Effect of metal contamination on health of two Benthic fish species, Shorthorn Sculpin (<i>Myoxocephalus scorpius</i> Linnaeus, 1758) and Southern Sand Flathead (<i>Platycephalus bassensis</i> Cuvier, 1829)

<p dir="ltr">Environmental pollution is a global issue and metals are one of the most significant pollutants. Exposure to metals can cause adverse effects on fish health, leading to diseases and even death to fish. The effects of pollutants, such as metals, on the health of aquatic organisms, particularly fish health, have been evaluated using biomarkers. <br>The important purpose of biomarkers is to reflect the relationship between levels of toxicant exposure, contamination levels of tissues or organs, and adverse effects on biota. An effect or biological response at sub-organismic levels (including organ or tissue level, cellular level, and molecular level) of target organism usually occurs prior to one being detected at the population, community, or ecosystem level. Benthic fish species are generally used as indicator species for biomarker studies in various aquatic ecosystems. <br>Shorthorn sculpin (<i>Myoxocephalus scorpius</i>) has been used as a sentinel species for environmental monitoring, including heavy metal contamination from mining activities in the Arctic. The lead–zinc (Pb–Zn) mines in Greenland resulted in elevated environmental concentrations of heavy metals, especially Pb, which is a non-essential element, in local marine biota. Yet, the potential accumulation of Pb in fish organs and their effects on the health of sculpins observed in the field studies have not been validated in controlled laboratory. There are no reports on the impact of heavy metals on the expression of genes related to metal-related stress and immune response in shorthorn sculpin. <br>Southern sand flathead, <i>Platycephalus bassensis</i>, is a popular fish species with Tasmanian recreational fishers. Muscle melanisation is the presence of dark pigments in skeletal muscle of the fish, which can have a range of causes, including infections or excessive metal concentrations. Cases of muscle melanisation were reported in farmed and wild fish, including wild southern sand flathead. Significant metal concentrations, especially Zn, were found in the muscle samples from melanised sand flatheads, although it is yet unknown if the severity of the melanisation was due to metal pollution.<br>The aims of this thesis were to assess effects of metal pollution on the overall fish health of benthic fish species, including shorthorn sculpin and sand flathead, and to evaluate for those two species potential biomarkers of pollutant exposure for an environmental risk assessment. These aims were achieved by 1) determining whether the field-observed effects of Pb on shorthorn sculpin (<i>M. scorpius</i>), including changes in morphology of gills and liver, could be confirmed under controlled conditions, 2) assessing the potential of stress and immune-related gene expressions in shorthorn sculpin (<i>M. scorpius</i>) as biomarkers of Pb exposure, 3) assessing the health of the southern sand flathead (<i>P. bassensis</i>) from metal polluted area using gross morphology, histology, and transcriptomics, 4) assessing relationship between muscle melanisation severity, metal concentrations, and histology in organs of sand flathead (P. bassensis), and 5) identifying the underlying molecular markers of immune responses associated with different levels of muscle melanisation in sand flathead (<i>P. bassensis</i>). <br>The results from the short-term (28 days) exposure to environmentally relevant concentrations of dissolved Pb in sculpins suggested that not only liver, but blood and gills were suitable for assessing Pb accumulation and exposure. The results of histopathological examinations showed that chronic Pb exposure may contribute to liver necrosis, gill telangiectasis, and increased number of mucous cells in gills. Upregulation of hepatic genes, including <i>metallothionein (mt)</i> and <i>immunoglobulin M (igm)</i>, indicated that exposure to Pb concentrations similar to those reported in Greenland Pb–Zn mines could influence the mt and immune responses in shorthorn sculpins. A positive correlation observed between micro RNA (<i>mir</i>155) transcript levels and Pb liver levels suggested that transcripts of <i>mir</i>155 could be induced by metal stress. This is thus the first study to identify some candidate molecular markers in the shorthorn sculpins exposed to waterborne environmentally relevant Pb, suggesting <i>mt</i> and<i> igm</i> as potential molecular markers of exposure to be applied in future assessments of the marine environment near Arctic mining sites. The results also validated field observations for the effects of Pb on wild sculpin and contributed to the improved use of the shorthorn sculpin as sentinel species for monitoring contamination from Pb mines in the Arctic. <br>The sand flathead were sampled at Deceitful Cove, the Tamar Estuary, northern Tasmania, where the fish had a significant difference in the percentage surface area of melanised muscle and a greater prevalence of muscle melanisation of affected fish when compared to other locations in the Tamar Estuary. The fish fillets were checked for the presence of melanisation and were scored from 0-3 for melanisation based on the percentage surface area of melanised muscle. Elemental analyses showed that concentrations of Zn in muscle of melanised fish were significantly higher than in muscle of non-melanised fish. Histopathological investigations revealed that the intensity of infection with gill monogeneans, severity of gill telangiectasis, and the number of gill mucous cells/interlamellar unit were significantly greater in the melanised fish than those in non-melanised fish. The density of melanomacrophage centres (MMCs) and surface area of melanin in MMCs were significantly greater in anterior kidney and spleen of the highest score melanised fish (melanisation score 3). The powerful high-throughput sequencing (RNA sequencing; RNA?seq) method was used to identify genes in sand flathead and specific genes as potential biomarkers for immune response in wild sand flathead. Transcriptome profiling revealed biological processes (gene ontology) were enriched in anterior kidney and liver of melanised fish relative to non-melanised fish, including immune functions. A number of genes encoding proteins involved in adaptive immune response (such as tyrosine kinase), complement pathways, and antigen processing and presentation were differentially expressed in anterior kidney and liver. Overall, the results provide further insight into the relationship between gill parasites, histopathology, gene transcripts, and muscle melanisation. <br>In summary, this study focused on examining effects of metal pollution on fish health as well as identification and validation of potential biomarkers in benthic fish species (shorthorn sculpin and sand flathead). In general, the findings suggested that various gene transcripts, histology, and presence of parasites in gills potentially could serve as suitable biomarkers for environmental monitoring program.<br></p>