title: The response of Isidorella newcombi to copper exposure: Using an integrated biological framework to interpret transcriptomic responses from RNA-seq analysis.
abstract: This study describes the transcriptomic response of the Australian endemic freshwater gastropod Isidorella newcombi exposed to 80±1μg/L of copper for 3days. Analysis of copper tissue concentration, lysosomal membrane destabilisation and RNA-seq were conducted. Copper tissue concentrations confirmed that copper was bioaccumulated by the snails. Increased lysosomal membrane destabilisation in the copper-exposed snails indicated that the snails were stressed as a result of the exposure. Both copper tissue concentrations and lysosomal destabilisation were significantly greater in snails exposed to copper. In order to interpret the RNA-seq data from an ecotoxicological perspective an integrated biological response model was developed that grouped transcriptomic responses into those associated with copper transport and storage, survival mechanisms and cell death. A conceptual model of expected transcriptomic changes resulting from the copper exposure was developed as a basis to assess transcriptomic responses. Transcriptomic changes were evident at all the three levels of the integrated biological response model. Despite lacking statistical significance, increased expression of the gene encoding copper transporting ATPase provided an indication of increased internal transport of copper. Increased expression of genes associated with endocytosis are associated with increased transport of copper to the lysosome for storage in a detoxified form. Survival mechanisms included metabolic depression and processes associated with cellular repair and recycling. There was transcriptomic evidence of increased cell death by apoptosis in the copper-exposed organisms. Increased apoptosis is supported by the increase in lysosomal membrane destabilisation in the copper-exposed snails. Transcriptomic changes relating to apoptosis, phagocytosis, protein degradation and the lysosome were evident and these processes can be linked to the degradation of post-apoptotic debris. The study identified contaminant specific transcriptomic markers as well as markers of general stress. From an ecotoxicological perspective, the use of a framework to group transcriptomic responses into those associated with copper transport, survival and cell death assisted with the complex process of interpretation of RNA-seq data. The broad adoption of such a framework in ecotoxicology studies would assist in comparison between studies and the identification of reliable transcriptomic markers of contaminant exposure and response.
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Rod.Ubrihien@canberra.edu.au.
authors.forename: Rodney P
authors.initials: RP
authors.lastname: Ubrihien
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Tariq.Ezaz@canberra.edu.au.
authors.forename: Tariq
authors.initials: T
authors.lastname: Ezaz
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Anne.Taylor@canberra.edu.au.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Taylor
authors.affiliation: NSW Department of Primary Industries, Yanco Agricultural Institute, Private Mail Bag, Yanco, NSW, 2703, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Australia. Electronic address: Mark.Stevens@dpi.nsw.gov.au.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Stevens
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Frank.Krikowa@canberra.edu.au.
authors.forename: Frank
authors.initials: F
authors.lastname: Krikowa
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Simon.Foster@canberra.edu.au.
authors.forename: Simon
authors.initials: S
authors.lastname: Foster
authors.affiliation: Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia. Electronic address: Bill.Maher@canberra.edu.au.
authors.forename: William A
authors.initials: WA
authors.lastname: Maher
mesh_terms: D000818:Animals; D003300:Copper / Q000378:metabolism / Q000633:toxicity*; D004781:Environmental Exposure / Q000032:analysis*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D056726:Genetic Association Studies; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D058977:Molecular Sequence Annotation; D017423:Sequence Analysis, RNA / Q000379:methods*; D012908:Snails / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D014874:Water Pollutants, Chemical; D003300:Copper
keywords: Bioaccumulation; Gastropod; Gene expression; Lysosomal membrane destabilisation; Mollusc