Faster monitoring of the invasive alien species (IAS) Dreissena polymorpha in river basins through isothermal amplification

Abstract

Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.

eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity

“…A previous study on lotic environments targeting Dressenia species 23 proved LAMP to be efficient in detecting invasive organisms in this type of habitat. However, mussel species release very high numbers of eggs in the water (from 40 000 to one million per female) and, accordingly, also exhibit high larval abundances 26 . This likely produces more abundant DNA release than crayfish and, more generally, crustaceans, which are known to shed much lower amounts of DNA and in which larvae are not dispersed into the environment 27 , 28 , 32 .…”

“…Advances have been made in using LAMP assays to process environmental samples such as hive debris 24 and water samples 23 , 25 , 26 . Three of the LAMP studies based on eDNA detection in water samples were targeted at mussel species from the Dreissena genus 23 , 26 , and one was developed to detect the presence of a parasite’s intermediate host (a snail species) in drinking spots for livestock 25 . The coupling of eDNA sampling with a LAMP detection approach was thus proven operational.…”

“…The coupling of eDNA sampling with a LAMP detection approach was thus proven operational. However, (1) those assays targeted high-density population mussel species that shed high amounts of DNA into the environment through external fecundation and (2) the snail survey was undertaken in small water volumes with low stream velocity, thus limiting the impact of the dilution factor 25 , 26 .…”

Shining a LAMP on the applications of isothermal amplification to monitoring illegal wildlife trade

“…Confirming the presence of aquatic pest species has been explored through the development of LAMP based assays for monitoring quagga and zebra mussels in river basins (Carvalho et al 2021;Williams et al 2017). The first study addressing LAMP development aimed at streamlining the eDNA detection of quagga and zebra mussels in Michigan lakes (Williams et al 2017).…”

“…A further two CO1 assays targetting quagga and zebra mussels seperately, with a sensitivity of 0.001 pg and 0.01 pg respectively (Williams et al 2017). A subsequent novel zebra mussel assay targetted the CO1 gene with a detection limit of 1.12 pg, which was also developed and field tested for a range of sample types collected from Portuguese, Spanish and French sources (Carvalho et al 2021). An additional application has been the delimitation of eels in the genus Anguilla, with a focus on Anguilla anguilla, a critically endangered species (Spielmann et al 2019).…”