Ecology and detection of harmful freshwater fish ciliate parasites Chilodonella spp. in aquaculture

Freshwater fish farming contributes to more than two thirds of global aquaculture production. This rapidly growing agribusiness can be seriously compromised by diseases which can constrain industry development and profitability. In fact, disease problems account for forty percent of global aquaculture production loss. Parasitic ciliates are one of the largest causes of production loss in aquaculture and evidence suggests that they serve as the first portal of entry for many secondary infections in farmed fishes. Ciliate protozoan species of the genus Chilodonella are problematic and ubiquitous in global freshwater aquaculture. While Chilodonella spp. are important components of the 'free-living' fauna of freshwater ecosystems, some species have been implicated in outbreaks resulting in fish mortalities, particularly in high density aquaculture. A comprehensive review on the impacts and current status of parasitic Chilodonella spp. in freshwater fish aquaculture is presented in Chapter 2. Limited information exists on the ecology, infection dynamics and potential triggers leading to outbreaks of Chilodonella spp. in fish production. To assist with preventative management practices and sustainability of freshwater fish aquaculture, this research addressed key knowledge gaps related to the biology, ecology, geographic distribution, and identity of harmful Chilodonella spp. in Australia and examined new non-invasive diagnostic methods. Collectively, the research reported in this thesis provides fundamental baseline understanding of Chilodonella spp. ecology and their detection in fish aquaculture. While Chilodonella piscicola ((Zacharias, 1894; syn. C. cyprini (see Moroff, 1902)) and Chilodonella hexasticha (Kiernik, 1909) have been reported to globally infect wild and farmed fishes, the diversity of Chilodonella spp. parasitising Australian freshwater farmed fishes was unknown prior to this study. Chilodonella cells were isolated from infected barramundi, Lates calcarifer (Bloch, 1790) and Murray cod, Maccullochella peelii (Mitchell, 1838) from fish farms in tropical north Queensland (QLD), temperate Victoria (VIC) and New South Wales (NSW) for genetic and morphological analysis (Chapter 3). Parasites were stained and measured for morphological description and comparative phylogenetic analyses were performed using three markers: ITS1-5.8S-ITS2, small subunit (SSU) rRNA and the mitochondrial small subunit (mtSSU) rDNA marker. However, only mtSSU rDNA revealed significant genetic variation between Chilodonella species. Morphological analyses revealed four distinct morphotypes of Chilodonella infecting farmed barramundi and Murray cod. However, phylogenetic analyses detected only three distinct genotypes, with the morphotypes putatively identified as C. hexasticha and C. piscicola sharing 100% sequence identity. This suggests that Australian isolates identified as C. hexasticha or C. piscicola likely represent the same species and exhibit marked phenotypic plasticity. Ciliate protozoans cause rapid epidemic events with little or no warning, resulting in mortalities and considerable economic losses in affected farms. Changes in environmental conditions (temperature, sunlight, oxygen levels and pH) have been associated with changes in ciliate population numbers as these conditions may affect life cycles, reproduction, and their ability to obtain food from the environment. To identify and treat protozoan infections prior to harmful outbreaks, novel methods to determine which combination of environmental factors can trigger outbreaks were applied. Environmental DNA (eDNA) is a technique suited to rapidly assess the background presence of pathogens in water in fish farms, thereby providing managers with critical information on pathogens' threshold, which can be used to mitigate disease threats. In Chapter 4, the ciliate protozoan Chilodonella hexasticha was used as a model to examine the relationship between environmental DNA of C. hexasticha, critical water parameters and the occurrence of disease outbreaks on a commercial barramundi farm. Production pond water was sampled monthly over a one-year timeframe and a qPCR assay based on the SSU-rDNA gene was used to monitor the abundance (SSU-rDNA copies/μl) of C. hexasticha. Increased C. hexasticha eDNA levels correlated with occurrence of subsequent fish mortality events (r = 0.402; P < 0.001), with smaller fish more prone to epidemics (r = - 0.189; P < 0.05). However, there was no correlation between water quality parameters (rainfall, water temperature and dissolved oxygen) and parasite abundance, although there were significantly more fish mortalities observed during the warmer, wetter monsoonal season compared to the cooler, dry season (1,280 vs. 135 total fish mortalities, respectively; P < 0.05). These findings demonstrate that eDNA based techniques have the potential to be used on farms as a management tool to rapidly assess parasite loads in water and minimize the risk of disease outbreaks in aquaculture systems. The management of ciliate protozoans in aquaculture ponds is complex. Ciliates naturally consume bacteria, algae and other organic or cellular material (e.g. decomposing eukaryote cells) and constantly interact with other microorganisms in the aquatic environment. The association and interaction among different microorganisms in finfish aquaculture systems are rarely explored. In Chapter 5 the microbiome profile from ponds and barramundi gills were characterised and the relationship of this fauna with C. hexasticha abundance and fish mortalities were explored. Water samples from four barramundi ponds with a history of C. hexasticha fish infections and twenty infected fish were collected from October 2013 to September 2014. Presence of C. hexasticha infecting fish gills was confirmed by microscopic and morphological analyses. The abundance of C. hexasticha in water samples was estimated by SSU-rDNA qPCR and the relative abundance of bacterial communities from water and gills was estimated by deep amplicon sequencing of the V3 region of the 16S rRNA gene. Additionally, fish mortalities were also monitored daily on the farm. Flavobacterium columnare was significantly (P < 0.05) associated with C. hexasticha abundance in water (eDNA) and fish mortalities. However, Bdellovibrio bacteriovorus was the most significant bacteria correlated (P < 0.05) to eDNA. This study demonstrated, for the first time, a possible link between infections caused by the ciliate C. hexasticha, fish mortalities and the pathogenic F. columnare in an aquaculture environment. In summary, this research advanced knowledge on the morphology and genetic characteristics of Chilodonella spp. in Australian freshwater fish farming, revealed the applicability of eDNA techniques to predict fish mortalities in aquaculture and indicated the potential association of Chilodonella spp. outbreaks with bacterial communities on fish farms.