarkdown file, including the data wrangling,
 statistics, and assumptions test performed for all analysis included in
 the manuscript. This HTML file includes the code used as well as the
 output generated from the code. This output can be re-created with RMD
 file included as software in this dataset, using the data files described
 below. All packaged used are included in the RMD file.  MT_meta.csv — Meta
 data for all samples included in the analysis. This includes all field
 collected data that were compared to the microbial community data. A blank
 cell indicates data point did not apply in that category, a “.” Indicates
 missing data. These data are: *animal.type *- category indicating
 development stage sample was taken from: mom = maternal adult , egg =  egg
 on the day of oviposition, hatchling = hatchling on the day of hatching.
 *broad.type* - Category of maternal tissue or hatchling sample type:
 mom.int = maternal intestine, ovi = maternal oviduct, mom.claoca =
 maternal cloaca, shell = portion of eggshell taken on day of oviposition,
 contents = entire contents of egg taken on day of oviposition, hatch.int =
 entire length of intestine taken from hatchling on day of hatching. 
 *egg.place *- position in the laying order (either first or last), only
 applied to egg samples taken on day of oviposition, cells for other
 samples types (including hatchling tissue and all maternal tissues) were
 intentionally left blank.  *tc *- toe clip number, used to permanently
 identify adult lizards. For offspring samples, tc is the dam ID. 
 *sample.mg* - mass of tissue samples in milligrams, does not apply to egg
 contents or eggshells, and cells were intentionally left blank.
 *treatment* - incubation environment, indicating fungal taxa or control
 (sterile). C =Control, A = *Aspergillus* sp, F = *Fusarium*, NR =
 *Neocosmospora rubicola,* PL = *Purpureocillium lilacinum*. Only applies
 to hatchling tissue samples, cells for other samples types (including
 eggshells, egg contents, and all maternal tissues) were intentionally left
 blank.  MT_counts.csv — Amplified sequence variants (ASVs, rows: ASV_XX),
 and recovered reads for each sample (columns), as determined by the DADA2
 pipeline (see manuscript methods section). A “0” indicates that no reads
 for that ASV were recovered from the sample.  MT_tax.csv— Taxonomy
 assigned to each ASV, determined by the DADA2 pipeline (see manuscript
 methods section). An NA indicates that the sequence could not be
 classified at that taxonomic level.  MT_tree — Phylogenetic tree of ASVs
 recovered from all samples (see manuscript methods section).  \**Other
 associated datasets: ** Raw Illumina files have been uploaded to NCBI and
 can be found here:  
 (bioproject ID: PRJNA1023543) Data were not derived from any other source.