Methods for sampling and manipulating the microbiome of reptile eggs at oviposition

Effective methods for microbiome sampling will allow efficient microbial recovery and lysis from host tissues or surface types. Although most vertebrates are oviparous, little is known about the taxonomic and functional diversity of microbiota that colonize and develop on eggshells, particularly on eggs of non-avian reptiles. Our primary goal was to develop methods to effectively sample and manipulate the eggshell microbiome of reptiles. First, to identify effective microbiome sampling methods, we compared the efficacy of whole-egg sonication versus swabbing of lizard (Anolis sagrei) eggs and elucidated bacterial taxa using 16S rRNA gene sequencing. Second, to determine an effective approach to manipulate the eggshell microbiome while minimizing egg mortality, we assessed the use of a broad-spectrum antimicrobial and a medium typically used to incubate microbes (i.e., tryptic soy broth) on eggshell bacterial community abundance and egg survival. We found that eggshell swabbing provided similar alpha diversity measurements compared to whole-egg sonication, but different bacterial community structures based on sampling method. Sonication also reduced egg survival and hatchling size. Bacterial community load was reduced using a broad-spectrum antimicrobial solution, with little impact on egg survival. However, antimicrobial eggshell treatment increased hatchling size. Overall, our results provide useful guidelines for future experiments that examine the source and function of the eggshell microbiome, which will help to advance our growing knowledge about host-microbe interactions in reptile species.