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High throughput transcriptomics: A multi-species approach
Citation:
Flynn, K., A. Biales, D. Bencic, R. Flick, J. Martinson, Dan Villeneuve, K. Jensen, J. Cavallin, R. Hockett, T. Norberg-King, M. Le, K. Bush, K. Santana-Rodriguez, AND M. Hazemi. High throughput transcriptomics: A multi-species approach. EPA Board of Scientific Councilors (BOSC) Virtual Meeting, Duluth, MN, February 02 - 05, 2021. https://doi.org/10.23645/epacomptox.13623977
Impact/Purpose:
This presentation provides the CSS Board of Scientific Counselors (BOSC) examples of research being conducted by EPA ORD to development of high throughput transcriptomic-based assays with four ecologically relevant species (Pimephales promelas, Daphnia magna, Chironomus dilutus, and Raphidocelis subcapitata) focused on linking apical and transcriptomic responses. Specifically, the audience is the CSS Board of Scientific Counselors. However, as a public meeting it is also intended for those interested in the development and use of transcriptomic data in ecological risk assessments. There is interest by regulatory agencies (ex, USEPA and Environment and Climate Change Canada), international organizations (ex., OECD), and the regulated community in leveraging high throughput assays to increase the efficiency of chemical risk assessments. The current battery of assays is focused on mammalian toxicological pathways. This presentation will summarize recent efforts to develop a high throughput assays with ecologically relevant species. These types of assays offer the possibility to increase the efficiency of ecological risk assessment by providing a tool capable of screening large numbers of chemicals.
Description:
While the US Environmental Protection Agency’s (USEPA) implementation of high-throughput (HTP) screening, as represented by the ToxCast and Tox21 efforts, have largely focused on human health, there is a need for HTP assays that address pathways and physiology unique to aquatic organisms representing different trophic levels. An aim of the present work was to modify standard protocols and methods to allow rapid toxicity tests with small aquatic organisms to be conducted on 96-well plates. HTP assays were developed for four ecologically relevant and taxonomically diverse species, Pimephales promelas (fathead minnow), Daphnia magna (a freshwater crustacean), Chironomus dilutus (midge; an insect), and Raphidocelis subcapitata (a green algae). Across all four species, the assays consisted of 24-hour exposures in 96-deep well plates which provide 12 chemicals concentrations and 8 replicates per concentration on each plate. Lifestages were chosen to be amenable to limited volumes (1 ml per well), to show high baseline survival, and to provide enough tissue for downstream endpoints. Therefore, the final assays were initiated with 24-hour post-hatch fathead minnows, 72-hour old daphnids, 3rd instar midge, and algae in log-phase of growth. After exposure, data was collected that could be anchored to phenotypic responses directly applicable to standard aquatic toxicology assays including survival, behavior, and photopigment concentrations (specific to algae). In addition, whole transcriptome RNA-seq data was collected in parallel, providing an opportunity to anchor transcriptomic responses to these phenotypic responses. Preliminary results suggest that standard toxicity estimates like LC50’s are comparable in these 24-hour exposures conducted on 96-deep well plates to traditional toxicity designs. A plate of fathead minnows, daphnids, or midge can be manually loaded with one individual per well and exposure started in less than 45 minutes, providing an opportunity to efficiently screen large numbers of chemicals. Algal plates can be processed in even less time. Finally, efforts to calculate transcriptomic-based points-of-departure and compare them to traditionally derived points-of-departure have been initiated. Overall, these results suggest HTP assays with ecologically relevant species can provide useful toxicological information in an efficient and cost-effective manner. The contents of this abstract neither constitute nor necessarily reflect US EPA policy.
