Advanced molecular-based surveillance of quagga and zebra mussels: A review of environmental DNA/RNA (eDNA/eRNA) studies and considerations for future directions

Sensitive methods, capable of rapidly and accurately detecting aquatic invasive species, are in demand. Molecular-based approaches, such as environmental DNA (eDNA) surveys, satisfy these requirements and have grown in popularity. As such, eDNA surveys could aid the effort to combat the colonisation and spread of two notoriously invasive freshwater mussel species, the quagga mussel (Dreissena rostriformis bugensis) and zebra mussel (D. polymorpha), through improved surveillance ability. Here, we provide a review of dreissenid eDNA literature (both grey and published), summarising efforts involved in the development of various assays for use in multiple different technologies (e.g. quantitative PCR, high-throughput sequencing and loop-mediated isothermal amplification) and sampling scenarios. We discuss important discoveries made along the way, including novel revelations involving environmental RNA (eRNA), as well as the advantages and limitations of available methods and instrumentation. In closing, we highlight critical remaining gaps, where further investigation could lead to advancements in dreissenid monitoring capacity.

Heteroplasmy and repeat expansion in the plant-like mitochondrial genome of a bivalve mollusc

Background: Animal mitochondrial genomes are typically circular, 14-20 kb in length, maternally inherited, contain 13 coding genes, two ribosomal genes and are homoplasmic. In contrast, plant mitogenomes display frequent gene rearrangements, often contain greatly expanded repetitive regions, encode various open reading frames of unknown function and may be heteroplasmic due to differential repeat expansions between molecules. Error correction by recombination is common in plant mitochondria and has been proposed as the driver behind the rearrangements and repeat expansions that are often observed. In contrast, most animal mitochondria never or only very seldomly recombine and their utilisation of other repair mechanisms for mitochondrial genome error correction is a potential driver of their non-coding DNA reduction. Results: Using PacBio long reads for genome assembly and structural variant detection, we identify evidence of heteroplasmy in the form of variable repeat lengths within two blocks of repetitive DNA within the expanded 46 kb mitochondrial genome of the bivalve mollusc, quagga mussel, Dreissena rostriformis. The quagga mussel also has a greatly expanded repertoire of coding genes in comparison to most animals which includes an additional nine open reading frames (ORFs) encoding predicted transmembrane peptides of unknown orthology. Conclusions: The genome size, repeat content and coding gene repertoire of the quagga mussel mitogenome closely resemble those of plants and the identification of repeat-associated heteroplasmy is consistent with the utilisation of plant-like recombination-based error correction mechanisms. Given the frequency of mitochondrial repeat expansions within the Bivalvia, recombination may be an underappreciated mechanism for mitogenomic error correction within this and other animal lineages.

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels, Dreissena rostriformis bugensis in flowing water

The early detection of invasive non-native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS – particularly during the early stages of an invasion.Here, we compared the use of traditional kick-net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR, and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis; a high priority INNS, along a density gradient on the River Wraysbury, UK.All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples, and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.Synthesis and application. All three molecular approaches were more sensitive than traditional kick-net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community, and consequently impacts of INNS.

The quagga mussel genome and the evolution of freshwater tolerance

Freshwater dreissenid mussels evolved from marine ancestors during the Miocene ∼30 million years ago and today include some of the most successful and destructive invasive species of freshwater environments. Here, we sequenced the genome of the quagga mussel Dreissena rostriformis to identify adaptations involved in embryonic osmoregulation. We provide evidence that a lophotrochozoan-specific aquaporin water channel, a vacuolar ATPase subunit and a sodium/hydrogen exchanger are involved in osmoregulation throughout early cleavage, during which time large intercellular fluid-filled ‘cleavage cavities’ repeatedly form, coalesce and collapse, expelling excess water to the exterior. Independent expansions of aquaporins coinciding with at least five freshwater colonization events confirm their role in freshwater adaptation. Repeated aquaporin expansions and the evolution of membrane-bound fluid-filled osmoregulatory structures in diverse freshwater taxa point to a fundamental principle guiding the evolution of freshwater tolerance and provide a framework for future species control efforts.