Fig 1.
Sampling sites were distributed along 36 km of the High Rhine in Switzerland between Basel (most downstream locations: sites #2a, #2b, and #3) and Mumpf (most upstream location: site #12). For detailed coordinates of sampling sites, see Table 1. In 2013, Ponto-Caspian gobies were reported, at very low densities, at a location midway between site #7 and site #8. Yellow bars, hydropower stations. Expansion of River Rhine opposite of site #7: former side arm of the river, today natural reserve. The map is oriented from North (top) to South (bottom).
Fig 2.
A commercially available water sampler was adapted for sampling benthic water. (A) Sampler as purchased. (B) Modified sampler, with plunger closed by a spring. (C) Sampler with bottle attached, plunger closed. (D) Bottle screw cap closed, plunger closed. (E) When the bottle touches ground, the plunger can be opened by pulling the attached cable. (F) When the plunger is opened, water can enter the bottle through holes in the bottle holder. See S2 Supplementary Material for building instructions.
Table 1.
Sampling sites.
Fig 3.
Ponto-Caspian goby, round goby N. melanostomus and bighead goby P. kessleri primers were tested on samples from Ponto-Caspian gobies, from other goby species, and on fish native in Switzerland. Cytochrome Oxidase I (COI) was amplified as positive control from all samples.
Fig 4.
Three water samples were taken from the invaded commercial harbor at site #2b. DNA was isolated, and each sample was subjected to conventional PCR and touchdown PCR with or without BSA. Primers: Ponto-Caspian goby primers. Experimental control: Carp pond (site #1) sampled after the field trip. Positive control: 5 ng N. melanostomus DNA as template. Negative control: ultrapure water as template.
Fig 5.
Nine samples from a heavily invaded site at the local commercial harbor (site #2b) were assigned randomly to one of three extraction methods (EtOH precipitation, glass fiber filtration followed by Qiagen Blood and Tissue (B+T) kit lysis, and glass fiber filtration followed by CTAB lysis). Control samples were processed with glass fiber filtration followed by Qiagen Blood and Tissue kit lysis. Control sample 1: tap water, re-bottled at the sampling site. Control sample 2: uninvaded carp pond (site #1), sampled after the field trip. Primers: Ponto-Caspian goby primers. Positive control: 5 ng N. melanostomus DNA as template. Negative control: ultrapure water as template.
Fig 6.
None of the primer pairs amplifies bands on ultrapure water (negative control), or on DNA isolated from tap water (1), tap water rebottled in the harbor (2), samples taken from an uninvaded carp pond (site #1) before (3) or after (4) a field trip to an invaded site (site #2b). Positive control: 5ng of DNA from Neogobius melanostomus (Ponto-Caspian goby primers, N. melanostomus primers) or from Ponticola kessleri (P. kessleri primers) as used as template.
Fig 7.
Sampling depth impacts signal strength.
Nine samples were collected from the invaded commercial harbor on Wednesday (weekday) or Sunday (weekend) and from the surface or the bottom of the harbor at site #2b. 1–3: experimental controls. 1: Carp pond (site #1) sampled after weekday field trip. 2: Carp pond (site #1) sampled after weekend field trip. 3: Tap water, re-bottled during weekend field trip. Primers: Ponto-Caspian goby primers. Positive control: 5ng N. melanostomus DNA as template. Negative control: ultrapure water as template.
Fig 8.
Abundance is not correlated with signal strength.
(A) Abundance. Catch data from 2012 to 2014 for sites #2a and #2b. (B) Signal strength from the same site. 1–3: field controls. 1: tap water, rebottled in harbor, site #2a. 2: tap water, rebottled in harbor, site #2b. 3: carp pond (site #1) sampled after field trip. Primers: Ponto-Caspian goby primers. Positive control: 5ng N. melanostomus DNA as template. Negative control: ultrapure water as template.
Fig 9.
Ponto-Caspian gobies can be detected from river water.
Numbers correspond to sampling sites in Fig 1, with site #1 as experimental control from the carp pond, and sites #2b-12 as field samples extending upstream from the heavily invaded harbor (site #2b). Primers: Ponto-Caspian goby primers. Positive control: 5ng N. melanostomus DNA as template. Negative control: ultrapure water as template.
Fig 10.
Testing environmental samples for PCR inhibition.
(A) Conceptual illustration of an inhibition testing PCR. An inhibition testing PCR is set up so that its endpoint is placed in the linear phase of a PCR amplification. Changes in the degree of PCR inhibition, template or cycle number will therefore result in changes in signal intensity. For a PCR with an endpoint as indicated by a black dot, more inhibition would result in lower signal intensity, and less inhibition would result in higher signal intensity. (B) Determination of the optimal cycle number for the inhibition testing PCR. Between cycles 10 to 28, the amplification curve is in the dynamic range. (C) Inhibition tests using environmental samples. Three microliters of the indicated environmental sample were added to the inhibition testing PCR, and PCR was run for 22 cycles in the absence (left) or presence (right) of BSA as an anti-inhibitory agent. Positive control, 70% EtOH. Negative control 1, ddH2O. Negative control 2, independent DNA preparation isolated with the Qiagen Blood and Tissue kit.