Dead reckoning of protist viability with propidium monoazide (PMA)-quantitative PCR; a case study using Neoparamoeba perurans.

The ability to distinguish between viable and non-viable protozoan parasites is central to improved human and animal health management. While conceptually simple, methods to differentiate cell viability in situ remain challenging. Amoebic gill disease, caused by Neoparamoeba perurans is a parasitic disease impacting Atlantic salmon aquaculture globally. Although commercial freshwater treatments alleviate AGD, viable amoebae remain on gills or in used treatment water. Existing PCR-based assays are able to quantify N. perurans abundance but cannot discriminate amoeba viability. We investigated the use of propidium monoazide (PMA) application, prior to real-time PCR, to distinguish between alive and dead cells. We demonstrate that 200 µM PMA can significantly reduce amplification from non-viable (isopropanol treated) cultured amoebae across at least three logs of cell concentrations. Using a serial dilution of viable and non-viable cells, we show that non-PMA PCR amplifies both viable and non-viable amoebae, while PMA exposure suppresses (but does not completely inhibit) amplification from non-viable amoebae. The effect of freshwater treatment on N. perurans viability was assessed using the PMA-PCR. Following PMA exposure, amplification from freshwater treated amoebae was reduced by approximately 94-97 %. Taken together this study demonstrates that PMA combined with traditional real-time PCR can estimate amoeba viability.

Gill Mucus and Gill Mucin O-glycosylation in Healthy and Amebic Gill Disease-Affected Atlantic Salmon.

Amoebic gill disease (AGD) causes poor performance and death in salmonids. Mucins are mainly comprised by carbohydrates and are main components of the mucus covering the gill. Since glycans regulate pathogen binding and growth, glycosylation changes may affect susceptibility to primary and secondary infections. We investigated gill mucin O-glycosylation from Atlantic salmon with and without AGD using liquid chromatography-mass spectrometry. Gill mucin glycans were larger and more complex, diverse and fucosylated than skin mucins. Confocal microscopy revealed that fucosylated mucus coated sialylated mucus strands in ex vivo gill mucus. Terminal HexNAcs were more abundant among O-glycans from AGD-affected Atlantic salmon, whereas core 1 structures and structures with acidic moieties such as N-acetylneuraminic acid (NeuAc) and sulfate groups were less abundant compared to non-infected fish. The fucosylated and NeuAc-containing O-glycans were inversely proportional, with infected fish on the lower scale of NeuAc abundance and high on fucosylated structures. The fucosylated epitopes were of three types: Fuc-HexNAc-R, Gal-[Fuc-]HexNAc-R and HexNAc-[Fuc-]HexNAc-R. These blood group-like structures could be an avenue to diversify the glycan repertoire to limit infection in the exposed gills. Furthermore, care must be taken when using skin mucus as proxy for gill mucus, as gill mucins are distinctly different from skin mucins.

Effects of Size and Geographical Origin on Atlantic salmon, Salmo salar, Mucin O-Glycan Repertoire.

Diseases cause ethical concerns and economic losses in the Salmonid industry. The mucus layer comprised of highly O-glycosylated mucins is the first contact between pathogens and fish. Mucin glycans govern pathogen adhesion, growth and virulence. The Atlantic salmon O-glycome from a single location has been characterized and the interindividual variation was low. Because interindividual variation is considered a population-based defense, hindering the entire population from being wiped out by a single infection, low interindividual variation among Atlantic salmon may be a concern. Here, we analyzed the O-glycome of 25 Atlantic salmon from six cohorts grown under various conditions from Sweden, Norway and Australia (Tasmania) using mass spectrometry. This expanded the known Atlantic salmon O-glycome by 60% to 169 identified structures. The mucin O-glycosylation was relatively stable over time within a geographical region, but the size of the fish affected skin mucin glycosylation. The skin mucin glycan repertoires from Swedish and Norwegian Atlantic salmon populations were closely related compared with Tasmanian ones, regardless of size and salinity, with differences in glycan size and composition. The internal mucin glycan repertoire also clustered based on geographical origin and into pyloric cecal and distal intestinal groups, regardless of cohort and fish size. Fucosylated structures were more abundant in Tasmanian pyloric caeca and distal intestine mucins compared with Swedish ones. Overall, Tasmanian Atlantic salmon mucins have more O-glycan structures in skin but less in the gastrointestinal tract compared with Swedish fish. Low interindividual variation was confirmed within each cohort. The results can serve as a library for identifying structures of importance for host-pathogen interactions, understanding population differences of salmon mucin glycosylation in resistance to diseases and during breeding and selection of strains. The results could make it possible to predict potential vulnerabilities to diseases and suggest that inter-region breeding may increase the glycan diversity.

Effects of an unprecedented summer heatwave on the growth performance, flesh colour and plasma biochemistry of marine cage-farmed Atlantic salmon (Salmo salar).

Global seawater temperatures are increasing and becoming more variable, with consequences for all marine animals including those in food production systems. In several countries around the world,arming of Atlantic salmon (Salmo salar) occurs towards the upper end of the thermal tolerance window for this species, and marked effects on salmon production during summers have been experienced but never empirically investigated. This project tracked the effects of an extreme summer heatwave on two different cohorts of fish stocked into farm cages either during early winter (EW) or late winter (LW). The farm site experienced an unprecedented high water temperature event, with a peak water temperature of 22.9 °C and 117 days above 18 °C. Fish in both EW and LW cohorts experienced a temperature-induced cessation of voluntary feed intake as well as inefficient osmoregulatory, liver and renal function during high temperature periods. Flesh colour declined primarily in the dorsal and ventral regions of the fillet and secondarily along the midline, with over 20% of fish demonstrated a complete loss of flesh colour during the months of March and April. A return to feeding in autumn occurred faster in some fish and caused a marked bimodal size distribution to appear within both the EW and LW cohorts as autumn progressed. However, the LW cohort returned to feeding at seawater temperatures of 20.2 °C, compared with 18.6 °C for the EW cohort. There was a strong positive relationship between fillet colour recovery and residual condition index (RCI). These findings identified alkaline phosphatase as a potential marker to non-destructively track individual fish for signs of recovery after a thermal stress event, and shed light on the physiological consequences of marine heatwaves on fishes. This study also identified that supporting feed intake or promoting a return to feeding may help mitigate the negative impacts of climate warming on cultured Atlantic salmon.

Mitochondrial DNA sequence and gene organization in the [corrected] Australian blacklip [corrected] abalone Haliotis rubra (leach).

The complete mitochondrial DNA of the blacklip abalone Haliotis rubra (Gastropoda: Mollusca) was cloned and 16,907 base pairs were sequenced. The sequence represents an estimated 99.85% of the mitochondrial genome, and contains 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes found in other metazoan mtDNA. An AT tandem repeat and a possible C-rich domain within the putative control region could not be fully sequenced. The H. rubra mtDNA gene order is novel for mollusks, separated from the black chiton Katharina tunicata by the individual translocations of 3 tRNAs. Compared with other mtDNA regions, sequences from the ATP8, NAD2, NAD4L, NAD6, and 12S rRNA genes, as well as the control region, are the most variable among representatives from Mollusca, Arthropoda, and Rhynchonelliformea, with similar mtDNA arrangements to H. rubra. These sequences are being evaluated as genetic markers within commercially important Haliotis species, and some applications and considerations for their use are discussed.