Technical note

One of the major threats to global biodiversity is invasive exotic species. The golden apple snail, Pomacea canaliculata (Lamarck, Gastropoda, Ampullariidae) is native to South America but was introduced to East and South-East Asia during the 1980s for commercial production and as a dietary protein supplement (Anderson 1993; Matienzo 1984; Mochida 1991). P. canaliculata feed on aquatic flora and inhabit slow-moving rivers, dams and irrigation channels. They are tolerant to variable temperatures, salinities and humidity making them eminently suited for successful dispersal (Cowie 2002). Since their introduction to East and South-East Asia, P. canaliculata have escaped, becoming a major pest to rice crops and other freshwater environments across Asia reaching Japan, the Philippines, New Guinea and Malaysia (Pain 1946; Carlsson et al. 2004; Hirai 1988; Rejesus et al. 1990; Halwart 1994). There are serious concerns that P. canaliculata will also establish and spread throughout much of northeast Australia, Bangladesh and India (Department of Agriculture, Forestry and Fisheries, Australia).

One of the major challenges in an effective management strategy across Australasia has been the development of a definitive diagnosis of exotic ampullarids. Indeed, there are no clear taxonomic criteria for distinguishing among species since the external differences are subtle; the within-species variation is high (e.g. Estebenet and Martin 2003), and separation of the highly invasive P. canaliculata from other ampullarid snails can only be accomplished by a trained malacologist. Yet, for urgent diagnoses, such as quarantine operations, timely and correct identifications are critical.

Molecular diagnostic techniques such as DNA ‘barcoding’ can circumvent problems associated with morphological identification (e.g. Herbert et al. 2003). For example, Cytochrome Oxidase Subunit I (COI) is easily amplified with universal primer sets across different animal species and has high phylogenetic resolution between species compared to other mitochondrial genes (Folmer et al. 1994). Here, using COI as the marker, we develop a rapid DNA-based diagnostic protocol that identifies P. canaliculata, from the non-invasive but phenotypically similar species P. insularum and P. bridgesii without the use of DNA sequencing.

DNA was extracted from 16 Pomacea samples from 10 to 50 mg of frozen tissue or eggs using the DNeasy® Blood and Tissue kit (Qiagen). COI was amplified by PCR in P. canaliculata, P. insularum and P. bridgesi, in reactions containing 1× CL buffer (Qiagen), 0.5× Q solution (Qiagen), 3.5 mM MgCl2, 0.05 mM of each dNTP, 10 μm of each primer LCO1490 and HCO2198 (Table 1), 2× BSA, and 5U of Taq polymerase (Qiagen). PCR cycling conditions were 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and a final extension of 72°C for 5 min. PCR product was checked using 1% TAE agarose gel electrophoresis and purified using ExoSap-IT® (USB Corporation, USA). Purified PCR product was sequenced by the Australian Genome Research Facility (AGRF) using an AB-3730xl (Applied Biosystems, USA).

Table 1 Primers used in multiplex PCR to amplify the mtDNA COI gene in Pomacea sp.
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Sequences were aligned using Sequencher (Gene Codes Corporation, USA) and checked for the absence of stop codons. The universal COI primers amplified ~658 base pairs (bp) fragments for all tested snails with suitable DNA quality. It should be noted that DNA extracted from degraded tissue samples did not amplify or produced low quality sequence data that was unsuitable for further analyses. Pomacea species identities were established or confirmed using GenBank® Blast and the Barcode of Life Data System (Bold). Samples that were identified with 99–100% confidence were used for Neighbour-joining phylogentic analysis using Paup v.4.0 to assess the resolution of COI to distinguish between Pomacea species of interest.

Generally, nucleotide divergence was low within-species (0–5%) and moderate between species ranging from 10% between P. insularum and P. canaliculata to 17% between P. insularum and P. bridgesii. Neighbor-joining phylogenetic analysis resulted in three well supported monophyletic clades representing each species. Species identities within the phylogenetic tree were consistent with GenBank and BOLD database results.

Visual comparison of the nucleotide sequences between each species, in combination with genetic distance and phylogenetic analysis suggested that nucleotide differences in COI were valid differentiators between species (Fig. 1). To create a single rapid molecular method to distinguish P. canaliculata, P. insularum and P. bridgesi, we developed a multiplex PCR protocol employing species specific forward primers in combination with the universal reverse primer. Here, forward primers from Matsukura et al. (2008) for P. canaliculata and P. insularum that amplify a 666 bp and a 390 bp fragment of the COI, respectively were used (Table 1). For P. bridgesi, a forward primer was designed in Oligo ® v.7 (MBI, USA) for a 250 bp fragment of COI. The universal reverse primer HCO2198 was used in the multiplex PCR. Amplification of COI fragments in the multiplex PCR used the same reaction conditions as above with 10 μm of each primer HCO2198, PcanCOI, PinsCOI and PbridCOI (Table 1). Multiplex PCR products were then assessed using 1% TAE agarose gel electrophoresis. In all amplifications, multiple fragments were not detected simultaneously in any snail. It must be noted however, if another ampullariid species is sampled, this multiplex PCR will produce uninformative results. Here, the new sample should be identified via PCR and DNA sequencing using primers LCO1490 and HCO2198 and sequence results can be used to design a new primer that is compatible with the multiplex PCR described here.

Fig. 1
figure 1

a Neighbour-joining phylgenetic tree showing relationships between P. canaliculata, P. insularum, and P. bridgesii inferred from 658 bp of COI mtDNA gene. Numbers below branches are bootstrap support values based on 1,000 bootstrap replicates, b Image of agarose gel electrophoresis showing length differences of the amplified fragments between P. canaliculata, P. insularum, and P. bridgesii. Fragments amplified were 666, 390 and 250 bp, respectively

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We have developed a fast and reliable molecular method that easily discriminates P. canaliculata from its most common congeners without requiring DNA sequencing. This approach is superior to traditional barcoding which also requires DNA purification, COI sequencing and sequence analysis. Thus, for rapid diagnostic purposes, the use of species-specific amplified fragments of COI in a multiplex PCR provides suitable resolution between target species without the need for DNA sequencing.