Acta Limnologica Brasiliensia
Acta Limnologica Brasiliensia
Thematic Section: Methods

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

Protocolo otimizado e validado para detecção do bivalve invasor Limnoperna fortunei de amostras de DNA ambiental de plâncton

Josiane Ribolli; Sophia Cassol; Samara Hermes Silva; Evoy Zaniboni Filho; Flávia Lucena Zacchi; Jacó Joaquim Mattos; Grasiela Fagundes Minatto Cardoso; Alex Pires de Oliveira Nuñer

Downloads: 1
Views: 852


Abstract: : 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.


biological invasion, biomonitoring, golden mussel, qPCR, reservoir


Resumo: : Nós otimizamos uma metodologia para detecção do mexilhão-dourado em amostras de DNA ambiental de plâncton e a validamos em amostras de um reservatório no sul do Brasil. Limnoperna fortunei é uma espécie exótica invasora bem-sucedida que promove impactos significativos nos ecossistemas de água doce. Nós ajustamos e validamos a metodologia para detectar L. fortunei em amostras de plâncton em reservatório, através de ensaios com SYBR Green. O nível teórico mínimo de detecção de qPCR observado, com base na análise de curva padrão, foi de 0,0005625 ng.µL-1 (R2 = 0,99) em um ciclo de quantificação de 14,09–29,56. Nós também apresentamos um guia prático a ser utilizado no monitoramento da invasão de L. fortunei em bacias hidrográficas. A metodologia ajustada foi eficiente na detecção de L. fortunei e, portanto, pode ser usada para monitorar ambientes já infestados ou invasões em novos ambientes em larga escala.


invasão biológica, biomonitoramento, mexilhão-dourado, qPCR, reservatórios


ALJANABI, S.M. and MARTINEZ, I. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 1997, 25(22), 4692-4693. PMid:9358185.

ANDRADE, J.T., CORDEIRO, N.I., MONTRESOR, L.C., LUZ, D.M., LUZ, R.C., MARTINEZ, C.B., PINHEIRO, J., PAGLIA, A.P. and VIDIGAL, T.H. Effect of temperature on behavior, glycogen content, and mortality in Limnoperna fortunei (Dunker, 1857)(Bivalvia: Mytilidae). Journal of Limnology, 2018, 77(2).

BALASINGHAM, K.D., WALTER, R.P. and HEATH, D.D. Residual eDNA detection sensitivity assessed by quantitative real-time PCR in a river ecosystem. Molecular Ecology Resources, 2017, 17(3), 523-532. PMid:27617668.

BALDWIN, B.S., BLACK, M., SANJUR, O., GUSTAFSON, R., LUTZ, R.A. and VRIJENHOEK, R.C. A diagnostic molecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves: mitochondrial COI. Molecular Marine Biology and Biotechnology, 1996, 5(1), 9-14. PMid:8869514.

BARBOSA, N.P.U., SILVA, F.A., OLIVEIRA, M.D., SANTOS NETO, M.A., CARVALHO, M.D. and CARDOSO, A.V. Limnoperna fortunei (Dunker, 1857) (Mollusca, Bivalvia, Mytilidae): first record in the São Francisco River basin, Brazil. Check List, 2016, 12(1), 1846.

BENG, K.C. and CORLETT, R.T. Applications of environmental DNA (eDNA) in ecology and conservation: opportunities, challenges and prospects. Biodiversity and Conservation, 2020, 29(7), 2089-2121.

BOEGER, W.A., PIE, M.R., FALLEIROS, R.M., OSTRENSKY, A., DARRIGRAN, G., MANSUR, M.C.D. and BELZ, C.E. Testing a molecular protocol to monitor the presence of golden mussel larvae (Limnoperna fortunei) in plankton samples. Journal of Plankton Research, 2007, 29(11), 1015-1019.

CALLIL, C.T., TEIXEIRA, A.L., PINILUS, A.C. and SOARES, V. A gametogênese em Limnoperna fortunei (Dunker, 1857). In: M.C.D. MANSUR, C.P. SANTOS, D. PEREIRA, I.C.P. PAZ, M.L.L. ZURITA, M.T.R. RODRIGUEZ, M.V. NEHRKE and P.E.A. BERGONCI, orgs. Guia prático: moluscos límnicos Invasores do Brasil: biologia, prevenção e controle. Porto Alegre: Redes Editora, 2012, pp. 111-118.

CATALDO, D.H. Larval Development of Limnoperna fortunei. In: D. BOLTOVSKOY, ed. Limnoperna fortunei: the ecology, distribution and control of a swiftly spreading invasive fouling mussel. London: Springer, 2015, pp. 43-53.

CORDEIRO, N.I., ANDRADE, J.T., MONTRESOR, L.C., LUZ, D.M., MARTINEZ, C.B., DARRIGRAN, G., PINHEIRO, J. and VIDIGAL, T.H.D.A. Effect of starvation and subsequent feeding on glycogen concentration, behavior and mortality in the golden mussel Limnoperna fortunei (Dunker, 1857)(Bivalvia: Mytilidae). Journal of Limnology, 2016, 75(3), 618-625.

DARRIGRAN, G. Potential impact of filter-feeding invaders on temperate inland freshwater environments. Biological Invasions, 2002, 4(1/2), 145-156.

DE PAULA, R.S., REIS, M.P., DE OLIVEIRA JÚNIOR, R.B., ANDRADE, G.R., DE CARVALHO, M.D., CARDOSO, A.V. and JORGE, E.C. Genetic and functional repertoires of Limnoperna fortunei (Dunker, 1857)(Mollusca, Mytilidae): a review on the use of molecular techniques for the detection and control of the golden mussel. Hydrobiologia, 2020, 847(10), 2193-2202.

DEINER, K., BIK, H.M., MÄCHLER, E., SEYMOUR, M., LACOURSIÈRE-ROUSSEL, A., ALTERMATT, F., CREER, S., BISTA, I., LODGE, D.M., DE VERE, N., PFRENDER, M.E. and BERNATCHEZ, L. Environmental DNA metabarcoding: transforming how we survey animal and plant communities. Molecular Ecology, 2017, 26(21), 5872-5895. PMid:28921802.

EICHMILLER, J.J., BAJER, P.G. and SORENSEN, P.W. The relationship between the distribution of common carp and their environmental DNA in a small lake. PLoS One, 2014, 9(11), e112611. PMid:25383965.

ENDO, N., SATO, K. and NOGATA, Y. Molecular based method for the detection and quantification of larvae of the golden mussel Limnoperna fortunei using real-time PCR. Plankton & Benthos Research, 2009, 4(3), 125-128.

GOLDBERG, C.S., SEPULVEDA, A., RAY, A., BAUMGARDT, J. and WAITS, L.P. Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshwater Science, 2013, 32(3), 792-800.

HARRISON, J.B., SUNDAY, J.M. and ROGERS, S.M. Predicting the fate of eDNA in the environment and implications for studying biodiversity. Proceedings of the Royal Society B, 2019, 286(1915), 20191409.

HEID, C.A., STEVENS, J., LIVAK, K.J. and WILLIAMS, P.M. Real time quantitative PCR. Genome Research, 1996, 6(10), 986-994. PMid:8908518.

HERMES-SILVA, S., RIBOLLI, J., ÁVILA-SIMAS, S., ZANIBONI-FILHO, E., CARDOSO, G.F.M. and NUÑER, A.P.O. Limnoperna fortunei - Updating the geographic distribution in the Brazilian watersheds and mapping the regional occurrence in the Upper Uruguay River basin. Biota Neotropica, forthcoming, 21(3), e20201175.

JERDE, C.L., MAHON, A.R., CHADDERTON, W.L. and LODGE, D.M. “Sight-unseen” detection of rare aquatic species using environmental DNA. Conservation Letters, 2011, 4(2), 150-157.

LUCY, F. Early life stages of Dreissena polymorpha (zebra mussel): the importance of long-term datasets in invasion ecology. Aquatic Invasions, 2006, 1(3), 171-182.

MACK, R.N., SIMBERLOFF, D., LONSDALE, W.M., EVANS, H., CLOUT, M. and BAZZAZ, F.A. Biotic invasions: causes, epidemiology, global consequences, and control. Ecological Applications, 2000, 10(3), 689-710.[0689:BICEGC]2.0.CO;2.

MACKENZIE, D.I., NICHOLS, J.D., ROYLE, J.A., POLLOCK, K.H., BAILEY, L.A. and HINES, J.E. Occupancy estimation and modeling: inferring patterns and dynamics of species occurrence. Amsterdam: Elsevier, 2006.

OLIVEIRA, M.D., CAMPOS, M.C.S., PAOLUCCI, E.M., MANSUR, M.C.D. and HAMILTON, S.K. Colonization and spread of Limnoperna fortunei in South America. In: D. BOLTOSVKOY, ed. Limnoperna fortunei: the ecology, distribution and control of a swiftly spreading invasive fouling mussel. London: Springer, 2015, pp. 333-355.

PASTORINO, G., DARRIGRAN, G., MARTÍN, S.M. and LUNASCHI, L. Limnoperna fortunei (Dunker, 1857) (Mytilidae), nuevo bivalvo invasor en aguas del Río de la Plata. Neotrópica, 1993, 39, 34.

PIE, M.R., STRÖHER, P.R., AGOSTINIS, A.R., BELMONTE-LOPES, R., TADRA-SFEIR, M.Z. and OSTRENSKY, A. Development of a real-time PCR assay for the detection of the golden mussel (Limnoperna fortunei, Mytilidae) in environmental samples. Annals of the Brazilian Academy of Sciences, 2017, 89(2), 1041-1045. PMid:28640350.

RAHEL, F.J. and OLDEN, J.D. Assessing the effects of climate change on aquatic invasive species. Conservation Biology, 2008, 22(3), 521-533. PMid:18577081.

RICCIARDI, A. Global range expansion of the Asian mussel Limnoperna fortunei (Mytilidae): Another fouling threat to freshwater systems. Biofouling, 1998, 13(2), 97-106.

ROUSSEL, J.M., PAILLISSON, J.M., TREGUIER, A. and PETIT, E. The downside of eDNA as a survey tool in water bodies. Journal of Applied Ecology, 2015, 52(4), 823-826.

RUPPERT, K.M., KLINE, R.J. and RAHMAN, M.S. Past, present, and future perspectives of environmental DNA (eDNA) metabarcoding: A systematic review in methods, monitoring, and applications of global eDNA. Global Ecology and Conservation, 2019, 17, e00547.

STOECKLE, M.Y., SOBOLEVA, L. and CHARLOP-POWERS, Z. Aquatic environmental DNA detects seasonal fish abundance and habitat preference in an urban estuary. PLoS One, 2017, 12(4), e0175186. PMid:28403183.

TABERLET, P., COISSAC, E., HAJIBABAEI, M. and RIESEBERG, L.H. Environmental DNA. Molecular Ecology, 2012, 21(8), 1789-1793. PMid:22486819.

TAKAHARA, T., MINAMOTO, T., YAMANAKA, H., DOI, H. and KAWABATA, Z. Estimation of fish biomass using environmental DNA. PLoS One, 2012, 7(4), e35868. PMid:22563411.

THOMAS, A.C., TANK, S., NGUYEN, P.L., PONCE, J., SINNESAEL, M. and GOLDBERG, C.S. A system for rapid eDNA detection of aquatic invasive species. Environmental DNA, 2020, 2(3), 261-270.

TSCHÁ, M.K., PATELLA, R., OSTRENSKY, A. and BOEGER, W.A. O método molecular de prospecção do mexilhão-dourado. In: M.C.D. MANSUR, ed. Moluscos límnicos invasores no Brasil: biologia, prevenção e controle. Porto Alegre: Redes, 2012, pp. 143-148.

VILÀ, M. and HULME, P.E. Impact of biological invasions on ecosystem services. Cham: Springer International Publishing, 2017.

WILCOX, T.M., MCKELVEY, K.S., YOUNG, M.K., JANE, S.F., LOWE, W.H., WHITELEY, A.R. and SCHWARTZ, M.K. Robust detection of rare species using environmental DNA: the importance of primer specificity. PLoS One, 2013, 8(3), e59520. PMid:23555689.

Submitted date:

Accepted date:

Publication date:

60ad0d5da9539538760a6873 alb Articles
Links & Downloads

Acta Limnol. Bras. (Online)

Share this page
Page Sections