Analysis of Bacterial Communities Associated with the Benthic Amphipod Diporeia in the Laurentian Great Lakes Basin Andrew D
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72 ARTICLE Analysis of bacterial communities associated with the benthic amphipod Diporeia in the Laurentian Great Lakes Basin Andrew D. Winters, Terence L. Marsh, Travis O. Brenden, and Mohamed Faisal Abstract: Bacterial communities play important roles in the biological functioning of crustaceans, yet little is known about their diversity, structure, and dynamics. This study was conducted to investigate the bacterial communities associated with the benthic amphipod Diporeia, an important component in the Great Lakes foodweb that has been declining over the past 3 decades. In this study, the combination of 16S rRNA gene sequencing and terminal restriction fragment length polymorphism revealed a total of 175 and 138 terminal restriction fragments (T-RFs) in Diporeia samples following treatment with the endonucleases HhaI and MspI, respectively. Relatively abundant and prevalent T-RFs were affiliated with the genera Flavobacterium and Pseudomonas and the class Betaproteobacteria. T-RFs affiliated with the order Rickettsiales were also detected. A significant difference in T-RF presence and abundance (P = 0.035) was detected among profiles generated for Diporeia collected from 4 sites in Lake Michigan. Comparison of profiles generated for Diporeia samples collected in 2 years from lakes Superior and Michigan showed a significant change in diversity for Lake Superior Diporeia but not Lake Michigan Diporeia. Profiles from one Lake Michigan site contained multiple unique T-RFs compared with other Lake Michigan Diporeia profiles, most notably one that represents the genus Methylotenera. This study generated the most extensive list of bacteria associated with Diporeia and sheds useful insights on the microbiome of Great Lakes Diporeia that may help to reveal potential causes of the decline of Diporeia populations. Key words: bacteria, Diporeia spp., Laurentian Great Lakes. Résumé : Les communautés bactériennes jouent un rôle important dans le fonctionnement biologique de crustacés, mais on sait peu de choses sur leur diversité, la structure et la dynamique. Cette étude a été menée pour étudier les communautés bactéri- ennes associées a` la Diporeia amphipode benthique, un élément important dans la chaîne alimentaire des Grands Lacs qui a été en baisse au cours des trois dernières décennies. Dans cette étude, la combinaison de séquençage du gène de l’ARNr 16S et « terminal restriction fragment length polymorphism » a révélé un total de fragments de 175 et 138 de restriction de la borne (« T-RFs ») dans des échantillons Diporeia après le traitement par les endonucléases HhaIetMspI, respectivement. T-RFs relative- ment abondantes et les plus répandus étaient affiliés a` des genres Flavobacterium et Pseudomonas, et la Betaprotéobactéries de classe. T-RFs affiliés a` l’ordre des Rickettsiales ont également été détectés. Une différence significative en présence T-RF et For personal use only. l’abondance (P = 0,035) a été détectée chez les profils générés pour Diporeia recueillies auprès de quatre sites dans le lac Michigan. La comparaison des profils générés pour les échantillons prélevés Diporeia en deux ans des lacs Supérieur et Michigan a montré un changement significatif dans la diversité pour le lac Supérieur diporeia mais pas du lac Michigan Diporeia. Profils d’un site du lac Michigan contenaient de multiples T-RFs uniques par rapport aux autres profils lac Michigan Diporeia, notamment celui qui représente le genre Methylotenera. Cette étude a généré la liste la plus complète des bactéries associées aux Diporeia et apporte des informations utiles sur le microbiome de Grande Lacs Diporeia qui peuvent aider a` révéler les causes possibles du déclin des populations de Diporeia. Mots-clés : bactéries, Diporeia spp., des Grands Lacs Laurentiens. Introduction 2002; Barbiero et al. 2011). As a result, there has been increased Detritivorous amphipods belonging to the genus Diporeia are interest in studying the biology of Diporeia in the Great Lakes. The important food resources for numerous Great Lakes fish species aim of the current study is make information on the bacterial (Barbiero et al. 2011) and, thus, serve as coupling mechanisms communities associated with Diporeia available to the scientific between pelagic and benthic zones of the Great Lakes. Histori- community to potentially shed light on the declines. cally, Diporeia have been the dominant benthic macroinvertebrate Considering the ubiquity and large abundances of crustaceans in the Great Lakes, averaging over 7000 individuals/m2, reaching in marine and freshwater environments, tight associations be- mean densities as high as 12 216 /m2, and accounting for approx- tween crustaceans and bacteria can widely affect bacterial behav- imately 70% of the macrobenthic community of the Great Lakes ior, growth, and biogeochemical activities (reviewed in Tang et al. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 (Nalepa 1987, 1989). However, Diporeia abundances have been de- 2010). Microbial communities within the Great Lakes ecosystem clining from the majority of its habitats throughout 4 of the have been studied for many years using basic microscopy and 5 Great Lakes (Dermott and Kerec 1997; Nalepa et al. 1998, 2005, culturing-based techniques (Inniss and Mayfield 1978; Maki and 2007; Dermott 2001; Lozano et al. 2001; Barbiero and Tuchman Remsen 1981; Ishii et al. 2006). However, since a large fraction of Received 3 July 2014. Revision received 15 October 2014. Accepted 25 October 2014. A.D. Winters and T.O. Brenden. Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA. T.L. Marsh. Center for Microbial Ecology and Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. M. Faisal. Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA; Department of Pathobiology and Diagnostic Investigation, 177K Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824, USA. Corresponding author: Mohamed Faisal (e-mail: [email protected]). Can. J. Microbiol. 61: 72–81 (2015) dx.doi.org/10.1139/cjm-2014-0434 Published at www.nrcresearchpress.com/cjm on 3 November 2014. Winters et al. 73 Fig. 1. Map of the Laurentian Great Lakes and the Finger Lakes showing sampling sites for this study. A circle indicates a sample was analyzed with terminal restriction fragment length polymorphism (T-RFLP), a triangle indicates a sample was analyzed with 16S rDNA sequencing, and a square indicates a sample was analyzed with both T-RFLP and 16S rDNA sequencing. microbial life cannot be cultured, a severe dearth regarding the ing from 40 to 186 m. For T-RFLP analysis, replicate samples were taxonomic diversity of these microbial communities currently collected at each site in August 2007, and one site in Lake Superior exists. Because microbial communities can evolve rapidly in re- (SU-23B) and one site in Lake Michigan (MI-18M) were resampled in sponse to environmental perturbations and could be used as in- August 2008 (Table 1). To identify bacterial taxa present in Diporeia dicators of change, it is important to acquire baseline information samples, sequence analysis of cloned 16S rDNA clones was applied against which future changes could be identified. In recent years, to a single replicate from each of the lakes mentioned above. cultivation-independent surveys employing sequence analysis of Additionally, sequence analysis of 16S rDNA clones was applied to 16S small-subunit rRNA genes within freshwater sediments have a single sample collected from Lake Ontario. allowed for the identification of multiple bacterial phylotypes Sediment samples were collected using a Ponar grab (sampling For personal use only. that appear to be ubiquitous in freshwater ecosystems (Zwart area: 22.86 × 22.86 mm/8.2 L). Once a sample was returned to the et al. 2002; Briée et al. 2007; Rinta-Kanto et al. 2009; Tang et al. surface, it was sieved (mesh = 0.25 mm) and Diporeia were identi- 2009; Newton et al. 2011). Unfortunately, knowledge of the struc- fied according to Edmonson (1959). Diporeia were then pooled ture and function bacterial communities associated with Great (5 amphipods/pool), rinsed several times in sterile water to elimi- Lakes Diporeia is still very limited. nate “free-living” bacteria, placed in sterile 80% ethanol in a 1.5 mL One culture-independent method for investigating bacterial tube, and immediately stored at –20 °C until further processing. communities in samples through fingerprinting is terminal re- striction fragment length polymorphism (T-RFLP). T-RFLP is con- DNA isolation sidered a cost-effective tool for assessing the diversity of complex Genomic bacterial community DNA was extracted from Diporeia bacterial communities and for rapidly comparing community samples using the PowerSoil DNA Isolation kit (MO BIO Laborato- structure and diversity among different ecosystems that is capa- ries Inc., Carlsbad, California, USA) following the manufacturer’s ble of recovering a resolution of bacterial community structure protocol. DNA was quantified with a Qubit fluorometer and the highly comparable to that of pyrotag sequencing (Liu et al. 1997; Quant-it dsDNA BR Assay kit (Invitrogen Corp., Austin, Texas, Pilloni et al. 2012). In the present work, the diversity of bacterial USA). The isolated bacterial DNA was then used as a template communities associated with Diporeia collected from multiple for PCR amplification. The bacterial