Sublethal responses to ammonia exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

Abstract

The delta smelt ( Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin Estuary in Northern California, which acts as an indicator of ecosystem health in its habitat range. Interrogative tools are required to successfully monitor effects of contaminants upon the delta smelt, and to research potential causes of population decline in this species. We used microarray technology to investigate genome-wide effects in fish exposed to ammonia; one of multiple contaminants arising from wastewater treatment plants and agricultural runoff. A 4-day exposure of 57-day old juveniles resulted in a total ammonium (NH 4 +–N) median lethal concentration (LC50) of 13 mg/L, and a corresponding un-ionized ammonia (NH 3) LC50 of 147 μg/L. Using the previously designed delta smelt microarray we assessed altered gene transcription in juveniles exposed to 10 mg/L NH 4 +–N from this 4-day exposure. Over half of the responding genes were associated with membrane integrity and function, however, neurological and muscular function was also affected. Amongst the notable pathways affected by ammonium exposure, directly associated with cellular membranes, are energy metabolism through oxidative phosphorylation, cellular responses to environmental stimuli, highlighted through signal transduction and molecular interactions, cellular processes encompassing transport and catabolism, along with cell motility, development, communication and cell death. To assess these impacts further, key genes were selected as potential biomarkers and investigated using quantitative PCR analysis on fish exposed to 2.5, 5, 10, 20 and 40 mg/L NH 4 +–N. Quantitative PCR results indicate biphasic responses, pivoting around the estimated no-observed effect concentration (NOEC; 5.0 mg/L NH 4 +–N) and below. Genes significantly affected by ammonia exposure include claudin-10, Keratin-15, Septin-3, Transmembrane protein 4, superfamily 4 (membrane), Tropomyosin, Myosin light chain, Calmodulin (muscular), Tubulin cofactor beta (neurological), Sirtuin-6 (development), and Rhesus associated type C glycoprotein 1 (gill- and skin-specific ammonium transporter). The quantitation of the ammonium transporter may highlight the capacity of delta smelt to contend with elevated levels of ammonia, the peak response of which may be indicative of short-term thresholds of tolerance. Our study supports the notion that exposure to ammonia results in cell membrane destabilization, potentially affecting membrane permeability, enhancing uptake and thus synergistic effects of multiple-contaminant exposure.