Chelated copper and copper sulfate are used to prevent parasitic infections and excessive algal growth. Studies have shown that copper treatment can alter the fish skin microbiota by encouraging growth of microorganisms with metal resistance mechanisms and causing an increase in the abundance of opportunistic pathogens. However, none of these studies conducted environmental microbiome analysis or incorporated metagenomic shotgun sequencing to explore functional diversity of the microorganisms. This is an important gap to fill because metal resistance genes (MRGs) and antibiotic resistance genes (ARGs) are often co-selected in many environments including those influenced by agriculture and aquaculture runoff . Copper treatment leading to increases in microorganisms with metal resistance mechanisms may cause increases in microorganisms with antibiotic resistance mechanisms as well. Metal e fflux systems can provide cross-resistance to antibiotics since they can also extrude the antibiotics from the cell. In addition, co-selection occurs due to MRGs and ARGs being located next to each other on a mobile genetic element (genetic cross-resistance), which is capable of being transferred between bacteria via horizontal gene transfer. Determining whether this co-selection occurs is significant because increases in ARGs or opportunistic pathogens due to copper treatment can have negative implications for fish health and survival.
The study presented here was an opportunistic sampling of a tank (12,870L ) containing twenty Florida pompano ( Trachinotus carolinus) after a mortality with confirmed Amyloodinium ocellatum infection. Triplicate biofilm samples for DNA extraction along with water samples for copper analysis were taken during nine time points before and after chelated copper treatment. P urified DNA extracts were sent to GeneWiz (South Plainfield, NJ) for metagenomic shotgun sequencing (Ilumina HiSeq 2500) . DNA sequences were quality filtered, assembled, binned, annotated, and quantified. Taxonomic and functional diversity was analyzed with a focus on exploring the relationships between the abundance of opportunistic pathogens, ARGs, and MRGs in relation to copper concentrations. Water copper concentrations were measured on a Perkin Elmer 8300 inductively coupled plasma optical emission spectrometer .
We hypothesized that: 1) the microbial beta and alpha diversity at lower copper levels will be more statistically similar to the pre-treatment diversity than to the diversity associated with higher levels of copper, 2) there will be a positive correlation between abundances of opportunistic pathogens, ARGs/MRGs, and the copper levels . This study will help establish potential risks associated with copper treatment due to changes in the functional and taxonomic diversity of the biofilm microbiome. Our findings will also inform project design of a more controlled, replicated study to further explore these risks. U nderstanding the risks associated with copper treatment will allow farmers to take appropriate action to mitigate the effects on fish health and survival.