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

A baseline study of the occurrence of non-indigenous species in Danish harbours

Project Manager/Main Author Jesper H. AndersenWe report the first ever nation-wide study of the occurrence of non-indigenous species in Danish harbours. The sampling was car-ried out using both conventional and biomolecular methods (eDNA). In total, 16 harbours were covered – Esbjerg and Aarhus, the two largest harbours in Denmark, with intensive sampling and 14 harbours with a reduced programme. 26 non-indigenous species were recorded using conventional sampling and 13 species were recorded using eDNA-based methods. Excluding overlapping rec-ords, we have recorded a total of 34 non-indigenous species in the 16 harbours studied. Based on the results, we conclude the following: 1) more non-indigenous species are found in the western parts of Denmark (North Sea region) then in the eastern parts (Baltic Sea), and 2) a few species previously unseen in Danish marine waters were recorded, i.e. the two bristle worms Eteone het-eropoda (fam. Phyllodocidae) and Streblospio benedicti (fam. Spionidae). Further, we provide a proof-of-concept regarding the overarching objectives of the MONIS 1-3 projects and the eDNA-based test systems developed. The results constitute a baseline for future studies in Danish ports and other hotspot areas.publishedVersio

Comparative genomics and signatures of selection in North Atlantic eels

Comparative genomic approaches can identify putative private and shared signatures of selection. We performed a comparative genomic study of North Atlantic eels, European eel (Anguilla Anguilla) and American eel (A. rostrata). The two sister species are nearly undistinguishable at the phenotypic level and despite a wide non overlapping continental distribution, they spawn in partial sympatry in the Sargasso Sea. Taking advantage of the newly assembled and annotated genome, we used genome wide RAD sequencing data of 359 individuals retrieved from Sequence Nucleotide Archive and state-of-the-art statistic tests to identify putative genomic signatures of selection in North Atlantic eels. First, using the F-ST and XP-EHH methods, we detected apparent islands of divergence on a total of 7 chromosomes, particularly on chromosomes 6 and 10. Gene ontology analyses suggested candidate genes mainly related to energy production, development and regulation, which could reflect strong selection on traits related to eel migration and larval duration time. Gene effect prediction using SNPeff showed a high number of SNPs in noncoding regions, pointing to a possible regulatory role. Second, using the iHS method we detected shared regions under selection on a total of 11 chromosomes. Several hypotheses might account for the detection of shared islands of selection in North Atlantic eels, including parallel evolution due to adaptation to similar environments and introgression. Future comparative genomic studies will be needed to further clarify the causes and consequences of introgression, including the directionality of these introgression events

Biodiversity monitoring of offshore structures in the Danish North Sea using environmental DNA (eDNA)

Environmental DNA (eDNA) is a cheap and accurate method for biodiversity monitoring based on sampling and analyzing the DNA that all living organisms shed to the environment. To investigate biodiversity at sites of interest in the sea, such as at and around platforms and natural reefs, it is necessary to understand the spatial and temporal variation of eDNA observations to accurately interpret and evaluate the results. Here, we used a metabarcoding approach to analyze top and bottom water samples collected along a transect from the DanF platform to Esbjerg harbor, which included both natural reefs and control stations. Temporal samples collected across 3 days using an onsite robotic sampler (ESP) were also analyzed to investigate short-term temporal variations. While general data analysis was performed using Illumina sequencing, a subset of data was further re-sequenced on the transportable ‘MinION’ sequencer from Oxford Nanopore to assess the suitability of this new technology for future on- site data generation

Speciation history of European (<i>Anguilla anguilla</i>) and American eel (<i>A. rostrata</i>), analyzed using genomic data

International audienceSpeciation in the ocean could differ from terrestrial environments due to fewer barriers to gene flow. Hence, sympatric speciation might be common, with American and European eel being candidates for exemplifying this. They show disjunct continental distributions on both sides of the Atlantic, but spawn in overlapping regions of the Sargasso Sea from where juveniles are advected to North American, European and North African coasts. Hybridization and introgression are known to occur, with hybrids almost exclusively observed in Iceland. Different speciation scenarios have been suggested, involving either vicariance or sympatric ecological speciation. Using RAD sequencing and whole‐genome sequencing data from parental species and F1 hybrids, we analysed speciation history based on the joint allele frequency spectrum (JAFS) and pairwise sequentially Markovian coalescent (PSMC) plots. JAFS supported a model involving a split without gene flow 150,000–160,000 generations ago, followed by secondary contact 87,000–92,000 generations ago, with 64% of the genome experiencing restricted gene flow. This supports vicariance rather than sympatric speciation, likely associated with Pleistocene glaciation cycles and ocean current changes. Whole‐genome PSMC analysis of F1 hybrids from Iceland suggested divergence 200,000 generations ago and indicated subsequent gene flow rather than strict isolation. Finally, simulations showed that results from both approaches (JAFS and PSMC) were congruent. Hence, there is strong evidence against sympatric speciation in North Atlantic eels. These results reiterate the need for careful consideration of cases of possible sympatric speciation, as even in seemingly barrier‐free oceanic environments palaeoceanographic factors may have promoted vicariance and allopatric speciation

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