The reproductive microbiome and maternal transmission via eggs in Sceloporus virgatus
Data files
Feb 05, 2024 version files 2.93 MB
Abstract
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.
README: 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 (https://benjjneb.github.io/dada2/tutorial.html). The Decontam package was used to remove contaminants, with a threshold of 0.1. Further, any ASVs that had < 27 reads total were removed from the datasets, as was any sample with <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 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: http://www.ncbi.nlm.nih.gov/bioproject/1023543 (bioproject ID: PRJNA1023543)
Data were not derived from any other source.
Methods
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.