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Management of non-indigenous invasive species (NIS) is challenging owing in part to limitations of early detection and identification. The advent of environmental DNA (eDNA) techniques provides an efficient way to detect NIS when their abundance is extremely low. However, eDNA-based methods often suffer from uncertain detection sensitivity, which requires detailed testing before applying these methods in the field. Here we developed an eDNA tool for early detection of the highly invasive golden mussel, Limnoperna fortunei, based on the mitochondrial cytochrome c oxidase subunit I gene (COI). Further, we tested technical issues, including sampling strategy and detection sensitivity, based on a laboratory experiment. We then applied the method to field samples collected from water bodies in China where this mussel has or is expected to colonize. Results showed that the detection limit varied extensively among our newly developed primer pairs, ranging from 4 × 10−2 to 4 × 10−6 ng of total genomic DNA. Laboratory detection was affected by the availability of eDNA (i.e., both mussel abundance and incubation time). Detection capacity was higher in laboratory samples containing re-suspended matter from the bottom layer versus that collected from the surface. Among 25 field sites, detection was 100% at sites with high mussel abundance and as low as 40% at sites with low abundance when tested using our most sensitive primer pair. Early detection of NIS present at low abundance in nature requires not only sensitive primers, but also an optimized sampling strategy to reduce the occurrence of false negatives. Careful selection and detailed testing of primer pairs ensures effective eDNA-based species detection in surveillance and management programs.

This work evaluates the tolerance of adult Limnoperna fortunei to aerial exposure at different temperatures. The aim was to contribute knowledge about factors, such as the mussel’s capacity for survival out of water over long periods, allowing the invasion of new environments after terrestrial transport. The analysis of the results revealed that the time of exposure to air influenced mortality at different temperatures throughout the experimental period (P < 0.001). The time to reach 100% mortality at temperatures of 10°C, 20°C, and 30°C were 11, 6, and 3 days, respectively. Mussel mortality outside the water is directly related to air temperature, so the higher the temperature is, the greater the mortality. The results suggest that in the case of the land transport of structures biofouled with L. fortunei, this species can remain alive for 2 to 10 days, depending on the ambient temperature. Thus, aerial exposure may be used as a strategy to control the dispersal of the golden mussel. However, the time required for effective elimination of all individuals depends on the air temperature. The aerial exposure can also be used to periodically clean industrial systems and to eliminate the dispersion of propagules by vectors (e.g., transport of equipments/watercrafts).

Zhan et al. (Biological Invasions, 2015, 17:3073–3080) stressed that China’s South-to-North Water Transfer Project (SNWTP)—the world’s largest constructed water diversion—could create an invasion highway by facilitating spread of non-native species, including invasive golden mussel Limnoperna fortunei. However, most available literature indicated that golden mussels could not survive the cold winter in Northern China. We proposed that phenotypic plasticity and rapid environmental adaptation, combined with relatively high water temperature derived from wastewater treatment plant effluents and a large potential inoculum continuously transported from southern source populations, could jointly contribute to golden mussel spread into northern locations. We conducted surveillance for the species both before and after the waterway was opened in late 2014 in the diversion destination—Beijing. While all surveys in the whole area were negative between 2014 and 2018, we detected rapid geographical expansions in 2019–2021 across multiple waterbodies based on traditional field surveys and environmental DNA (eDNA)-based methods. Surprisingly, we subsequently observed populations that had successfully survived a cold winter in Beijing. The SNWTP may facilitate further spread of cold-adapted populations, placing high-latitude areas at risk. This case study highlights the need for robust scientific assessment and management to predict and mitigate non-native species’ distributional changes that may accompany large-scale hydraulic projects.

We review the research on L. fortunei over the past 22 years, systematically elucidating its impacts on ecological environments and water engineering structures. We explored the effects of external factors on the invasion and spread of L. fortunei, as well as the internal factors that impact the ecological environment and water engineering structures. We also provide new perspectives and directions for L. fortunei control. The major research findings include the following: (1) L. fortunei negatively impacts hydraulic structures, being hard to remove and capable of damaging them, disrupting normal operations. (2) L. fortunei’s ecological impact is multifaceted: it reduces water cloudiness and organic matter by filtering suspended particles and depositing feces, but its decay after death consumes dissolved oxygen, increasing chemical oxygen demand and lowering water quality. (3) L. fortunei control techniques are effective for localized use in small bodies of water and aqueducts, but their control in large open reservoirs is difficult to achieve with one method. Existing control methodologies for L. fortunei were systematically evaluated across multiple dimensions, including engineering applicability and feasibility, technical advantages and limitations, and economic cost-effectiveness. This comprehensive analysis establishes a decision-support framework for optimizing control strategy selection in diverse engineering scenarios and application contexts.

River–floodplain systems are characterized by high connectivity, which favours the spread of non-native species. In floodplain, floods increase connectivity, which increases the similarity of abiotic conditions among environments. High connectivity and low environmental variability may favour the establishment of non-native species such as Limnoperna fortunei, but this has not yet been tested. We sampled L. fortunei larvae in nine connected lakes and nine isolated lakes to rivers in the upper Paraná River floodplain to evaluate how spatial (connection) and abiotic (environmental variability) factors affect the larvae density of L. fortunei. We considered the rivers as propagule source of L. fortunei because this invasive species has successfully established in rivers, but not in lakes. Our findings revealed that connected lakes had a high larval density of L. fortunei, while isolated lakes had a low density. Isolated lakes presented a high multi-environmental variability, which was strong negatively related with the larval density of L. fortunei. However, the connectivity decreased the multi-environmental variability, indirectly increasing the larval density of L. fortunei. Our study illustrates that permanent connectivity with invaded environments increase the larvae density of L. fortunei in non-invaded environments, which occurs both directly (through propagule dispersion) and indirectly (by decreasing multi-environmental variability).

Limnoperna fortunei, an invasive mussel altering the structure of benthic communities, is preyed upon by several fish species in South America. To investigate the impact of predatory fish on populations of this mussel, and the effects of this top-down interaction on native macroinvertebrates, we performed an in situ experiment in the Uruguay River. By comparing benthic communities of artificial substrates colonised in treatments allowing or excluding fish access, it was demonstrated that fish significantly reduce colonisation by L. fortunei (i.e. its density, biomass and body size). In the absence of mussels (before settlement), fish preyed upon the native macroinvertebrate community inhabiting artificial substrates, reducing their densities compared to the fish exclusion treatment. However, it was found that in the presence of golden mussel (after settlement), the total density of benthic macroinvertebrates, densities of scrapers, and densities of dipterans and gastropods strongly decreased in fish exclusion treatments, suggesting a strong effect of L. fortunei in reducing the native fauna. By preying on L. fortunei, fish favoured the persistence of native macroinvertebrates. Further studies focused on predatory fish species and their efficiency in removing L. fortunei may contribute to advancing towards using native fish as mitigating agents.

Detection of species in nature at very low abundance requires innovative methods. Conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) are two widely used approaches employed in environmental DNA (eDNA) detection, though lack of a comprehensive comparison of them impedes method selection. Here we test detection capacity and false negative rate of both approaches using samples with different expected complexities. We compared cPCR and qPCR to detect invasive, biofouling golden mussels (Limnoperna fortunei), in samples from laboratory aquaria and irrigation channels where this mussel was known to occur in central China. Where applicable, the limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate of each PCR method were tested. Quantitative PCR achieved a lower LoD than cPCR (1 × 10−7 vs. 10−6 ng/μl) and had a higher detection rate for both laboratory (100% vs. 87.9%) and field (68.6% vs. 47.1%) samples. Field water samples could only be quantified at a higher concentration than laboratory aquaria and total genomic DNA, indicating inhibition with environmental samples. The false negative rate was inversely related to the number of sample replicates. Target eDNA concentration was negatively related to distance from sampling sites to the water (and animal) source. Detection capacity difference between cPCR and qPCR for genomic DNA and laboratory aquaria can be translated to field water samples, and the latter should be prioritized in rare species detection. Field environmental samples may involve more complexities—such as inhibitors—than laboratory aquaria samples, requiring more target DNA. Extensive sampling is critical in field applications using either approach to reduce false negatives. This manuscript presents a comprehensive comparison of conventional PCR and real‐time quantitative PCR for detecting rare species from environmental DNA (eDNA) samples, with detection optimization and method verification using samples from different sources. We recommend real‐time qPCR as a prioritized method for eDNA‐based rare species detection, as well as the use of replicate samples to reduce false negatives.

Limnoperna fortunei, a problematic freshwater invasive mussel in South America, was first detected in 1991 at Bagliardi Beach, Río de la Plata (Argentina). Since then, there has been a high increase in population density, reaching up to 150,000 ind. m−2. The distribution, density, individual sizes, and associated mollusk assemblages of L. fortunei were evaluated 30 years after its first detection. Seven sites along Río de la Plata River were sampled between 2018 and 2020. The highest density was recorded in the La Balandra Beach (above the stabilization value) and the lowest density in the Martín García Island (IMG—by its acronym in Spanish—, below the stabilization value). Two reproductive events were observed: late spring and late summer. Our results showed different sets of species associated with Limnoperna fortunei, with protected areas such as the IMG standing out, showing greater species richness, including first records, versus other coastal environments. We recommend increasing conservation efforts given the constant advance of urbanization in the coastal sites of the province of Buenos Aires, with environmental impact studies prior to coastal reforms, and implementation of density control strategies for Limnoperna fortunei in protected areas.

Many countries/areas are experiencing or may soon experience water scarcity owing to rapid population growth, urbanization and/or climate change. Currently, almost one-fifth of the world’s population (1.2 billion) live in areas of physical water scarcity. Water diversions have become a commonplace solution proposed by governments for alleviation of physical water scarcity. Thus far, more than 80 major projects are completed or under construction globally, including the world’s largest diversion: South-to-North Water Transfer Project (SNWTP) in China. Negative effects associated with water diversions, such as habitat loss and transfer of pollutants, have been recognized. However, it has been largely overlooked that “invasion highways” are created when water diversions link biogeographic regions. These “invasion highways” can facilitate spread of an array of non-native species. Although previous experiences have provided clear warnings regarding spread of non-native invasive species through artificial waterways, these lessons have been largely ignored by governments when resolving water scarcity problems. Here we use SNWTP, which will likely facilitate spread of invasive golden mussels, as well as many known examples of non-native invasive species spread through artificial waterways in other water systems, to call on governments to formally establish policy and seek management solutions to considering spread of non-native species when planning water diversions.

Among the invasive species known to occur in South America is the bivalve mollusc Limnoperna fortunei, whose presence is linked to several environmental and economic problems. Early detection and mitigation actions are needed to limit its impact in the remaining L. fortunei-free areas. PCR-based molecular methods have become the gold standard methodology for L. fortunei detection. However, PCR-based methods require complex logistics from field sampling to laboratory processing. Thus, the use of methods that can be directly applied in the field can speed up the detection process. This work aimed to establish, for the first time, the loop-mediated isothermal amplification (LAMP) method for the detection of L. fortunei, with perspectives for in situ application. A set of primers designed for LAMP was tested for amplification of DNA from L. fortunei adult tissues and environmental samples containing bivalve larvae. The test showed a limit of detection as low as 0.01 ng of DNA obtained from adult tissue samples and a minimum reaction time of 60 min. The set of primers used seems to be specific for L. fortunei, since there was no cross-amplification with other bivalve or invasive molluscs that co-occur with the golden mussel in the same environment. The LAMP technique also proved to be efficient in amplifying DNA derived from L. fortunei larvae, demonstrating it to be a robust method regarding potential environmental reaction inhibitors. Although the results obtained here were acquired under controlled laboratory conditions, the LAMP method is a promising tool to integrate L. fortunei invasion monitoring protocols.