The Impacts of Sublethal Nitrite Exposure on Goldfish (Carassius auratus) Microbiomes

Abstract

Around 50% of fish consumption comes from aquaculture. An increase in world population and food demands requires fish production to dramatically increase over the next decade. One of the major obstacles of increasing aquaculture production is disease. Thus, a key aspect of aquaculture research is understanding immune health and the spread of disease within farmed populations and into wild stocks. A major portal for pathogen entry in fish is the nose; nasal infections tend to be severe and result in damage to the olfactory and nervous systems. Immune health is impacted by numerous systems including the microbiome. The microbiome trains the immune system, provides a barrier for pathogens, and helps maintain homeostasis. Therefore, dysbiosis, a disruption of the microbiome due to changes in diet, exposure to pollutants, antibiotic consumption, and physiological stress, can contribute to disease. One common pollutant in water is agricultural runoff containing nitrates and nitrites, which are known to cause stress in fish and can be deadly in some concentrations. However, it is unknown how nitrites impact the microbiomes of sensitive tissues and how these changes impact immune health. I hypothesize that increased nitrite concentrations will cause dysbiosis of the microbiome resulting in changing abundance and diversity of the microbiome. This work aims to determine the impacts of sublethal levels of nitrite on the nasal microbiome and compare it to the microbiomes of the gills and gut. For 2 months goldfish (10 fish/30L tank) were held under various nitrite concentrations, 0.0mM (control), 0.01mM, 0.1mM, and 1.0mM. The system utilized continuous aerated water flow at 24°C to maintain the desired concentrations of nitrite. Tissue samples were collected from each fish with water collected to serve as a control to determine the host‐specific microbiome. The DNA from the tissues were extracted, amplified, and prepared as a library for sequencing using 16S rRNA gene primers for Illumina MiSeq. The sequences were analyzed using the DADA2 pipeline in R and MaAsLin from Galaxy. The gill microbiome showed no significant changes in abundance compared to the control treatment. The nose microbiome appears to show an increase in diversity compared to the control microbiome; however, these changes were not determined to be significant. Both the gut and water microbiomes showed significant changes in abundance compared to their control levels, with the gut microbiome changing the most. Sublethal levels of nitrite exposure significantly increase the diversity of the microbial communities of the water and the gut microbiomes. There are also some indications that the nose and gill microbiomes are impacted, however, small sample sizes could be obscuring the changes. These impacts imply that sublethal nitrite concentrations can cause dysbiosis of the fish microbiomes and could have negative impacts of fish health.