Moving towards improved surveillance and earlier diagnosis of aquatic pathogens: from traditional methods to emerging technologies

Early and accurate diagnosis is key to mitigating the impact of infectious diseases, along with efficient surveillance. This however is particularly challenging in aquatic environments due to hidden biodiversity and physical constraints. Traditional diagnostics, such as visual diagnosis and histopathology, are still widely used, but increasingly technological advances such as portable next generation sequencing (NGS) and artificial intelligence (AI) are being tested for early diagnosis. The most straightforward methodologies, based on visual diagnosis, rely on specialist knowledge and experience but provide a foundation for surveillance. Future computational remote sensing methods, such as AI image diagnosis and drone surveillance, will ultimately reduce labour costs whilst not compromising on sensitivity, but they require capital and infrastructural investment. Molecular techniques have advanced rapidly in the last 30 years, from standard PCR through loop‐mediated isothermal amplification (LAMP) to NGS approaches, providing a range of technologies that support the currently popular eDNA diagnosis. There is now vast potential for transformative change driven by developments in human diagnostics. Here we compare current surveillance and diagnostic technologies with those that could be used or developed for use in the aquatic environment, against three gold standard ideals of high sensitivity, specificity, rapid diagnosis, and cost‐effectiveness

Vitellogenin is not an appropriate biomarker of feminisation in a Crustacean

The expression of the yolk protein vitellogenin (Vtg) has been used as a biomarker of feminisation in multiple fish species throughout the world. Since the late 1990s, researchers have attempted to develop similar biomarkers to address whether reproductive endocrine disruption also occurs in the males of invertebrate groups such as the Crustacea. To date, the vast majority of studies investigating Vtg induction in male Crustacea have resulted in negative or inconclusive results, leading researchers to question the utility of Vtg expression as a biomarker in this taxon. This study measured the expression of Vtg genes in two intersex phenotypes (termed internal and external) found in the male amphipod, Echinogammarus marinus, and compared them with those of normal males and females. Males presenting the external intersex phenotype are infected with known feminising parasites and display a variety of feminised traits including oviduct structures on their testes and external female brood plates (oostegites). The internal intersex male phenotype, that displays a pronounced oviduct structure on the testes without the external intersex characteristics, is not parasite infected and it is thought to be a result of environmental contamination. Given their morphology, these phenotypes might be considered highly ‘feminised’ or ‘de-masculinised’ and can be utilised to test the suitability of feminisation biomarkers. The E. marinus transcriptome was searched for genes resembling Vtg and two sequences were revealed, that we subsequently refer to as Vtg1 and Vtg2. Results from a high-throughput transcriptomic sequencing screen of gonadal cDNA libraries suggested that very low expression (in this manuscript gene transcription is taken to represent gene expression, although it is acknowledged that in addition to transcription, translation, transcript processing, mRNA stability and protein stability can regulate gene expression) of Vtg1 and Vtg2 in normal males (ESTs = 1 and 0 for Vtg1 and Vtg2, respectively), internal intersex males (ESTs = 0 for both Vtg sequences) and external intersex males (ESTs = 5 and 0 for Vtg1 and Vtg2, respectively). In contrast, the sequencing suggested notable levels of expression of both Vtg genes in females (ESTs = 1133 and 84 for Vtg1 and Vtg2, respectively). Subsequent qPCR analysis validates these expression levels, with the signal for Vtg1 and Vtg2 transcripts in all male phenotypes being indistinguishable from that caused by contamination of trace levels of genomic DNA or the low-level amplification non-target sequences. These findings suggest that Vtg expression is not notably induced in highly feminised amphipods and is therefore not an appropriate biomarker of feminisation/de-masculination in crustaceans. We discuss our findings in the context of previous attempts to measure Vtg in male crustaceans and suggest a requirement for more appropriate taxon-specific biomarkers to monitor feminisation in these groups

DNA-based methods: technology solutions to evaluate ecosystem function (Part of 'Understanding ecosystems and resilience using DNA: Chief Scientist’s Group report')

Isolation of genetic material from environmental samples (soil, water sediment), so called eDNA is now routine. These samples provide insight into the macro and micro ecology of the ecosystem from which they are isolated. Targeting specific sequences from specific species allows non-invasive sampling of organisms of interest. Information derived from DNA can be used to support conservation efforts and it also allows the tracking of pathogens and invasive species. More generic approaches allow us to profile bacterial, fungal, algal, plant or animal species. From these data we can perform multi-dimensional analysis, addressing key environmental questions. For example, a bacterial profile can report on the presence of pathogens or the impact of pollutants. Quantitative genetic approaches and Next Generation Sequencing technology that supports the use of eDNA, represents a mature technology with validated applications throughout the healthcare industry. New innovations will provide additional, enhanced utility and cost efficiencies, supporting increased spatial and temporal resolution monitoring with the potential to provide real time surveillance. Currently, these eDNA approaches have been deployed as adjuncts to established approaches limiting the potential benefits gained. Our recommendation is for a new generation of biomonitoring approaches to be adopted based on the full potential proved by eDNA. The potential of these novel eDNA approaches combined with good ecological knowledge and interpretation will provide the tools needed to realise ‘A Green Future’, and deliver the “Plan to Improve the Environment”. Since the technology is well established, the major hurdles to exploiting eDNA tools are in transferring from research tools to regulatory and industrial implementation. Our recommendation therefore are focused around a four step process to fast-track this objective: 1. Define and specify the explicit need or question. 2. Establish a transparent ‘AGILE’ assessments and validation processes. 3. Liaise between UKRI and BEIS to establish funding pipeline from research concept to product. 4. Engage with the industrial sectors to deliver products and services for the environmental sector

Metallothioneins may not be enough - the role of phytochelatins in invertebrate metal detoxification

'Viewpoint' article

The regulation of copper stress response genes in the Polychaete Nereis diversicolor during prolonged extreme copper contamination

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their conspecifics

A large set of microsatellites for the highly invasive earthworm Amynthas corticis predicted from low coverage genomes

Invasive species can significantly affect local biodiversity and create important challenges for conservation. They usually present an outstanding plasticity that permits the adaptation to the new environments. Understanding their genetic background is fundamental to better comprehend invasion dynamics and elaborate proper management plans as well to infer population and evolutionary patterns. Here, we present a reasonable set of tools for the study of a highly invasive earthworm, the megascolecid Amynthas corticis. We designed in silico a large set of primers targeting microsatellite regions (ca. 9400) from two low coverage genomes presented here. This study provides 154 high quality primer pairs targeting polymorphic repeats conserved in two Amynthas corticis mitochondrial lineages. From this dataset, a set of primer pairs (15) was validated by polymerase chain reaction with 86% consistent amplification, confirming the accuracy of the in silico prediction. Nine of the primer pairs tested were selected for population genetics and presented polymorphism in the studied populations, thus showing promising potential for future studies of this global invasive species. The nuclear markers used in this study appear to recapitulate and complement the mitochondrial relationships found in a previous study. Interestingly, all genotyped individuals showed at least one triploid locus profile among the tested loci, which may be evidence of polyploidy associated to their life history, in particular to asexual reproduction by parthenogenesis

Population screening and transmission experiments indicate paramyxid-microsporidian co-infection in Echinogammarus marinus represents a non-hyperparasitic relationship between specific parasite strains

Phylogenetically distant parasites often infect the same host. Indeed, co-infections can occur at levels greater than expected by chance and are sometimes hyperparasitic. The amphipod Echinogammarus marinus presents high levels of co-infection by two intracellular and vertically transmitted parasites, a paramyxid (Paramarteilia sp. Em) and a microsporidian strain (Dictyocoela duebenum Em). This co-infection may be hyperparasitic and result from an exploitative ‘hitchhiking’ or a symbiotic relationship between the parasites. However, the best-studied amphipod species are often collected from contaminated environments and may be immune-compromised. Immune-challenged animals frequently present co-infections and contaminant-exposed amphipods present significantly higher levels of microsporidian infection. This suggests the co-infections in E. marinus may result from contaminant-associated compromised immunity. Inconsistent with hyperparasitism, we find that artificial infections transmit Paramarteilia without microsporidian. Our population surveys reveal the co-infection relationship is geographically widespread but find only chance co-infection between the Paramarteilia and another species of microsporidian, Dictyocoela berillonum. Furthermore, we identify a haplotype of the Paramarteilia that presents no co-infection, even in populations with otherwise high co-infection levels. Overall, our results do not support the compromised-immunity hypothesis but rather that the co-infection of E. marinus, although non-hyperparasitic, results from a relationship between specific Paramarteilia and Dictyocoela duebenum strains

The genome sequence of the common earthworm, Lumbricus terrestris (Linnaeus, 1758)

We present a genome assembly from an individual Lumbricus terrestris (the common earthworm; Annelida; Clitellata; Haplotaxida; Lumbricidae). The genome sequence is 1,056.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.93 kilobases in length

Quantitative analysis of gene expression changes in response to genotoxic compounds

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n = 18) chosen to represent compounds that are genotoxic (n = 8), non-genotoxic non-carcinogenic (n = 2) or have a less well defined mechanism of action with respect to genotoxicity (n = 8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1 fmol but often 10–50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology