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

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.publishedVersio

An evaluation of corallophelia pertusamucus as an analytical matrix for environmental monitoring: A preliminary proteomic study

For the environmental monitoring of coral, mucus appears to be an appropriate biological matrix due to its array of functions in coral biology and the non-intrusive manner in which it can be collected. The aim of the present study was to evaluate the feasibility of using mucus of the stony coral Lophelia pertusa (L. pertusa) as an analytical matrix for discovery of biomarkers used for environmental monitoring. More specifically, to assess whether a mass-spectrometry-based proteomic approach can be applied to characterize the protein composition of coral mucus and changes related to petroleum discharges at the seafloor. Surface-enhanced laser desorption/ionization–time of flight mass spectrometry (SELDI-TOF MS) screening analyses of orange and white L. pertusa showed that the mucosal protein composition varies significantly with color phenotype, a pattern not reported prior to this study. Hence, to reduce variability from phenotype difference, L. pertusa white individuals only were selected to characterize in more detail the basal protein composition in mucus using liquid chromatography, mass spectrometry, mass spectrometry (LC-MS/MS). In total, 297 proteins were identified in L. pertusa mucus of unexposed coral individuals. Individuals exposed to drill cuttings in the range 2 to 12 mg/L showed modifications in coral mucus protein composition compared to unexposed corals. Although the results were somewhat inconsistent between individuals and require further validation in both the lab and the field, this study demonstrated preliminary encouraging results for discovery of protein markers in coral mucus that might provide more comprehensive insight into potential consequences attributed to anthropogenic stressors and may be used in future monitoring of coral health

LOD determined for SL2 assay.

LOD was determined from dilution series, 8 replicates were amplified at concentration of 550000, 110000, 22000, 4400, 880, 176, 35.2, 7.04, 1.408, 0.2816, 0.05632 and O copies 1μl-1. The proportion of positive amplifications are plotted against the standard concentrations (x- axis logarithmic). LOD was determined as the minimum concentration of 95% replicates amplified (95% threshold is shown as a line). (TIF)</p

SL2 and SL1 primers and probes specificity characteristics based on the in-silico evaluation using blastn.

The table includes Lepeophtheirus BLAST hits and the best non-Lepeophtheirus hits (on bold).</p

Number of estimated MGC in field samples (per 1 mL of seawater) collected in October 2019, May 2020, and September 2020.

The field data include results obtained for samples collected from 1m, 5m, 10m depth determined by using the MC assay (orange) and the SL2 assay (blue). For the comparison, the total number of MGC corresponding to 1–2 individuals (nauplii stage) are also included in the graph. Sample size (n) is depicted for each analyzed station.</p

The number of DNA copies per 1L in the control tank and in the experimental tanks with infested fish.

(TIF)</p

Distribution of estimated <i>L</i>. <i>salmonids</i> MGC at individual depths from samples collected in May and September.

N is number of samples included. (TIF)</p

LOQ-Limit of quantification for SL2 assay.

LOQ was determined from dilution series, 8 replicates were amplified at concentration of 550000, 110000, 22000, 4400, 880, 176, 35.2, 7.04, 1.408, 0.2816, 0.05632 1μl-1. The coefficient of variation (relative standard deviation) (CV = 100*(SD/mean)) was plotted against logarithmic transformed concentrations. (TIF)</p

Specificity of SL1 and SL2 assays, a result of Primer-BLAST search.

The figure includes hits up to 5 mismatches within the last 5 bps at the 3’ end.</p

Illustration of dissimilarities in the sequences between L. salmonids and <i>Caligus</i> sp.

Fragments of the alignment generated for the randomly picked 136 (from 256) L. salmonids mitochondrial 16S rRNA sequences with regions primers and probe of qPCR assay target (on yellow). The number on blue–the start position (including gaps) for the oligo binding. In addition, for L. salmonids number of mismatches are provided. (TIF)</p