Development and validation of a quantitative PCR assay for the invasive mussel, Mytella strigata (Hanley, 1843). Report to the Department of Agriculture, Water and the Environment.
Abstract
The charru mussel (Mytella strigata) is a fouling species that has formed invasive populations in the south-eastern United States, Philippines, Taiwan, Singapore, India and throughout the Gulf of Thailand. Due to the species’ rapid spread throughout Asia and the potential of introduction to Australia, M. strigata was added to the Australian Priority Marine Pest List. Surveillance for exotic pests such as M. strigata is important to facilitate early detection and effective management of incursions. SARDI has developed a molecular surveillance system using plankton tows tested using qPCR assays for pests of concern. This molecular method provides greater survey confidence, while requiring less than half the staff time and equipment costs for sample collection and processing, than traditional survey methods (dive visual, traps, trawl sampling) for detection of target pests. qPCR assays are available for many pests of concern, but not for Mytella strigata. We therefore developed and laboratory validated nine putative qPCR assays for Mytella strigata. Putative assays targeting gene regions other than CO1 were found by initial validation testing to not be sufficiently specific. Of the five putative qPCR assays targeting CO1 gene regions, the two best performing, which each had assay efficiency > 90% and analytical limit of detection < 2 fg µL 1, were selected for further validation. DNA extracted from plankton samples collected around Australia was tested to assess field specificity, with no detections by the two selected assays occurring in these samples. Diagnostic performance of these selected assays for testing plankton and scrape samples was estimated using latent class modelling and the results of testing constructed samples, i.e. samples to which M. strigata tissue was added prior to extraction. The best performing assay showed diagnostic sensitivity (DSe) of 72.6% in plankton and 71.6% in scrape samples, with the other candidate assay having a similar DSe (71.9%) in scrapes, but slightly lower DSe (65.5%) in plankton. Both assays had high diagnostic specificity (99.7%) in both sample types. PCR inhibition as measured by a scale factor negatively impacted DSe of both assays, with higher scale factor resulting in increased CT value. The effect was, however, relatively minor, with detections occurring reliably even at scale factor > 10. We assessed a high throughput sequencing approach used for CO1 barcoding and established that is it suitable for confirmatory testing.
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The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation—from field sampling to the analysis of PCR‐based results—for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays, as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA‐based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks (e.g., “in silico analysis”), and arranged on a 5‐level validation scale from “incomplete” to “operational” with defined minimum validation criteria for each level. These variables were evaluated for 546 published single‐species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterized by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta‐analysis demonstrates the suitability of the 5‐level validation scale for assessing targeted eDNA assays. It is a user‐friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.
Considerable promise and excitement exist in the application of environmental DNA (eDNA) methods to environmental monitoring and species inventories as eDNA can provide cost‐effective and accurate biodiversity information. However, considerable variation in data quality, rigor, and reliability has eroded confidence in eDNA application and is limiting regulatory and policy uptake. Substantial effort has gone into promoting transparency in reporting and deriving standardized frameworks and methods for eDNA field workflow components, but surprisingly little scrutiny has been given to the design and performance elements of targeted eDNA detection assays which, by far, have been most used in the scientific literature. There are several methods used for eDNA detection. The most accessible, cost‐effective, and conducive to standards development is targeted real‐time or quantitative real‐time polymerase chain reaction (abbreviated as qPCR) eDNA analysis. The present perspective is meant to assist in the development and evaluation of qPCR‐based eDNA assays. It evaluates six steps in the qPCR‐based eDNA assay development and validation workflow identifying and addressing concerns pertaining to poor qPCR assay design and implementation; identifies the need for more fulsome mitochondrial genome sequence information for a broader range of species; and brings solutions toward best practices in forthcoming large‐scale and worldwide eDNA applications, such as at‐risk or invasive species assessments and site remediation monitoring.
High-throughput sequencing (HTS) technologies offer new promise to support surveillance programs targeting marine non-indigenous species (NIS). Metabarcoding might surpass traditional monitoring methods, for example through its ability to detect rare species, a key feature in early detection of NIS. Another interest of this approach is the identification of organisms difficult to identify based on morphology only (e.g., early developmental stages), making it relevant in the context of management programs. Because many marine benthic NIS have a biphasic bentho-pelagic life cycle, targeting their pelagic larval stages in zooplankton may allow early detection and assessment of their establishment and potential spread. We illustrate this approach with an analysis of bulk-DNA retrieved from a time-series of zooplankton samples collected over 22 months in one bay in Brittany (France). Using HTS of amplicons obtained with two markers (COI and 18S) and a metabarcoding approach, 12 NIS were identified and their temporal larval dynamics were monitored. Importantly, we chose to focus on a closed list of species, from four metazoan classes encompassing 52 NIS reported within the study area or nearby seas, with molecular references available or obtained locally for 42 of them. The use of a custom-designed database allowed the detection of three NIS otherwise not detected when using public databases. Interestingly, NIS known to have a short-lived larval stage were detected (e.g., the bryozoan Bugula neritina or the tunicate Corella eumyota). For two molluscs Ruditapes philippinarum and Crepidula fornicata, metabarcoding results were compared to those obtained using traditional methods (i.e., barcoding of individual larvae and morphology, respectively) to show the reliability of the approach in detecting and assessing the extent of their reproductive periods. Our results also revealed that the Pacific oyster Crassostrea gigas, a notorious invasive species, failed to reproduce in the study bay, showing that metabarcoding on larval stages also provides information regarding the establishment success (or failure) of NIS. While metabarcoding has its limitations and biases, this study demonstrates its effectiveness for surveillance of targeted NIS, notably to support management strategies like the European Marine Strategy Framework Directive (MSFD).
Successful detection of introduced marine pests (IMP) relies upon effective surveillance. However, the expedience of responding following IMP detection is often dependent upon the relationship between regulators and stakeholders.
Effective detection of IMP in areas such as commercial ports requires a collaborative approach, as port environments can be highly complex both above and below the water. This complexity can encompass physical, logistical, safety and legislative issues. With this in mind, the aquatic pest biosecurity section within the Department of Primary Industries and Regional Development (DPIRD) developed the State‐Wide Array Surveillance Program (SWASP) in collaboration with Western Australian Port Authorities and port industry stakeholders.
The SWASP is primarily based on passive settlement arrays for IMP detection. Arrays are deployed at strategic locations within Ports. Marine growth samples collected from the arrays are processed using Next‐Generation Sequencing (NGS) to identify the presence of IMP within a specific geographical location.
Over 8 years, participation in SWASP has grown from 3 to 11 ports, spanning over 11,000 km, from the tropical north to temperate south of Western Australia. The programme has proven to be highly effective as a means of fostering stakeholder involvement and, importantly for IMP surveillance. The growth and success of SWASP has continued primarily because of the commitment and farsightedness of the ports involved. The regular presence of the biosecurity regulator as a partner in SWASP has provided a consistent face for biosecurity and fostered good stakeholder relationships, ensuring there is a reliable and effective ongoing marine surveillance programme for the state.
Synthesis and applications. Through a united and collaborative approach to marine biosecurity surveillance, port authorities, industry and biosecurity regulators have developed the State‐Wide Array Surveillance Program (SWASP) and closed a major gap in biosecurity surveillance. The SWASP collaboration uses passive settlement arrays and molecular analyses to provide regular marine pest surveillance from the tropics to temperate regions of Western Australia. The continued commitment has embedded valuable relationships between stakeholder and regulator ensuring ongoing surveillance in marine biosecurity for the state. The Western Australian SWASP example has inspired other jurisdictions around Australia to develop similar collaborative approaches which will have far‐reaching marine biosecurity benefits.
Background
In the present study, phylogenetic relationships within Heterobranchia in particular to Pulmonata were evaluated by means of Histone-3 (H3) gene sequence information. H3 gene is a slow evolving marker and is useful in resolving the deep level relationships. This is the first study to report the phylogeny of Pulmonata with more number of representatives from the group on the basis of H3 gene.
Results
The major groups within Heterobranchia viz. Lower Heterobranchia, Opisthobranchia, and Pulmonata were non-monophyletic. A few of the pulmonate groups’ viz. Planorbidae, Lymnaeidae, Siphonariidae, Veronicellidae, and Stylommatophora were recovered as monophyletic. The concepts of Eupulmonata and Geophila were not observed in the present study.
Conclusions
The present study was undertaken with an objective to study the phylogeny of Pulmonata reconstructed on the basis of H3 gene and its ability to resolve the deeper divergences in Pulmonata. However, the resolution at the deeper nodes is limited. There is a good resolution at the level of genera. In the future, inclusion of more number of taxa with increased sequence length of H3 marker may yield resolved topologies that may shed more insights into the phylogeny of Pulmonata.
The invasive tropical American brackish water mussel Mytella strigata is recorded for the first time from the Indian sub-continent and this is the fourth report from the Indo-Pacific. The mussels were found attached in high densities (120 ± 24 ind. 25 cm-2) to floating plastic bottles, wooden pilings, walls of fish cages, hulls of boats and
bottom sediment in the Cochin backwater, Kerala, India during summer 2019. The only other bivalve species found with this population was Perna viridis. The mean length to height ratio for the Cochin population of M. strigata was 2.06 ± 0.26 cm with the largest individual having a length of 5.9 cm. The general external colour of the shells in Cochin population was uniformly black when wet but they exhibit dark
green over the dorsal and posterior areas with faint paler streaks when dry. In addition some of the individuals rarely form bright green colour and pattern as seen in Singapore populations. The mitochondrial DNA cytochrome c oxidase subunit I gene sequences of mussels from this study were consistent with specimens in their native range, from Colombia, and from Singapore where it has recently been reported
as invasive. The invasion of this species in Cochin may be through ballast water or fouling on ships hulls from its native range or from Singapore where it has been established recently. Their rapid growth, early maturity and wide salinity tolerance make them a potentially alarming fouling species in all brackish waters of India and
neighbouring countries.
The diagnostic accuracy of a test or rater has a crucial impact on clinical decision making. The assessment of diagnostic accuracy for multiple tests or raters also merits much attention. A Bayesian hierarchical conditional independence latent class model for estimating sensitivities and specificities for a large group of tests or raters is proposed, which is applicable to both with-gold-standard and without-gold-standard situations. Through the hierarchical structure, not only are the sensitivities and specificities of individual tests estimated, but also the diagnostic performance of the whole group of tests. For a small group of tests or raters, the proposed model is further extended by introducing pairwise covariances between tests to improve the fitting and to allow for more modeling flexibility. Correlation residual analysis is applied to detect any significant covariance between multiple tests. Just Another Gibbs Sampler (JAGS) implementation is efficiently adopted for both models. Three real data sets from literature are analyzed to explicitly illustrate the proposed methods.
In 2019, a survey of hard clam-cultured ponds along the southwestern coast of Taiwan revealed an unknown, almost ubiquitous, brackish water mussel. The mussels were attached to the concrete walls and drainage systems of the clam ponds, hulls of boats, bottom sediment, and riverbanks of the estuary. The largest individual had a shell length of over 6 cm. The general external color of the shells was uniformly dark brown or black, but shells with a dark greenish color were also found. The mitochondrial DNA cytochrome c oxidase gene sequences obtained from specimens were consistent with Mytella strigata from Singapore, India, and the Philippines. Based on interviews with farmers, the occurrence of this species has become common since 2014 and is increasing in abundance and density, suggesting that its first invasion may have occurred before 2014. The introduction vector of M. strigata may be ballast water discharged from ships or bio-fouled ship hulls from its native range or somewhere else in Asia. This report represents the first time this invasive tropical mussel (M. strigata) has been recorded in Taiwan and the fifth time it has been reported from the Indo-Pacific region. The rapid growth, high fecundity, and broad salinity tolerance of this species makes it a competitive threat to clams in the ponds as well as other native species.
This study investigated the distribution and utilization of Mytella strigata in the Philippines thru site surveys, focus group discussions, and interviews. Initially reported in Manila Bay in 2014, results showed that this mussel species had spread to provinces mainly in the western and northern sections of Luzon Island. Interviews and focus groups revealed that Asian green mussel and oyster farming were affected by the non-indigenous species resulting in a reduction in harvest of farmed shellfish. As a result, local communities in traditional shellfish farming sites generally expressed negative perception towards the non-indigenous species as this was not regarded as a food item. In contrast, local fishers in non-traditional shellfish farming sites generally welcomed M. strigata as it provided food and livelihood opportunities. These results show that the emergence of M. strigata in the Philippines had both positive and negative consequences on the livelihoods of affected local communities. However, local and national agencies had been slow in recognizing the threat of M. strigata on Asian green mussel and oyster industries. These results, therefore, provide useful information to address issues related to the non-indigenous mytilid species and signify the need to review local and national policies in response to the emergence of potentially invasive species.
Calonectria pseudonaviculata and C. henricotiae are the causal agents of boxwood blight, a devastating disease of boxwood that has caused significant economic impact on the nursery and landscape industries in the U.S. and in Europe. The two species are genetically distinct and are found in different geographic areas but are difficult to distinguish based on morphology and pathogenicity. Fast, accurate, and inexpensive methods to detect and differentiate these species is critical in stopping the spread of the disease. We designed primer pairs based on available sequences of four conserved regions - calmodulin, histone H3, Internal Transcribed Spacer, and β-tubulin – and tested their ability to differentiate the two Calonectria species. Here we report three primer pairs derived from sequence differences in the Histone H3 region that can be used to specifically detect C. pseudonaviculata, C. henricotiae, or both species. Specificity of these primers was tested against nine isolates of C. pseudonaviculata, three isolates of C. henricotiae, 13 other Calonectria species, and five isolates from related genera using conventional and real-time PCR. These are the first primers available that can be used with either a multiplexed conventional PCR or SYBR-based real-time PCR to specifically detect and differentiate the two fungal species.