title: A novel and versatile flash-freezing approach for evaluating the health of Delta Smelt.
abstract: A common approach used to assess environmental impacts in aquatic environments is to measure indicators of stress (biomarkers) and condition of fish within ecosystems. Particularly in estuarine ecosystems with multiple stressors, it is often desirable to quantify a suite of biological endpoints that (1) reflect fish condition at several levels of biological organization and time scales and (2) are sensitive to a range of environmental stressors. However, established methods of preservation and processing of fish for specific endpoints are often incompatible. Here, we developed a novel flash-freezing approach for assessing the health of a small, sensitive fish, the endangered Delta Smelt (Hypomesus transpacificus) after collections from the San Francisco Estuary (SFE). We assess whether flash-freezing the entire fish ensures effective preservation of multiple tissues for subsequent biomarker analyses by comparing measurements of fresh to frozen tissue. Tissues included brain, gill, and liver for enzyme activity, kidney and spleen for pathogens, and gills, liver, and gonads for histopathology and reproduction. Although flash-freezing in liquid nitrogen altered the length, weight, and condition factor of Delta Smelt, the percent changes were small (<1.5%). Histological analyses of the cellular morphology of gills, liver, and gonads were similar between both methods. Freezing artefacts were observed in ovaries, but did not hinder the identification and interpretation of cell types and oocyte stages. Freezing did not alter bacterial isolation or the activities of ethoxyresorufin-0-deethylase (EROD) or acetylcholinesterase (AChE), but had a small, negative influence on sodium potassium adenosine triphosphatase (ATPase) activity. Thus, flash-freezing in the field is a versatile preservation method for Delta Smelt, allowing for multiple tissue collections and bioassays from an individual tiny fish exposed to a wide range of natural and anthropogenic stressors. Similar methodology may be applicable to other species for which a range of biological endpoints and histopathology data are needed.
authors.affiliation: Aquatic Health Program, School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, One Shields Avenue, Davis, CA 95616, USA. Electronic address: sjteh@ucdavis.edu.
authors.forename: Swee J
authors.initials: SJ
authors.lastname: Teh
authors.affiliation: Aquatic Health Program, School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Dolores V
authors.initials: DV
authors.lastname: Baxa
authors.affiliation: Aquatic Health Program, School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Hammock
authors.affiliation: Aquatic Health Program, School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Saikrithika A
authors.initials: SA
authors.lastname: Gandhi
authors.affiliation: Aquatic Health Program, School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Kurobe
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D005615:Freezing; D005880:Gills / Q000201:enzymology / Q000473:pathology; D006066:Gonads / Q000473:pathology; D008099:Liver / Q000473:pathology; D009865:Oocytes / Q000378:metabolism / Q000473:pathology; D036441:Osmeriformes / Q000254:growth & development / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D015415:Biomarkers; D019363:Cytochrome P-450 CYP1A1; D000110:Acetylcholinesterase; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Biomarker; Delta Smelt; Flash-freezing; Necropsy; San Francisco Estuary