Gonad metabolomics and blood biochemical analysis reveal differences associated with testicular oocytes in wild largemouth bass (Micropterus salmoides)

Abstract

Adverse reproductive effects associated with gonadal intersex among freshwater fish could hold considerable implications for population sustainability. Presence of testicular oocytes (TO) is the most common form of intersex and is widespread among centrarchids (sunfishes) of North America and other freshwater teleosts. Placing TO within the toxicological context of adverse outcome pathways (AOPs) to assess ecological risk is a priority for ecotoxicologists due to the association of TO with harmful chemical exposure and adverse reproductive effects in some cases. However, key event relationships between EDC exposure, incidence of TO, and apical outcomes have yet to be fully elucidated - in part due to a lack of knowledge of relationships between intersex gonad physiology and fish health. Understanding the physiological status of intersex fish is critical to assess ecological risk, understand mechanisms of induction, and to establish biomarkers of intersex in fish. In the present study, features of gonad metabolite profiles associated with TO in largemouth bass (LMB, Micropterus salmoides) from an impoundment in Georgia (USA) were determined using GC-MS-based metabolomics. Clinical blood biochemical screens were used to evaluate markers of fish health associated with TO. Results suggest that physiological changes in energy expenditure as well as relatively 'feminized' gonad lipid and protein metabolism may be related to the occurrence of TO in male LMB, and highlight the need to understand relationships between intersex and physical stressors such as elevated temperature and hypoxia. These results provide novel insight to AOPs associated with TO and identify candidate analytes for biomarker discovery.

Keywords: Aquatic toxicology; Biomarkers; Ecotoxicogenomics; Ecotoxicology; Endocrine disruption; Intersex.

Fig. 1.

Receiver operating characteristic (ROC) curve and 95% confidence band for the ratio of alkaline phosphatase (ALP) and aspartate phosphatase (AST) activity (generalized log transformed) in blood plasma of male largemouth bass with high TO (> 20 testicular oocytes) and low TO (≤ 20 testicular oocytes; A). e.g., Curves passing through the upper left corner have 100% diagnostic accuracy. Boxplot representing differences between high and low TO groups in plasma ALP/AST ratio (B). The red dot on the ROC curve, as well as the red dotted line in the corresponding boxplot represents the optimal cutoff value.

Fig. 2.

Example gonad metabolomics total ion chromatogram from largemouth bass collected from impoundment in Northeast Georgia (USA) aligned and processed, along with select analyte annotations.

Fig. 3.

PLS-DA scores plot representing differences in individual gonad metabolite profiles of female (red) and male largemouth bass with varying levels of testicular oocytes (‘Low TO’ - males with low testicular oocyte count [≤ 20, blue]; ‘High TO’- males with high testicular oocyte count [> 20, green]) from a Georgia (USA) impoundment. Each point represents an individual fish, and distance between points represents overall differences in gonad metabolome.