The rate of non-native species introductions has increased dramatically in modern times, correlating with human population growth, advances in transportation, and increased international trade (Mack et al., 2000). In aquatic ecosystems, new species are often introduced accidentally via ballast waters from ships or in some cases introductions are intentional efforts organized by local governments to improve fisheries (see Gaither et al. 2010, 2012 for examples in Hawaiʻi). Aquarium releases are a common invasion pathway, with numerous examples of popular aquarium fish ending up in freshwater and marine systems (Padilla & Williams 2004). While most introduced species never become established, those that persist and thrive can have serious economic impacts (Pimentel et al., 2005) and can pose a significant threat to biodiversity and ecosystem function (Simberloff, 2003; Burgiel et al., 2006; Albins & Hixon, 2013; Gaither et al., 2013a; Reaser et al., 2020). Documenting the spread and range of introduced species is essential to understanding their overall consequences on the ecosystems they invade (Gaither et al., 2013b; Coleman et al., 2014) and is necessary to plan effective management strategies.
The Indo-Pacific lionfish Pterois volitans (family Scorpaenidae) was introduced in the western Atlantic Ocean in the early 1980s (Whitfield et al., 2002). Later, genetic analyses also identified the presence of P. miles mitochondrial haplotypes in the invasive population, but only at low frequency, indicating the introduction of both species (Hamner et al., 2007). Since the first report of lionfish in southern Florida in 1985 (Schofield, 2009), they have spread throughout much of the Caribbean. Individuals have been caught as far north as Massachusetts and are well-established along most coastal regions between North Carolina and Venezuela (Schofield et al, 2022), with an individual captured as far south as southeastern Brazil (Ferreira et al., 2015, Luiz et al., 2021). They are typically found at depths between 2 and 55 m but have been recorded at 304 m off the coast of Bermuda (Nuttall et al., 2014; Gress et al., 2017). While an accidental or intentional release from aquaria is the most often cited source for the Atlantic introduction of lionfish (Whitfield et al., 2002, Semmens et al., 2004), ballast transport cannot be ruled out as the invasion pathway, nor as a factor contributing to their spread (Wonham et al., 2000; Whitfield et al., 2002; Semmens et al., 2004).
Lionfish are generalist predators that consume a wide variety of fishes (Côté et al., 2013), including rare and endemic species such as the critically endangered social wrasse, Halichoeres socialis (Rocha et al., 2015). A decline in the recruitment of native fish species has been attributed to lionfish predation (Benkwitt, 2015), and while they are a marine species, their broad salinity tolerance may allow them to invade sensitive estuarine environments (Barbour et al., 2010; Jud et al., 2014; Schofield et al., 2015). For instance, in central Florida, lionfish are suspected to have entered the Indian River Lagoon (IRL), which spans nearly 400 km of Florida's eastern coastline. The IRL, like other estuaries throughout the region, supports a diversity of wildlife (Gilmore, 1995). While observations recorded on iNaturalist (https://www.inaturalist.org/) and the United States Geological Survey (Schofield et al., 2022) Nonindigenous Aquatic Species (NAS; https://nas.er.usgs.gov/) database show that lionfish are found at IRL inlets, the extent of encroachment into this and other estuaries is unknown as the rocky substrates suitable for lionfish in these ecosystems largely go unmonitored.
Traditional approaches for monitoring marine invasive species typically involve either direct observation or the capture of individuals. However, these techniques are time consuming, expensive, and often impractical (Eble et al., 2020). An attractive alternative is the use of environmental DNA (eDNA) for the detection and tracking of invasive species (Fediajevaite et al., 2021; Forsman et al., 2022). Environmental DNA is the genetic material shed by organisms into their surrounding environment. This DNA can be extracellular or contained within whole cells sloughed from an organism (Ficetola et al., 2008; Goldberg et al., 2011; Taberlet et al., 2012; Kumar et al., 2020). By isolating DNA from environmental samples and utilizing PCR methods, it is possible to detect the presence of an organism in the nearby environment. These approaches are now widely used to track invasive, at-risk, and rare organisms across a diversity of habitats (Ficetola et al., 2008; Goldberg et al., 2011; Taberlet et al., 2012; Goldberg et al., 2013; Takahara et al., 2013; Roux et al., 2020; Rodgers et al., 2020; Kumar et al., 2022a; Gaither et al., 2022). Moreover, in many cases, eDNA approaches have been shown to be more sensitive and time-efficient compared to traditional monitoring methods (Fediajevaite et al., 2021).
Due to the increasing interest in eDNA approaches for biodiversity monitoring, there are a growing number of species-specific assays designed to detect and monitor marine species, including one for lionfish. Whitaker et al. (2021) is the first and only published PCR assay for the detection of lionfish P. volitans and P. miles based on the mitochondrial cytochrome oxidase I (COI) gene. However, the specificity of this assay is unverified, and the critical performance parameters limit of detection (LOD) and limit of quantification (LOQ) have not been established. Here we evaluate the method of Whitaker et al. (2021) and design and test a new qPCR TaqMan® probe-based assay targeting the mitochondrial cytochrome b (Cytb) gene. We determine the specificity of these assays using tissues collected from 23 non-target species (primarily from the family Scorpaenidae) known to inhabit the western Atlantic and report LOD and LOQ for the new TaqMan® assay.