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

Bui, Samantha; Dalvin, Sussie; Vågseth, Tone; Oppedal, Frode; Fossøy, Frode; Brandsegg, Hege; Jacobsen, Ása; Norði, Gunnvør á; Fordyce, Mark John; Michelsen, Helena Kling; Finstad, Bengt; Skern‐Mauritzen, Rasmus

doi:10.1111/are.15202

Cited by 9 publications

(14 citation statements)

References 50 publications

(77 reference statements)

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“…The issue has prompted the search for alternative adequate monitoring methods, ideally providing results in real time to be able to immediately introduce suitable actions to prevent large scale salmon lice invasion and infection. In a recent study [ 49 ] none of available methods passed the comparative test of salmon louse enumeration in plankton samples. These included visual-based -fluorescence microscopy and automated fluid imaging and molecular-based—droplet digital PCR (ddPCR), quantitative fraction PCR and qPCR.…”

Section: Discussionmentioning

confidence: 99%

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA

et al. 2022

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The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions.

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Section: Discussionmentioning

confidence: 99%

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA

et al. 2022

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“…The fluorescence-aided method for rapid identification of salmon lice can provide results at 82% accuracy, using far less time, effort and resources than alternative methods. In a comparison of lice enumerating methods, Bui et al (2021) found light microscopy to be 86% accurate and it took 87 min to process each sample which contained no more than 1531 total animals. Here, the samples contained an average of 11,600 animals and if including the processing time took 31 min to enumerate.…”

Section: Accuracy and Enumeration Timementioning

confidence: 99%

“…Finding planktonic salmon lice within a zooplankton assemblage is a needle in a haystack problem and the traditional method is too labour‐intensive for most endeavours. Bui et al (2021) explored several alternative methods for identification and enumeration including some molecular techniques but found limited success in terms of throughput, accuracy and cost. Although one of the attempted methods utilized fluorescence, the filter wavelengths chosen followed work done by Fordyce (2017) for which the range of filters available was limited.…”

Section: Introductionmentioning

confidence: 99%

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

Thompson

Bron

Bui

et al. 2022

Aquaculture Research

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“…The negative signal was due to fluorescence of chlorophyll consumed by the grazing zooplankters in contrast to the non-feeding L. salmonis. A negative signal might be useful in an automated process as argued by Nielsen et al (2019), but it would not be useful in enumerating animals via a fluorescence modification of traditional light microscopy methods in which the animal must standout against the darkfield (as described by Bui et al, 2020). Thus, wavelengths above 600 nm, where chlorophyll fluoresces, were not examined here and the spectrum section analysis identified the wavelengths with greatest positive peak intensity distance.…”

Section: Spectrum Section Analysis Of Eem Measurementsmentioning

confidence: 99%

“…Since identifying and enumerating planktonic L. salmonis stages in a zooplankton sample are a laborious task, several methodologies have been employed to that effect, with mixed results (Bui et al, 2020). Amongst them, fluorescence microscopy has been shown to increase the visibility of L. salmonis copepodids in comparison with other species (Bui et al, 2020;Fordyce, 2017). However, the reliability of the fluorescence signal has not been investigated nor has the optimal method been described in detail.…”

Section: Introductionmentioning

confidence: 99%

Illuminating the planktonic stages of salmon lice: A unique fluorescence signal for rapid identification of a rare copepod in zooplankton assemblages

Thompson

Bron

Bui

et al. 2021

Journal of Fish Diseases

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Monitoring of planktonic salmon louse (Lepeophtheirus salmonis salmonis) abundance and parameterization of key life‐history traits has been hindered by labour‐intensive and error‐prone quantification using traditional light microscopy. Fluorescence illumination has been proposed as a means of improving visualization, but prior to this study adequate investigation of the relevant fluorescence profiles and measurement conditions has not been undertaken. We investigated the fluorescence profiles of L. salmonis and non‐target copepod spp. with excitation and emission matrices (200–600 nm) and identified unique fluorescence signals. Fluorescence microscopy using excitation wavelengths of 470 ± 40 nm, and emission wavelengths of 525 ± 50 nm, showed that after 90 days of formalin storage salmon lice have a mean fluorescence intensity that is 2.4 times greater than non‐target copepods (copepodid and adult stages). A 7‐day heat treatment of 42°C in formalin increased the difference between salmon louse copepodids and non‐target copepods to a factor of 3.6, eliminating the need for prolonged storage. Differences in the fluorescence signal and endogenous fluorophores were investigated with respect to variation in sea lice species, age, stage and host fish origin. Under the conditions outlined in this paper, the fluorescence signal was found to be a reliable means of visualizing and differentiating salmon lice from non‐target zooplankters. Adaptation of the fluorescence signal would greatly expedite traditional methods of enumerating salmon louse larvae in plankton samples and could provide a means of automated detection.

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

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Cited by 9 publications

References 50 publications

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA

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

Illuminating the planktonic stages of salmon lice: A unique fluorescence signal for rapid identification of a rare copepod in zooplankton assemblages

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