Aquaculture-driven evolution of the salmon louse mtDNA genome.

Resistance toward the antiparasitic pyrethroid, deltamethrin, is reported in the Atlantic salmon louse (Lepeophtheirus salmonis salmonis), a persistent ectoparasite of farmed and wild salmonids. The resistance mechanism is linked to mitochondrial DNA (mtDNA), where genetic markers for resistance have been identified. Here, we investigated how widespread pyrethroid use in aquaculture may have influenced mtDNA variation in lice, and the dispersion of resistant haplotypes across the North Atlantic, using historical (2000-2002 "pre-resistance") and contemporary (2014-2017 "post-resistance") samples. To study this, we sequenced ATPase 6 and cytochrome b, genotyped two genetic markers for deltamethrin resistance, and genotyped microsatellites as "neutral" controls of potential population bottlenecks. Overall, we observed a modest reduction in mtDNA diversity in the period 2000-2017, but no reduction in microsatellite variation was observed. The reduction in mtDNA variation was especially distinct in two of the contemporary samples, fixed for one and two haplotypes, respectively. By contrast, all historical samples consisted of close to one mtDNA haplotype per individual. No population genetic structure was detected among the historical samples for mtDNA nor microsatellites. By contrast, significant population genetic differentiation was observed for mtDNA among some of the contemporary samples. However, the observed population genetic structure was tightly linked with the pattern of deltamethrin resistance, and we therefore conclude that it primarily reflects the transient mosaic of pyrethroid usage in time and space. Two historically undetected mtDNA haplotypes dominated in the contemporary samples, both of which were linked to deltamethrin resistance, demonstrating primarily two origins of deltamethrin resistance in the North Atlantic. Collectively, these data demonstrate that the widespread use of pyrethroids in commercial aquaculture has substantially altered the patterns of mtDNA diversity in lice across the North Atlantic, and that long-distance dispersion of resistance is rapid due to high level of genetic connectivity that is observed in this species.

The salmon louse genome may be much larger than sequencing suggests.

The genome size of organisms impacts their evolution and biology and is often assumed to be characteristic of a species. Here we present the first published estimates of genome size of the ecologically and economically important ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae). Four independent L. salmonis genome assemblies of the North Atlantic subspecies Lepeophtheirus salmonis salmonis, including two chromosome level assemblies, yield assemblies ranging from 665 to 790 Mbps. These genome assemblies are congruent in their findings, and appear very complete with Benchmarking Universal Single-Copy Orthologs analyses finding > 92% of expected genes and transcriptome datasets routinely mapping > 90% of reads. However, two cytometric techniques, flow cytometry and Feulgen image analysis densitometry, yield measurements of 1.3-1.6 Gb in the haploid genome. Interestingly, earlier cytometric measurements reported genome sizes of 939 and 567 Mbps in L. salmonis salmonis samples from Bay of Fundy and Norway, respectively. Available data thus suggest that the genome sizes of salmon lice are variable. Current understanding of eukaryotic genome dynamics suggests that the most likely explanation for such variability involves repetitive DNA, which for L. salmonis makes up ≈ 60% of the genome assemblies.

The information continuum model of evolution.

Most biologists agree that evolution is contingent on inherited information shaped by natural selection. This apparent consensus could be taken to indicate agreement on the forces shaping evolution, but vivid discussions reveal divergences on how evolution is perceived. The predominant Modern Synthesis (MS) paradigm holds the position that evolution occurs through random changes acting on genomic inheritance. However, studies from recent decades have revealed that evolutionary inheritance also includes DNA-methylation, RNA, symbionts, and culture, among other factors. This has fueled a demand of a broader evolutionary perspective, for example from the proponents of the Extended Evolutionary Synthesis (EES). Despite fundamental disagreements the different views agree that natural selection happens through dissimilar perpetuation of inheritable information. Yet, neither the MS, nor the ESS dwell extensively on the nature of hereditary information. We do - and conclude that information in and of itself is immaterial. We then argue that the quality upon which natural selection acts henceforth is also immaterial. Based on these notions, we arrive at the information-centric Information Continuum Model (ICM) of evolution. The ICM asserts that hereditary information is embedded in diverse physical forms (DNA, RNA, symbionts etc.) representing a continuum of evolutionary qualities, and that information may migrate between these physical forms. The ICM leaves theoretical exploration of evolution unrestricted by the limitations imposed by the individual physical forms wherein the hereditary information is embedded (e.g. genomes). ICM bestows us with a simple heuristic model that adds explanatory dimensions to be considered in the evolution of biological systems.

The salmon louse genome: Copepod features and parasitic adaptations.

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

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

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.

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation.

Vaccination against salmon lice (Lepeophtheirus salmonis) is a means of control that averts the negative effects of chemical approaches. Here, we studied the immunogenicity and protective effect of a vaccine formulation (based on a salmon lice-gut recombinant protein [P33]) against Lepeophtheirus salmonis infestation in Atlantic salmon in a laboratory-based trial. Our findings revealed that P33 vaccine can provide a measure of protection against immature and adult salmon lice infestation. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. We also provide immunological evidence confirming that P33-specific immune response can be triggered in Atlantic salmon after P33 vaccination, and that production of P33-specific antibodies in blood can be detected in vaccinated fish. The negative correlation between P33-specific IgM in salmon plasma and salmon lice numbers on vaccinated fish suggests that protection against lice can be mediated by the specific antibody in salmon plasma. The success of P33 vaccination in protecting salmon against lice confirms the possibility of employing the hematophagous nature of the parasite to deliver salmon-specific antibodies against lice-gut proteins.

The effect of temperature on ability of Lepeophtheirus salmonis to infect and persist on Atlantic salmon.

The salmon louse (Lepeophtheirus salmonis) is an ecologically and economically important parasite of salmonid fish. Temperature is a strong influencer of biological processes in salmon lice, with development rate increased at higher temperatures. The successful attachment of lice onto a host is also predicted to be influenced by temperature; however, the correlation of temperature with parasite survival is unknown. This study describes the effects of temperature on infection success, and survival on the host during development to the adult stage. To accurately describe infection dynamics with varying temperatures, infection success was recorded on Atlantic salmon (Salmo salar) between 2 and 10°C. Infection success ranged from 20% to 50% and was strongly correlated with temperature, with the highest success at 10°C. Parasite loss was monitored during development at eight temperatures with high loss of lice at 3 and 24°C, whilst no loss was recorded in the temperature range from 6 to 21°C. Sea temperatures thus have large effects on the outcome of salmon louse infections and should be taken into account in the management and risk assessment of this parasite. Improving understanding of the infection dynamics of salmon lice will facilitate epidemiological modelling efforts and efficiency of pest management strategies.

Whole genome resequencing reveals diagnostic markers for investigating global migration and hybridization between minke whale species.

BACKGROUND: In the marine environment, where there are few absolute physical barriers, contemporary contact between previously isolated species can occur across great distances, and in some cases, may be inter-oceanic. An example of this can be seen in the minke whale species complex. Antarctic minke whales are genetically and morphologically distinct from the common minke found in the north Atlantic and Pacific oceans, and the two species are estimated to have been isolated from each other for 5 million years or more. Recent atypical migrations from the southern to the northern hemisphere have been documented and fertile hybrids and back-crossed individuals between both species have also been identified. However, it is not known whether this represents a contemporary event, potentially driven by ecosystem changes in the Antarctic, or a sporadic occurrence happening over an evolutionary time-scale. We successfully used whole genome resequencing to identify a panel of diagnostic SNPs which now enable us address this evolutionary question. RESULTS: A large number of SNPs displaying fixed or nearly fixed allele frequency differences among the minke whale species were identified from the sequence data. Five panels of putatively diagnostic markers were established on a genotyping platform for validation of allele frequencies; two panels (26 and 24 SNPs) separating the two species of minke whale, and three panels (22, 23, and 24 SNPs) differentiating the three subspecies of common minke whale. The panels were validated against a set of reference samples, demonstrating the ability to accurately identify back-crossed whales up to three generations. CONCLUSIONS: This work has resulted in the development of a panel of novel diagnostic genetic markers to address inter-oceanic and global contact among the genetically isolated minke whale species and sub-species. These markers, including a globally relevant genetic reference data set for this species complex, are now openly available for researchers interested in identifying other potential whale hybrids in the world's oceans. The approach used here, combining whole genome resequencing and high-throughput genotyping, represents a universal approach to develop similar tools for other species and population complexes.

Characterization of a novel RXR receptor in the salmon louse (Lepeophtheirus salmonis, Copepoda) regulating growth and female reproduction.

BACKGROUND: Nuclear receptors have crucial roles in all metazoan animals as regulators of gene transcription. A wide range of studies have elucidated molecular and biological significance of nuclear receptors but there are still a large number of animals where the knowledge is very limited. In the present study we have identified an RXR type of nuclear receptor in the salmon louse (Lepeophtheirus salmonis) (i.e. LsRXR). RXR is one of the two partners of the Ecdysteroid receptor in arthropods, the receptor for the main molting hormone 20-hydroxyecdysone (E20) with a wide array of effects in arthropods. RESULTS: Five different LsRXR transcripts were identified by RACE showing large differences in domain structure. The largest isoforms contained complete DNA binding domain (DBD) and ligand binding domain (LBD), whereas some variants had incomplete or no DBD. LsRXR is transcribed in several tissues in the salmon louse including ovary, subcuticular tissue, intestine and glands. By using Q-PCR it is evident that the LsRXR mRNA levels vary throughout the L. salmonis life cycle. We also show that the truncated LsRXR transcript comprise about 50% in all examined samples. We used RNAi to knock-down the transcription in adult reproducing female lice. This resulted in close to zero viable offspring. We also assessed the LsRXR RNAi effects using a L. salmonis microarray and saw significant effects on transcription in the female lice. Transcription of the major yolk proteins was strongly reduced by knock-down of LsRXR. Genes involved in lipid metabolism and transport were also down regulated. Furthermore, different types of growth processes were up regulated and many cuticle proteins were present in this group. CONCLUSIONS: The present study demonstrates the significance of LsRXR in adult female L. salmonis and discusses the functional aspects in relation to other arthropods. LsRXR has a unique structure that should be elucidated in the future.

Human-induced evolution caught in action: SNP-array reveals rapid amphi-atlantic spread of pesticide resistance in the salmon ecotoparasite Lepeophtheirus salmonis.

BACKGROUND: The salmon louse, Lepeophtheirus salmonis, is an ectoparasite of salmonids that causes huge economic losses in salmon farming, and has also been causatively linked with declines of wild salmonid populations. Lice control on farms is reliant upon a few groups of pesticides that have all shown time-limited efficiency due to resistance development. However, to date, this example of human-induced evolution is poorly documented at the population level due to the lack of molecular tools. As such, important evolutionary and management questions, linked to the development and dispersal of pesticide resistance in this parasite, remain unanswered. Here, we introduce the first Single Nucleotide Polymorphism (SNP) array for the salmon louse, which includes 6000 markers, and present a population genomic scan using this array on 576 lice from twelve farms distributed across the North Atlantic. RESULTS: Our results support the hypothesis of a single panmictic population of lice in the Atlantic, and importantly, revealed very strong selective sweeps on linkage groups 1 and 5. These sweeps included candidate genes potentially connected to pesticide resistance. After genotyping a further 576 lice from 12 full sibling families, a genome-wide association analysis established a highly significant association between the major sweep on linkage group 5 and resistance to emamectin benzoate, the most widely used pesticide in salmonid aquaculture for more than a decade. CONCLUSIONS: The analysis of conserved haplotypes across samples from the Atlantic strongly suggests that emamectin benzoate resistance developed at a single source, and rapidly spread across the Atlantic within the period 1999 when the chemical was first introduced, to 2010 when samples for the present study were obtained. These results provide unique insights into the development and spread of pesticide resistance in the marine environment, and identify a small genomic region strongly linked to emamectin benzoate resistance. Finally, these results have highly significant implications for the way pesticide resistance is considered and managed within the aquaculture industry.