Biol Invasions DOI 10.1007/s10530-014-0810-2 MOLECULAR TOOLS The influence of invasive jellyfish blooms on the aquatic microbiome in a coastal lagoon (Varano, SE Italy) detected by an Illumina-based deep sequencing strategy Caterina Manzari • Bruno Fosso • Marinella Marzano • Anita Annese • Rosa Caprioli • Anna Maria D’Erchia • Carmela Gissi • Marianna Intranuovo • Ernesto Picardi • Monica Santamaria • Simonetta Scorrano • Giuseppe Sgaramella • Loredana Stabili • Stefano Piraino • Graziano Pesole Received: 14 November 2013 / Accepted: 31 October 2014 Ó The Author(s) 2014. This article is published with open access at Springerlink.com Abstract The rapid expansion of multicellular microbial taxonomic and functional diversity. Here native and alien species outbreaks in aquatic and we used an effective and highly sensitive experimental terrestrial ecosystems (bioinvasions) may produce strategy, bypassing the efficiency bottleneck of the significant impacts on bacterial community dynamics traditional bacterial isolation and culturing method, to and nutrient pathways with major ecological implica- identify changes of the planktonic microbial commu- tions. In aquatic ecosystems, bioinvasions may cause nity inhabiting a marine coastal lagoon (Varano, adverse effects on the water quality resulting from Adriatic Sea) under the influence of an outbreak- changes in biological, chemical and physical proper- forming alien jellyfish species. Water samples were ties linked to significant transformations of the collected from two areas that differed in their level of confinement inside in the lagoon and jellyfish densities (W, up to 12.4 medusae m-3; E, up to 0.03 medusae Caterina Manzari, Bruno Fosso and Marinella Marzano have -3 contributed equally to this work. m ) to conduct a snapshot microbiome analysis by a metagenomic approach. After extraction of the genetic Stefano Piraino and Graziano Pesole are senior authors. material in the environmental water samples, we deep- sequenced metagenomic amplicons of the V5–V6 Electronic supplementary material The online version of region of the 16S rRNA bacterial gene by an Illumina this article (doi:10.1007/s10530-014-0810-2) contains supple- MiSeq platform. Experiments were carried out in mentary material, which is available to authorized users. C. Manzari Á B. Fosso Á A. Annese Á R. Caprioli Á S. Scorrano Á S. Piraino A. M. D’Erchia Á M. Intranuovo Á E. Picardi Á G. Pesole Consorzio Nazionale Interuniversitario per le Scienze del Dipartimento di Bioscienze, Biotecnologie e Mare (Co.N.I.S.Ma.), Via Isonzo 32, 00198 Rome, Italy Biofarmaceutica, Universita` degli Studi di Bari ‘‘Aldo Moro’’, Via Orabona 4, 70126 Bari, Italy R. Caprioli Á S. Piraino Dipartimento di Scienze e Tecnologie Biologiche e M. Marzano Á A. M. D’Erchia Á E. Picardi Á Ambientali (DiSTeBA), Universita` del Salento, M. Santamaria Á G. Sgaramella Á G. Pesole (&) Via Monteroni, 73100 Lecce, Italy Istituto di Biomembrane e Bioenergetica (IBBE), CNR, Via Amendola 165/A, 70126 Bari, Italy C. Gissi e-mail: [email protected]; [email protected] Dipartimento di Bioscienze, Universita` degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy R. Caprioli Dipartimento di Biologia Animale, Universita` degli Studi di Modena e Reggio Emilia, Via G. Campi 213/d, 41100 Modena, Italy 123 C. Manzari et al. triplicates, so six libraries of dual indexed amplicons they participate in essential biogeochemical processes of 420 bp were successfully sequenced on the MiSeq such as the carbon, nitrogen and sulphur cycles in platform using a 2 9 250 bp paired-end sequencing terrestrial and aquatic ecosystems, are producers and strategy. Approximately 7.5 million paired-end reads decomposers in food webs (Dorigo et al. 2005; Kisand (i.e. 15 million total reads) were generated, with an et al. 2012; Zehr 2010), and are primarily responsible average of 2.5 million reads (1.25 M pairs) per sample for degradation of a large variety of natural organic replicate. The sequence data, analyzed through a novel compounds (Ogawa et al. 2001). bioinformatics pipeline (BioMaS), showed that the Aquatic environments harbor abundant and diverse structure of the resident bacterial community was microbial communities that ensure their functioning and significantly affected by the occurrence of jellyfish sustainability (Azam and Malfatti 2007; Zinger et al. outbreaks. Clear qualitative and quantitative differ- 2012). In recent decades, the importance of bacterio- ences were found between the western and eastern plankton has been persuasively demonstrated in nutrient areas (characterized by many or few jellyfish), with 84 cycling and food-web structure in the marine environ- families, 153 genera and 324 species in the W samples, ment (Azam 1998;Cho1990) and a multitude of marine and 104 families, 199 genera and 331 species in the E bacteria have been isolated in ocean sites and different samples. Significant differences between the two coastal areas of temperate, tropical and polar zones sampling areas were particularly detected in the (Pommier et al. 2005; Rusch et al. 2007; Stabili and occurrence of 16 families, 22 genera and 61 species Cavallo 2011). Aquatic microbes are genetically, phys- of microbial taxa. This is the first time that a NGS iologically and ecologically diverse, and exhibit many platform has been used to screen the impact of jellyfish different physiological responses, adaptation and evo- bioinvasions on the aquatic microbiome, providing a lutionary patterns (Bahgat 2011; Kemp and Aller 2004). preliminary assessment of jellyfish-driven changes of The microbiota composition varies as a function of the functional and structural microbial biodiversity. water class (e.g. oceans, lakes, rivers, springs, ponds and ground water) and it is affected by different factors such Keywords Metagenomics Á Next-generation as water salinity, organic compound concentration, sequencing platforms Á Marine microbial ecology Á turbidity, temperature, and contamination sources Jellyfish biomass (Bahgat 2011). Study of the overall biodiversity is crucial for assessing and monitoring aquatic ecosystems; however, the composition, abundance, and distribution patterns of Introduction microbial communities usually remain poorly explored (Kisand et al. 2012; Zinger et al. 2012). The recent advent of next generation sequencing Previously, difficulties in collecting representative technologies has revolutionized microbiome research, samples in particular habitats (e.g. deep sea) and allowing unprecedented depth and resolution at experimental limitations related to the characteriza- affordable costs and, thus, enabling large-scale studies tion of the uncultivable microbes have been the two of microorganisms living in taxonomically and phys- main limitations for comprehensive assessments of the ically complex habitats. Because microbes have lived microbial communities living in certain environments on Earth for more than 3 billion years, they are (Kisand et al. 2012). This view recently changed with ubiquitous and play important roles in the Earth’s the development of culture-independent approaches, ecosystems (Kowalchuk et al. 2008). In particular, such as metagenomics. As defined by Thomas et al. (2012), metagenomics represent the direct genetic S. Scorrano analysis of genomes contained in an environmental Dipartimento di Scienze Ecologiche e Biologiche (DEB), sample, without isolation or culture of individual Universita` della Tuscia, Largo dell’ Universita`, organisms. The metagenomic approach is experienc- 01100 Viterbo, Italy ing an explosive improvement since the advent of L. Stabili high-throughput Next-Generation Sequencing (NGS) Istituto per l’Ambiente Marino Costiero (IAMC), CNR, technologies, which allow an unprecedented large- Via Roma 3, 74100 Taranto, Italy scale identification of organisms and communities 123 The influence of invasive jellyfish blooms through the production of an enormous amount of also the case for the Varano lagoon, a coastal lagoonal genetic data (Bourlat et al. 2013). Either of two habitat with a strong aquaculture vocation and tradi- experimental strategies can be applied starting from tion. The Varano ecosystem was invaded by the moon the total extracted DNA: (1) the entire metagenome is jellyfish Aurelia sp. more than 10 years ago and now sequenced by a shotgun approach, or (2) gene markers has a persistent population with high densities in some specific for a given taxon are selectively sequenced parts (Belmonte et al. 2011; Manini et al. 2005; after their amplification by PCR primer pairs able to Scorrano 2014). function over a broad taxonomic range (amplicon- The invasion of Aurelia jellyfish in coastal lagoons based approach). The second approach is the most often can be related to the introduction of mussel common strategy applied in various ecological set- culture rafts bearing the polyp stages, which asexually tings and is used to investigate prokaryotic diversity release great numbers of medusae into the water through analysis of hypervariable regions of the 16S column (Lo et al. 2008; Purcell, 2012). In Varano rRNA gene (Hajibabaei et al. 2011; Klindworth et al. lagoon, eventually a year-round resident population 2013). Illumina/Solexa represents the NGS sequenc- was established with high densities of jellyfish (mean ing system most widely used for amplicon-based 4.5 ind) in spring and summer months. Molecular COI metagenomic studies (Luo et al. 2012), providing barcoding assigned the Varano jellyfish population to optimal performances in terms of time, costs