Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Modern stromatolites represent ideal ecosystems to understand the biological processes required for the precipitation of carbonate due to their long evolutionary history and occurrence in a wide range of habitats. However, most of the prior molecular work on stromatolites has focused on understanding the taxonomic complexity and not fully elucidating the functional capabilities of these systems. Here, we begin to characterize the microbiome associated with stromatolites of Little Darby Island, The Bahamas using predictive metagenomics of the 16S rRNA gene coupled with direct whole shotgun sequencing. The metagenomic analysis of the Little Darby stromatolites revealed many shared taxa and core pathways associated with biologically induced carbonate precipitation, suggesting functional convergence within Bahamian stromatolites. A comparison of the Little Darby stromatolites with other lithifying microbial ecosystems also revealed that although factors, such as geographic location and salinity, do drive some differences within the population, there are extensive similarities within the microbial populations. These results suggest that for stromatolite formation, 'who' is in the community is not as critical as metabolic activities and environmental interactions. Together, these analyses help improve our understanding of the similarities among lithifying ecosystems and provide an important first step in characterizing the shared microbiome of modern stromatolites.

Performing gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no longterm harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.