Development and validation of a DNA-based multi-species biomonitoring toolkit using a high-throughput qPCR platform: A case study of Irish shellfish species.

Biomonitoring of marine life has been enhanced in recent years by the integration of innovative DNA-based approaches, which offer advantages over more laborious techniques (e.g. microscopy). However, trade-offs between throughput, sensitivity and quantitative measurements must be made when choosing between the prevailing molecular methodologies (i.e. metabarcoding or qPCR/dPCR). Thus, the aim of the present study was to demonstrate the utility of a microfluidic-enabled high-throughput quantitative PCR platform (HTqPCR) for the rapid and cost-effective development and validation of a DNA-based multi-species biomonitoring toolkit, using larvae of 23 commercially targeted bivalve and crustacean species as a case study. The workflow was divided into three main phases: definition of (off-) target taxa and establishment of reference databases (PHASE 1); selection/development and assessment of molecular assays (PHASE 2); and protocol optimization and field validation (PHASE 3). 42 assays were eventually chosen and validated. Genetic signal not only showed good correlation with direct visual counts by microscopy but also showed the ability to provide quantitative data at the highest taxonomic resolution (species level) in a time- and cost-effective fashion. This study developed a biomonitoring toolkit, demonstrating the considerable advantages of this state-of-the-art technology in boosting the developmental testing and application of panels of molecular assays for the monitoring and management of natural resources. Once developed, this approach provides a cost and time-effective alternative compared to other multi-species approaches (e.g. metabarcoding). In addition, it is transferable to a wide range of species and will aid future monitoring programmes.

Experimental infection trials with European North Atlantic ranavirus (Iridoviridae) isolated from lumpfish (Cyclopterus lumpus, L.).

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Investigation of the transcriptomic response in Atlantic salmon (Salmo salar) gill exposed to Paramoeba perurans during early onset of disease.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.

Environmental DNA sampling protocols for the surveillance of marine non-indigenous species in Irish coastal waters.

Understanding the spread and distribution of Non-Indigenous Species (NIS) is key when implementing legislation to maintain good ecosystem health. Environmental DNA (eDNA) has shown great potential to detect aquatic organisms in a rapid and cost-effective way, however their applicability to new environments must be validated prior to their implementation. Here, we tested different field sampling methods in combination with eDNA metabarcoding to develop a tool to detect NIS. Large and small volumes of seawater were filtered, in addition to the collection of sediment and horizontal tow net samples at 12 locations across four distinct geographic areas in Ireland. The biggest dissimilarity in the species recovered was found between sediment and town net samples. Tow nets showed to be the most efficient. A total of 357 taxa were identified, including 16 NIS. Fine mesh tow nets were identified as the most cost-efficient for large-scale monitoring and surveillance of NIS.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Don't catch me if you can - Using cabled observatories as multidisciplinary platforms for marine fish community monitoring: An in situ case study combining Underwater Video and environmental DNA data.

Cabled observatories are marine infrastructures equipped with biogeochemical and oceanographic sensors as well as High-Definition video and audio equipment, hence providing unprecedented opportunities to study marine biotic and abiotic components. Additionally, non-invasive monitoring approaches such as environmental DNA (eDNA) metabarcoding have further enhanced the ability to characterize marine life. Although the use of non-invasive tools beholds great potential for the sustainable monitoring of biodiversity and declining natural resources, such techniques are rarely used in parallel and understanding their limitations is challenging. Thus, this study combined Underwater Video (UV) with eDNA metabarcoding data to produce marine fish community profiles over a 2 months period in situ at a cabled observatory in the northeast Atlantic (SmartBay Ireland). By combining both approaches, an increased number of fish could be identified to the species level (total of 22 species), including ecologically and economically important species such as Atlantic cod, whiting, mackerel and monkfish. The eDNA approach alone successfully identified a higher number of species (59%) compared to the UV approach (18%), whereby 23% of species were detected by both methods. The parallel implementation of point collection eDNA and time series UV data not only confirmed expectations of the corroborative effect of using multiple disciplines in fish community composition, but also enabled the assessment of limitations intrinsic to each technique including the identification of false-negative detections in one sampling technology relative to the other. This work showcased the usefulness of cabled observatories as key platforms for in situ empirical assessment of both challenges and prospects of novel technologies in aid to future monitoring of marine life.

Quantifying dispersal between marine protected areas by a highly mobile species, the bottlenose dolphin, Tursiops truncatus.

The functioning of marine protected areas (MPAs) designated for marine megafauna has been criticized due to the high mobility and dispersal potential of these taxa. However, dispersal within a network of small MPAs can be beneficial as connectivity can result in increased effective population size, maintain genetic diversity, and increase robustness to ecological and environmental changes making populations less susceptible to stochastic genetic and demographic effects (i.e., Allee effect). Here, we use both genetic and photo-identification methods to quantify gene flow and demographic dispersal between MPAs of a highly mobile marine mammal, the bottlenose dolphin Tursiops truncatus. We identify three populations in the waters of western Ireland, two of which have largely nonoverlapping core coastal home ranges and are each strongly spatially associated with specific MPAs. We find high site fidelity of individuals within each of these two coastal populations to their respective MPA. We also find low levels of demographic dispersal between the populations, but it remains unclear whether any new gametes are exchanged between populations through these migrants (genetic dispersal). The population sampled in the Shannon Estuary has a low estimated effective population size and appears to be genetically isolated. The second coastal population, sampled outside of the Shannon, may be demographically and genetically connected to other coastal subpopulations around the coastal waters of the UK. We therefore recommend that the methods applied here should be used on a broader geographically sampled dataset to better assess this connectivity.

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.

Historical gene flow constraints in a northeastern Atlantic fish: phylogeography of the ballan wrasse Labrus bergylta across its distribution range.

The distribution and demographic patterns of marine organisms in the north Atlantic were largely shaped by climatic changes during the Pleistocene, when recurrent glacial maxima forced them to move south or to survive in northern peri-glacial refugia. These patterns were also influenced by biological and ecological factors intrinsic to each species, namely their dispersion ability. The ballan wrasse (Labrus bergylta), the largest labrid fish along Europe's continental margins, is a target for fisheries and aquaculture industry. The phylogeographic pattern, population structure, potential glacial refugia and recolonization routes for this species were assessed across its full distribution range, using mitochondrial and nuclear markers. The existence of a marked population structure can reflect both recolonization from three distinct glacial refugia and current and past oceanographic circulation patterns. Although isolated in present times, shared haplotypes between continental and Azores populations and historical exchange of migrants in both directions point to a common origin of L. bergylta. This situation is likely to be maintained and/or accentuated by current circulation patterns in the north Atlantic, and may lead to incipient speciation in the already distinct Azorean population. Future monitoring of this species is crucial to evaluate how this species is coping with current environmental changes.

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding.

This study examines the potential of next-generation sequencing based 'genotyping-by-sequencing' (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

Genetic and Morphological Characterization of Freshwater Shrimps (Caridina africana Kingsley, 1882) Reveals the Presence of Alien Shrimps in the Cape Floristic Region, South Africa.

Morphological identification and molecular data (mtDNA COI) were used to resolve the taxonomic identity of a non-native freshwater shrimp in the Cape Floristic Region (CFR) of South Africa and to evaluate levels of genetic diversity and differentiation in the species' core natural distribution. The species was morphologically and genetically identified as Caridina africana Kingsley, 1882, whose main natural distribution is in the KwaZulu-Natal (KZN) Province, more than 1200 km from the point of new discovery. Subsequently, sequence data from natural populations occurring in seven rivers throughout KZN showed the presence of nuclear copies of the mtDNA COI gene (NUMTs) in 46 out of 140 individuals. Upon removal of sequences containing NUMTs, levels of genetic diversity were low in the alien population (possibly as a consequence of a bottleneck event), while varying levels of genetic diversity and differentiation were found in natural populations, indicating habitat heterogeneity, fragmentation and restricted gene flow between rivers. Following the present study, the alien shrimp has survived the Western Cape's winter and dispersed into a nearby tributary of the Eerste River System, hence posing an additional potential threat to endangered endemics. Understanding the biology of this alien species will aid detection and eradication procedures.

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus F(ST) between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global F(ST) was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.

Insights into genetic diversity, parentage, and group composition of Atlantic white-sided dolphins (Lagenorhynchus acutus) off the west of Ireland based on nuclear and mitochondrial genetic markers.

The analysis of stranding events and the application of molecular markers can be powerful tools to study cryptic biological aspects of delphinid species that occur mainly in open ocean habitat. In the present study, we investigated nuclear and mitochondrial genetic variability of Atlantic white-sided dolphins that stranded from 1990 to 2006 (n = 42) along the west coast of Ireland, using 8 microsatellite loci and 599 bp of the mitochondrial DNA control region. Results from both classes of markers are concordant with the hypothesis of a large random-mating population of white-sided dolphins along the west coast of Ireland. In addition, the analyses of 2 live mass stranding events (19 and 5 individuals, respectively) revealed that dolphins within each group were mainly unrelated to each other, suggesting dispersal of both sexes from the natal group (i.e., no natal phylopatry). Parentage analyses allowed the identification of mother-offspring pairs but ruled out all adult males as possible fathers. In combination with data on age of individuals, these results confirmed previous knowledge on life-history parameters, with sexually mature females ranging between 11 and 15 years of age and an interbirth interval of at least 2 years. The present study provides novel information on population and group composition of Atlantic white-sided dolphins along the west coast of Ireland, where population and social structure of the species are still poorly understood.