Finding the needle in the haystack: Comparison of methods for salmon louse enumeration in plankton samples

The economic and social implications of salmon louse (Lepeophtheirus salmonis) epidemics in salmon aquaculture drive focus of the dispersal dynamics of the planktonic larval stages. The vast spatial scale and high connectivity of the marine environment creates difficult conditions to monitor the infective planktonic louse stage, whereby the number of samples required for a representative description is bottlenecked by processing capacity. This study assessed five quantification methods for accuracy and precision in enumeration of lice in plankton samples, validated against the benchmark method of light microscopy. Visual-based (fluorescence microscopy and automated fluid imaging) and molecular-based (droplet digital PCR, quantitative fraction PCR and quantitative PCR) were tested using high- and low-density plankton samples spiked with louse copepodids, with spike numbers blind to assessors. We propose an approach to comparative assessment that uses the collective bias and deviation of a test method to determine whether it is acceptably similar to the benchmark method. Under this framework, no methods passed the comparative test, with only ddPCR comparable to light microscopy (87% mean accuracy and 74% precision). qfPCR and fluorescence microscopy were moderately efficient (88% and 67% accuracy, and 36% and 52% precision respectively). Molecular techniques are currently restricted in distinguishing between larval stages, which is an essential distinction for some research questions, but can be economical in processing numerous samples. Overall method suitability will depend on the research objectives and resources available. These results provide evidence for operational accuracy for the tested methods and highlight the direction for further development to optimize their use

Workshop on the Norwegian Sea Aquaculture Overview

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A novel method for the rapid enumeration of planktonic salmon lice in a mixed zooplankton assemblage using fluorescence

The relative rarity of the planktonic larval stages of salmon lice in comparison to other animals captured in a zooplankton assemblage is an obstacle to estimating their abundance and distribution. Due to the labour intensiveness of standard plankton sorting approaches, the planktonic stages of salmon lice remain understudied and unmonitored despite their importance to the spread of the parasite between salmon farms and to wild salmonids. Alternative methods of identification have been investigated and in a previous study a fluorescence signal was identified. Using filters to target that signal with fluorescence microscopy (excitation/emission wavelengths of 470/525 nm), the salmon louse has a fluorescence intensity 2.4 times greater than non-target animals, which distinguishes it from the zooplankton assemblage and enables rapid enumeration. Here, we present a novel method for the enumeration of planktonic salmon lice larvae, nauplius and copepodid stages, in a mixed zooplankton sample using fluorescence-aided microscopy. Performance of the method was evaluated with a blind trial which found a median accuracy of 81.8% and a mean sample processing time of 31 min. Compared with previously published findings, the novel method provides satisfactory accuracy and enumeration that is more than 20 times faster than traditional light microscopy approaches. Factors influencing the performance of the method are identified and recommendations are made for targeted sampling and automated enumeration

Fluorolice - Final report for FHF project 901508

Salmon lice ( Lepeophtheirus salmonis ) represent a substantial obstacle for the sustainability of salmon farming (Torrissen, et al. 2013; Vollset, et al. 2018). As such, there are many avenues of research that aim to improve understanding of lice biology and ecology in order to combat the epidemic or predict the distribution of infective larvae. The latter remains increasingly important as passive non-chemical prevention measures, comprising cage structure modifications or new technologies (Grøntvedt, et al. 2015; Oppedal, et al. 2017; Stien, et al. 2018), become increasingly favored. Many of these technological innovations (e.g. skirts, snorkel cages, submerged cages, depth position of feeding and artificial lights) as well as area management and site locations are founded on knowledge of vertical and horizontal distribution of sea lice larvae. Understanding the fluctuations in abundance and behavior of lice larvae is also required to develop predictive tools to assist farmers in management of preventative measures, as well as for validating models of larval dispersion which in turn can be used for area management and choice of production site locations. However, larval distribution (both spatially and temporally) is heavily influenced by factors such as environmental conditions ( e.g . temperature, salinity, and light), hydrodynamics, connectivity between farms, and natural mortality (Kristoffersen, et al. 2014; Johnsen, et al. 2016; Samsing, et al. 2016; Brooker, et al. 2018). Addressing these parameters in the field requires enumeration of salmon lice larvae in complex plankton samples. Furthermore, to answer questions about larval distribution, large numbers of plankton samples are required to be processed (Nelson, et al. 2017). The ability to meet the desire for knowledge on the planktonic distribution of salmon lice has hitherto been restrained by the resource intensive effort required to identify copepodids in a plankton sample (Bui, et al. 2021). To circumvent this, indirect methods of enumerating lice abundance in coastal waters have been attempted, including assessment of site-specific lice abundance in farms (Jansen, et al. 2012), use of sentinel cages (Bjorn, et al. 2011), and enumeration of lice abundance on migrating wild salmonids (Serra-Llinares, et al. 2014). Ultimately, these approaches operate by using lice on fish as a proxy and do not fill the knowledge gap concerning actual planktonic abundance of sea lice larvae

Finding the needle in the haystack: Comparison of methods for salmon louse enumeration in plankton samples

The economic and social implications of salmon louse (Lepeophtheirus salmonis) epidemics in salmon aquaculture drive focus of the dispersal dynamics of the planktonic larval stages. The vast spatial scale and high connectivity of the marine environment creates difficult conditions to monitor the infective planktonic louse stage, whereby the number of samples required for a representative description is bottlenecked by processing capacity. This study assessed five quantification methods for accuracy and precision in enumeration of lice in plankton samples, validated against the benchmark method of light microscopy. Visual-based (fluorescence microscopy and automated fluid imaging) and molecular-based (droplet digital PCR, quantitative fraction PCR and quantitative PCR) were tested using high- and low-density plankton samples spiked with louse copepodids, with spike numbers blind to assessors. We propose an approach to comparative assessment that uses the collective bias and deviation of a test method to determine whether it is acceptably similar to the benchmark method. Under this framework, no methods passed the comparative test, with only ddPCR comparable to light microscopy (87% mean accuracy and 74% precision). qfPCR and fluorescence microscopy were moderately efficient (88% and 67% accuracy, and 36% and 52% precision respectively). Molecular techniques are currently restricted in distinguishing between larval stages, which is an essential distinction for some research questions, but can be economical in processing numerous samples. Overall method suitability will depend on the research objectives and resources available. These results provide evidence for operational accuracy for the tested methods and highlight the direction for further development to optimize their use

Finding the needle in the haystack: Comparison of methods for salmon louse enumeration in plankton samples

The economic and social implications of salmon louse (Lepeophtheirus salmonis) epidemics in salmon aquaculture drive focus of the dispersal dynamics of the planktonic larval stages. The vast spatial scale and high connectivity of the marine environment creates difficult conditions to monitor the infective planktonic louse stage, whereby the number of samples required for a representative description is bottlenecked by processing capacity. This study assessed five quantification methods for accuracy and precision in enumeration of lice in plankton samples, validated against the benchmark method of light microscopy. Visual-based (fluorescence microscopy and automated fluid imaging) and molecular-based (droplet digital PCR, quantitative fraction PCR and quantitative PCR) were tested using high- and low-density plankton samples spiked with louse copepodids, with spike numbers blind to assessors. We propose an approach to comparative assessment that uses the collective bias and deviation of a test method to determine whether it is acceptably similar to the benchmark method. Under this framework, no methods passed the comparative test, with only ddPCR comparable to light microscopy (87% mean accuracy and 74% precision). qfPCR and fluorescence microscopy were moderately efficient (88% and 67% accuracy, and 36% and 52% precision respectively). Molecular techniques are currently restricted in distinguishing between larval stages, which is an essential distinction for some research questions, but can be economical in processing numerous samples. Overall method suitability will depend on the research objectives and resources available. These results provide evidence for operational accuracy for the tested methods and highlight the direction for further development to optimize their use

Workshop on the Norwegian Sea Aquaculture Overviews (WKNORAO). ICES Scientific Reports. 3:116.

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Workshop on the Norwegian Sea Aquaculture Overview

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