Optimized and validated protocol to the detection of the invasive bivalve <i>Limnoperna fortunei</i> from eDNA plankton samples

We optimized a methodology for plankton environmental DNA detection of the invasive golden mussel and validated it in samples from a Southern Brazil reservoir. Limnoperna fortunei is a successful invasive alien species that causes significant impacts on freshwater ecosystems. We adjusted and validated the methodology to detect L. fortunei in plankton samples, with a SYBR Green assay. Based on the standard curve analysis, the observed theoretical minimal qPCR detection level was 0.0005625 ng.µL^-1 (R² = 0.99) at a PCR quantification cycle of 14.09–29.56. We also presented a practical guide to be used in monitoring and detection of L. fortunei. The optimized protocol was efficient in detecting L. fortunei and can be used to monitor already infested environments or invasions in new environments.

Keywords:
biological invasion; biomonitoring; golden mussel; qPCR; reservoir

Authorship

Josiane Ribolli ^**e-mail: josianeribolli@gmail.com

Laboratório de Biologia e Cultivo de Peixes de Água Doce, Universidade Federal de Santa Catarina – UFSC, Rodovia Francisco Thomaz dos Santos, 3532, CEP 88066-260, Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilLaboratório de Biologia e Cultivo de Peixes de Água Doce, Universidade Federal de Santa Catarina – UFSC, Rodovia Francisco Thomaz dos Santos, 3532, CEP 88066-260, Florianópolis, SC, Brasil

http://orcid.org/0000-0002-5553-9973

Sophia Cassol

http://orcid.org/0000-0002-9303-8870

Samara Hermes Silva

http://orcid.org/0000-0001-8549-8191

Evoy Zaniboni Filho

http://orcid.org/0000-0001-6457-2655

Flávia Lucena Zacchi

Laboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, Universidade Federal de Santa Catarina – UFSC, Servidão Caminho do Porto, S/N, CEP 88034-257, Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilLaboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, Universidade Federal de Santa Catarina – UFSC, Servidão Caminho do Porto, S/N, CEP 88034-257, Florianópolis, SC, Brasil

http://orcid.org/0000-0003-1808-1390

Jacó Joaquim Mattos

Núcleo de Estudos em Patologia Aquícola, Universidade Federal de Santa Catarina, Servidão Caminho do Porto, S/N, CEP 88034-257, Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilNúcleo de Estudos em Patologia Aquícola, Universidade Federal de Santa Catarina, Servidão Caminho do Porto, S/N, CEP 88034-257, Florianópolis, SC, Brasil

http://orcid.org/0000-0002-8766-4285

Grasiela Fagundes Minatto Cardoso

Engie Brasil Energia, Rua Paschoal Apóstolo Pítsica, 5064, Agronômica, CEP 88025-255, Florianópolis, SC, BrasilEngie Brasil EnergiaBrasilFlorianópolis, SC, BrasilEngie Brasil Energia, Rua Paschoal Apóstolo Pítsica, 5064, Agronômica, CEP 88025-255, Florianópolis, SC, Brasil

http://orcid.org/0000-0001-7505-3252

Alex Pires de Oliveira Nuñer

Programa de Pós-graduação em Aquicultura, Departamento de Aquicultura, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina – UFSC, Rodovia Admar Gonzaga, 1346, Itacorubi, CEP 88034-000, Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilPrograma de Pós-graduação em Aquicultura, Departamento de Aquicultura, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina – UFSC, Rodovia Admar Gonzaga, 1346, Itacorubi, CEP 88034-000, Florianópolis, SC, Brasil

http://orcid.org/0000-0002-4682-3307

*e-mail: josianeribolli@gmail.com

Associate Editor: Victor Satoru Saito.

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Figures

Figures (5)

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Figure 1
Sampling sites of eDNA plankton samples collected in the Itá reservoir, Upper Uruguay River, in Southern Brazil.

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Figure 2
(A) Quantitation curves of known amounts and (B) melting curves of known amounts to 45 (red), 4.5 (orange), 0.45 (blue), 0.045 (light green) 0.0045 (purple), 0.00225 (brown), 0.001125 (dark green), 0.0005625 (pink) ng of DNA/reaction of L. fortunei DNA. Negative controls are shown in black lines.

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Figure 3
A schematic figure with a practical guide to eDNA plankton sampling and qPCR analysis of Limnoperna fortunei: “A practical guide: eDNA plankton sampling and qPCR analysis of golden mussel, Limnoperna fortunei”. (1) Field sampling: Environmental DNA sampling using a plankton net (mesh opening of 53 μm), with a motor pump and a hose. Collect twice at the same sample site (filtering 200 L each) at two points (30 meters between each one) totalizing 400 L of water. Concentrate the sample in the same bottle; (2) Sample conservation in the field: Fix the collected sample with 96% ethanol in the proportion of 1:4 (sampling:ethanol). Keep the flasks with the samples in ice until getting to the laboratory. Store the sample in a freezer at -20 °C; (3) Sample filtration at the laboratory: Pre-filter the samples with a 100 μm net. Use the filtrate to filter in 0.22 μm smooth membrane filtrates, with the aid of a vacuum pump. Sterilize the materials between one filter and another (UV light); (4) DNA dilution: Extract the DNA from the membrane using a purification kit, following the manufacturer's recommendation. Quantify the total DNA extracted and standardize at 8.0 ng of DNA.µL^-1; (5) Real-time PCR: Perform the PCR reaction in a 20 µL in a final volume containing 10 µL of QuantiFast SYBR Green PCR Mix (Qiagen), 1.0 µL of Forward primer (20µM), 1.0 µL of Reverse primer (20 µM), 8.0 ng of eDNA/reaction, and 3.0 µL of RNAse free water (Qiagen) and the program as follows: 5 min at 95 °C, 35 cycles of 10 seconds at 95 °C, and 15 seconds at 60 °C.

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Figure 4
PCR Quantification cycle (Cq) vs. DNA concentration of known amounts (45, 4.5, 0.45, 0.045, 0.0045, 0.00225, 0.001125, 0.0005625 ng of DNA/reaction) of L. fortunei.

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Figure 5
Melting curves of eDNA plankton samples collected in the Itá reservoir, Upper Uruguay River, Southern Brazil. Blue lines represent positive samples, and red lines represent a negative sample for L. fortunei.

Figure 1 Sampling sites of eDNA plankton samples collected in the Itá reservoir, Upper Uruguay River, in Southern Brazil.

Figure 2 (A) Quantitation curves of known amounts and (B) melting curves of known amounts to 45 (red), 4.5 (orange), 0.45 (blue), 0.045 (light green) 0.0045 (purple), 0.00225 (brown), 0.001125 (dark green), 0.0005625 (pink) ng of DNA/reaction of L. fortunei DNA. Negative controls are shown in black lines.

Figure 3 A schematic figure with a practical guide to eDNA plankton sampling and qPCR analysis of Limnoperna fortunei: “A practical guide: eDNA plankton sampling and qPCR analysis of golden mussel, Limnoperna fortunei”. (1) Field sampling: Environmental DNA sampling using a plankton net (mesh opening of 53 μm), with a motor pump and a hose. Collect twice at the same sample site (filtering 200 L each) at two points (30 meters between each one) totalizing 400 L of water. Concentrate the sample in the same bottle; (2) Sample conservation in the field: Fix the collected sample with 96% ethanol in the proportion of 1:4 (sampling:ethanol). Keep the flasks with the samples in ice until getting to the laboratory. Store the sample in a freezer at -20 °C; (3) Sample filtration at the laboratory: Pre-filter the samples with a 100 μm net. Use the filtrate to filter in 0.22 μm smooth membrane filtrates, with the aid of a vacuum pump. Sterilize the materials between one filter and another (UV light); (4) DNA dilution: Extract the DNA from the membrane using a purification kit, following the manufacturer's recommendation. Quantify the total DNA extracted and standardize at 8.0 ng of DNA.µL^-1; (5) Real-time PCR: Perform the PCR reaction in a 20 µL in a final volume containing 10 µL of QuantiFast SYBR Green PCR Mix (Qiagen), 1.0 µL of Forward primer (20µM), 1.0 µL of Reverse primer (20 µM), 8.0 ng of eDNA/reaction, and 3.0 µL of RNAse free water (Qiagen) and the program as follows: 5 min at 95 °C, 35 cycles of 10 seconds at 95 °C, and 15 seconds at 60 °C.

Figure 4 PCR Quantification cycle (Cq) vs. DNA concentration of known amounts (45, 4.5, 0.45, 0.045, 0.0045, 0.00225, 0.001125, 0.0005625 ng of DNA/reaction) of L. fortunei.

Figure 5 Melting curves of eDNA plankton samples collected in the Itá reservoir, Upper Uruguay River, Southern Brazil. Blue lines represent positive samples, and red lines represent a negative sample for L. fortunei.

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Optimized and validated protocol to the detection of the invasive bivalve <i>Limnoperna fortunei</i> from eDNA plankton samples

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[1] *e-mail: josianeribolli@gmail.com