Analysis of Bacterial Communities Associated with the Benthic Amphipod Diporeia in the Laurentian Great Lakes Basin Andrew D

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 restriction 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 16S gene was amplified sites in lakes Superior, Michigan, and Huron, and the inland using the universal eubacterial primer set 27F–1387R (27F: 5=- Cayuga Lake (New York) were mapped and compared using AGAGTTTGATCMTGGCTCAG-3=) labeled with carboxyfluorescein T-RFLP of 16S rRNA genes to determine if these communities vary (6-FAM) and 1387R (5=-GGGCGGWGTGTACAAGGC-3=)(Marchesi spatially or temporally. In a second step, sequence analysis of et al. 1998). Each 50 ␮L PCR reaction contained 25 ␮L of 2× Green Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 16S rDNA clones was applied to a total of 5 Diporeia bacterial GoTaq Master Mix (Promega Corporation, Madison, Wisconsin, communities from lakes Superior, Michigan, and Huron, and the USA), 45 ␮mol/L each primer, and ϳ100 ng of template DNA. PCR inland Cayuga Lake (New York). Results of this study provide im- reaction conditions for amplification of partial 16S rRNA genes portant baseline information regarding the structure of bacterial were as follows: initial 94 °C for 4 min, followed by 29 cycles of communities associated with Diporeia in the Great Lakes ecosystem. 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final Materials and methods extension for 7 min at 72 °C. A negative control containing no DNA was included in each set of PCR reactions. Resulting PCR Sample collection products were visualized by agarose gel electrophoresis. Amplifi- For all analyses, Diporeia samples were collected from a total cation of the proper gene fragment (ϳ1.36 kb) was confirmed by of 8 locations in the Great Lakes basin and a single location in comparison with a DNA size ladder. The PCR reaction was carried Cayuga Lake, an inland lake in New York, (Fig. 1) at depths rang- out in triplicate for each sample and resulting products were

Published by NRC Research Press 74 Can. J. Microbiol. Vol. 61, 2015

Table 1. Name, location, and depth of sampling sites, year of collection, and number of consensus terminal restriction fragment length polymorphism (T-RFLP) proﬁles generated for Diporeia for each restriction endonuclease for this study. HhaI MspI Waterbody Site Latitude Longitude Depth (m) 2007 2008 2007 2008 Lake Michigan MI-18M 42.733 –87.000 161 3 4 4 4 MI-27M 43.600 –86.917 112 3 — 3 — MI-40 44.760 –86.967 160 3 — 3 — MI-47 45.178 –86.375 186 3 — 2 — Lake Huron HU-54M 45.517 –83.417 91 5 — 5 — Lake Superior SU-20B 46.883 –90.283 116 3 — 3 — SU-23B 46.598 –84.807 62 3 4 4 4 Cayuga Lake Cayuga 42.538 –76.553 40 3 — 3 — Note: —, indicates a sample was not taken.

pooled and purified using a Wizard SV Gel and PCR Clean-Up was the response variable and site or year was the independent System (Promega) following the manufacturer’s protocol. variable. The total number of permutations performed was 999. The PERMANOVA analysis was conducted in R using ADONIS T-RFLP analysis function in the VEGAN library. Additionally, permutation tests To construct profiles of Diporeia bacterial communities for each (999 permutations) were used to test for significant differences in restriction enzyme, 300 ng of purified fluorescently labeled PCR multivariate dispersion among sites and years using the BETADISPER product were cut individually with 5 units of HhaI and MspI (New function in VEGAN, which is a multivariate analogue of Levene’s England Biolabs, Beverly, Massachusetts, USA) for2hat37°C. test for homogeneity of variances. Since the Chao method takes Digested PCR products were precipitated with 3 volumes of abso- the fraction of rarely present/low abundant T-RFs into account lute ethanol. After8hofincubation at –20 °C, the precipitates (Chao et al. 2005), it was used to define ␤ diversity. To determine were pelleted by centrifugation at 14 000 r/min (16 000g) for whether community structure was significantly correlated with 15 min. Pellets were resuspended in 6 ␮L of sterile water (DEPC- depth we used Mantel tests (Legendre and Legendre 1998) based on treated, DNase, RNase-free). The DNA fragments were separated Pearson’s product–moment correlation. For Mantel tests, depth on an ABI 3100 Genetic Analyzer automated sequence analyzer values were Z-score standardized, and community dissimilarities (Applied Biosystems Instruments, Foster City, California, USA) in were standardized using Wisconsin standardization (dividing all GeneScan mode at Michigan State University’s sequencing facil- species by their maxima, and then standardizing sites to unit ity. The 5= terminal restriction fragments (T-RFs) were detected by totals). To test for differences in relative abundance of individual excitation of the 6-FAM molecule attached to the forward primer. T-RFs among samples, MANOVA tests were conducted using the The sizes and abundance of the fragments were calculated using PROC GLIMMIX procedure (SAS Institute Inc., Cary, North Caro- GeneScan 3.7 in relation to the MM1000 internal standard (Bio- lina, USA).

For personal use only. Ventures Inc., Murfreesboro, Tennessee, USA). T-RFLP data was analyzed with T-REX (http://trex.biohpc.org). T-REX software uses DNA sequence analysis the methodology described by Abdo et al. (2006) and Smith et al. The PCR products generated for a total of 5 samples from lakes (2005) to identify and align true peaks, respectively. We used one Superior, Michigan, Huron, and Ontario, and Cayuga Lake (SU-20B, standard deviation in peak area as the limit to identify true peaks MI-27M, HU-54M, ON-41, and Cayuga), were amplified with the and defined T-RFs by rounding off fragment sizes to nearest inte- unlabeled primer set (27F–1387R) and cloned using a TOPO TA ger. Additionally, only profiles with a cumulative peak height of Cloning kit (Invitrogen, Grand Island, Nebraska, USA) following ≥5000 fluorescence units were used in the analysis. the manufacturer’s protocol. A total of 399 clones (between For all analyses, abundance values for both T-RFLP data sets 93 and 95 for the Great Lakes and 23 for Cayuga Lake) were were standardized by expressing the abundance of each OTU as a cultured on Luria–Bertani agar plates (Fisher Scientific Inc., percentage of total abundance for each sample. Additionally, to Waltham, Massachusetts, USA) containing 50 ␮g/mL kanamycin, account for “blind sampling” and large numbers of absences in as directed by the manufacturer’s protocol, and screened for posi- the data sets, the data sets were transformed using the Hellinger tive transformation with PCR using the primer set M13F (5=- equation as suggested by Ramette (2007). Species richness and GTTTTCCCAGTCACGAC-3=) and M13R (5=-CAGGAAACAGCTATG- diversity for the T-RFLP community profiles at each sampl- ACC-3=). Transformants were then purified using a QIAprep ing site (for both the HhaI and MspI data sets) were calculated Spin Miniprep kit (Qiagen Inc., Valencia, California, USA), and in R (Pinheiro et al. 2010) with the Vegan package (Oksanen et al. the resulting purified plasmid DNA was partially sequenced 2013). For calculating species richness and diversity, we assumed from the 5= end using either the M13F or M13R primer. All that the number of T-RFs present in a profile represented the sequences generated in this study were screened for chimeras

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 operational taxonomic units (OTUs) and that the T-RF peak height with Pintail (Ashelford et al. 2005), and deposited in GenBank represented the relative abundance of each OTU. For characteriz- (accession Nos. JQ772645–JQ773027 and KC436139–KC436264). ing species diversity, we used the Shannon diversity index, which Phylogenetic assignments were determined using the Ribosomal accounts for both abundance and evenness of the community at Database Project (RDP) (Cole et al. 2014) “Classifier” using a 95% a particular site. For characterizing species evenness, we used confidence threshold and “Seqmatch” (Wang et al. 2007). Pielou’s Evenness index, which is based on the Shannon index and Comparison of Diporeia T-RFLP profiles with profiles generated is constrained between 0.0 and 1.0, where less variation in com- for individual 16S rDNA clones was conducted in an attempt to munities between the OTU abundance approaches 1.0. identify and quantify the presence of particular bacterial groups, We tested for significant differences in OTU composition and including potential pathogens, in Diporeia populations. T-RFLP abundance among sites and years using permutational multi- analysis was performed on a total of 10 clones, which showed high variate analysis (PERMANOVA). For the PERMANOVA, the Bray– similarity (≥98%) to bacterial sequences contained in GenBank. Curtis distance matrix of Hellinger transformed OTU composition The purified cloned DNA was digested and analyzed using T-RFLP

Published by NRC Research Press Winters et al. 75

as described above to both ensure that complete digestion of PCR (Shannon–Weiner diversity index and Pielou’s Evenness index) products was achieved and to help determine the placement of showed that diversity of profiles for Diporeia communities varied each represented bacterial group in the Diporeia community pro- among each sampling event (Fig. 2). For both the HhaI and MspI files. Additionally, to obtain corresponding T-RFLP fragments, an data sets, the MI-18M samples from 2008 had the highest diversity in silico digest of clones was carried out in MEGA 5.0 (Tamura et al. and relatively high species evenness compared with the other 2011). samples analyzed. Additionally, for both the HhaI and MspI data sets the MI-18M samples from 2008 had the highest number of Estimation of bacterial community coverage OTUs and the lowest species evenness. Analyzing the community structure of bacteria in Diporeia sam- For both the HhaI and MspI data sets, a significant difference in ples included estimating coverage, which is defined as an estima- ␤ diversity was observed among all Diporeia sampling events for tion of the percentage of bacterial groups or OTUs identified in a all indices of similarity (HhaI MANOVA: df = 9, F = 3.583, P = 0.001; sample out of the total number of groups or OTUs. Calculating MspI MANOVA: df = 9, F = 5.6686, P = 0.001). For the HhaI data set, distances between known and unknown 16S rRNA sequences is a significant difference in multivariate dispersions among all important in comparing such sequences. In previous studies, dis- Diporeia sampling events was observed (df = 9, F = 4.406, P = 0.015). tance values of 0.03 have been used to differentiate bacteria at the T-RF’s 489 HhaI, 496 HhaI, and 484 MspI occurred in 23.53%, 20.59%, species level, 0.05 at the genus level, 0.10 at the family and class level, and 0.20 at the phylum level (Stackebrandt and Goebel 1994; and 60.00% of samples, respectively, with mean relative abundances Hugenholtz et al. 1998; Hughes et al. 2001; Sait et al. 2002). With of 7.77%, 1.67%, and 1.96%, respectively. Although T-RF 846 HhaI ac- this in mind, bacterial community coverage was calculated based counted for 3.20% of the mean relative abundance for the T-RFLP on a distance value of 0.03 using the formula profiles, it only occurred in the 3 Cayuga Lake samples, with mean relative abundances ranging from 24.44% to 29.95%. For both the C ϭ [1 Ϫ (n/N)] × 100 entire HhaI and MspI data sets, a significant positive correlation (Man- tel test) between diversity and depth was observed for Diporeia (HhaI: P = 0.002; MspI: P = 0.001). where n is the number of unique clones, and N is the total number A number of interesting findings were observed among profiles of clones analyzed. To gain a better understanding of the bacterial generated for Diporeia samples collected from multiple locations diversity associated with Great Lakes Diporeia, an overall estimate of coverage was obtained by removing the Cayuga Lake sequences in Lake Michigan (MI-18M, MI-27M, MI-40, and MI-47). First, for the ␤ and analyzing a single composite library constructed from se- MspI data set, a significant difference in diversity was observed quences from the 4 Great Lakes Diporeia clone libraries (SU-20B, only among Lake Michigan Diporeia profiles (df = 3, F = 2.50, MI-27M, HU-54M, and ON-41). P = 0.035). Second, results of multivariate analysis of variance (ANOVA) showed that site MI-18M was unique in that it had higher Sequence alignment phylogenetic affiliation abundances of certain T-RFs compared with the other sites. Third, For phylogenetic analyses of cloned Diporeia sequences, for the HhaI and MspI data sets, 20 and 27 T-RFs were shown to be Methanocaldococcus jannaschii (accession No. L77117) was used as the unique to MI-18M profiles, respectively. For the T-RFs that were outgroup taxon. A total of 353 16S rRNA gene sequences and the unique to MI-18M in the HhaI data set, with the exception of T-RF single outgroup sequence were aligned with the Jukes–Cantor 366 HhaI, which had an mean relative abundance of 21.15% ± correction (Jukes and Cantor 1969) using RDP II. The mean length 4.68%, the overall mean relative abundance was 2.03% ± 1.09%. For personal use only. of sequences in the final alignment file was 719 bp. The alignment Similarly, for the T-RFs that were unique to MI-18M in the MspI file was visually checked for alignment gaps and missing data in data set, the overall mean relative abundance was 1.18% ± 1.09%. nucleotide positions using MEGA 5.0 (Tamura et al. 2011). The No significant association was found between depth and the di- phylogenetic tree containing all 16S rRNA sequences from this versity of Lake Michigan Diporeia profiles (HhaI Mantel: P = 0.898; study was generated using the neighbor-joining (Saitou and Nei MspI Mantel: P = 0.892). Additionally, for both the HhaI and MspI 1987) method included in MEGA 5.0 using Maximum Composite data sets, no significant difference in variance was detected Likelihood as a measure of genetic distance. among Lake Michigan Diporeia samples. To test for significant clustering of taxa among the 5 clone libraries based on phylogenetic relationships, the UniFrac lineage- Temporal shifts in bacterial diversity specific analysis was used to break the tree up into the lineages For the Lake Superior site that was sampled in both years, the at a specified distance from the root (Lozupone et al. 2006). To ␤ diversity in bacterial community structure in 2008 was found to determine taxonomic assignments and phylogenetic relation- be significantly greater than in 2007 for the HhaI data set but not ships, additional trees were generated for cloned sequences and the MspI data set (HhaI MANOVA: df = 1, F = 3.682, P = 0.021; MspI sequences identified as closely related reference sequences con- MANOVA: df = 1, F = 1.859, P = 0.101). However, for Lake Michigan tained in RDP. Sequences were aligned and trees were generated no significant differences in ␤ diversity were found for the revis- as described above. Trees were then annotated to indicate the ited site (HhaI MANOVA: df = 1, F = 0.171, P = 0.456; MspI MANOVA: environment of origin for each sequence. df=1,F = 1.766, P = 0.146). Significant differences in the abundance of particular T-RFs (MANOVA) were observed between 2007 and Results 2008 profiles for both SU-23B and MI-18M (Table 2). For the HhaI Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 Bacterial communities of Diporeia data set, of the 45 total T-RFs present for MI-18M, 27 were detected Analysis of T-RFLP data revealed the presence of diverse bacte- in samples from both 2007 and 2008, 12 were unique to 2007, and rial communities in Diporeia samples. For the HhaI data set, a total 6 were unique to 2008. For the MspI data set, of the 57 total T-RFs of 175 T-RFs ranging from 50 to 983 bp in size were identified present for MI-18M, 37 were detected in samples from both 2007 across all samples; for the MspI data set, a total of 138 T-RFs rang- and 2008, 11 were unique to 2007, and 9 were unique to 2008. For ing from 55 to 983 bp in size were identified across all samples. For the HhaI data set, of the 141 total T-RFs present in SU-23B, 41 were the HhaI data set, the mean (±SE) number of T-RFs identified at the detected in samples from both 2007 and 2008, 20 were unique to sampling sites was 23.91 (±2.00) and ranged from 5 to 53 across all 2007, and 80 were unique to 2008. For the MspI data set, of the sites. For the MspI data set, the mean (±SE) number of T-RFs iden- 75 total T-RFs present in SU-23B, 34 were detected in samples tified at the sampling sites was 21.46 (±1.25) and ranged from 7 to from both 2007 and 2008, 18 were unique to 2007, and 23 were 35 across all sites. Species richness values and diversity indices unique to 2008.

Published by NRC Research Press 76 Can. J. Microbiol. Vol. 61, 2015

Fig. 2. Mean diversity index values (±SE) for 2 terminal restriction fragment length polymorphism (T-RFLP) data sets (ampliﬁed 16S rDNA digested with HhaI and MspI) generated for replicate Diporeia samples collected from lakes Michigan (MI), Superior (SU), and Huron (HU), and Cayuga Lake (New York) between 2007 and 2008. Richness is the number of operational taxonomic units, diversity is the Shannon–Weiner Diversity index, and Evenness is Pielou’s Evenness.

Table 2. Signiﬁcant differences (P < 0.05) in relative peak heights (MANOVA) for both 16S rRNA terminal restriction fragment length polymorphism (T-RFLP) data sets (HhaI and MspI) generated for Diporeia samples collected from lakes Michigan and Superior between 2007 and 2008. Lake Michigan MI-18M SU-23B sites (2007) (2007–2008) (2007–2008) T-RFLP data set T-RF P value T-RF P value T-RF P value

For personal use only. HhaI 60 <0.0001 67 0.039 61 0.034 83 0.004 231 0.045 205 0.004 330 0.001 367 0.034 366 0.002 375 0.004 MspI 127 0.012 312 0.038 127 0.045 146 0.001 128 0.026 167 0.049 146 0.024 428 0.039 496 0.034 534 0.015 563 0.009

Table 3. Ribosomal Database Project (RDP) matches (≥98%) and terminal restriction fragment (T-RF) lengths based on virtual restriction digestion and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene clones from Great Lakes Diporeia. T-RF size (bp) with: HhaI MspI Acc. No.

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 Clone (clone) Phylogenetic group (RDP match) Virtual T-RFLP Virtual T-RFLP 1 JQ772925 Bacillus sp. (Firmicutes) 574 576 162 157 2 JQ772936 Comamonadaceae (Betaproteobacteria) 188 188 491 494 3 JQ772844 Flavobacterium sp. (Bacteroidetes)87868280 4 JQ772993 Methylotenera sp. (Betaproteobacteria) 366 365 488 491 5 JQ772911 Microbacteriaceae (Actinobacteria) 369 372 279 277 6 JQ772796 Pseudomonas sp. (Gammaproteobacteria) 205 205 488 489 7 JQ772985 Rhodobacteraceae (Alphaproteobacteria) 569 568 440 440 8 JQ772737 Rickettsiaceae (Alphaproteobacteria) 526 528 450 449 9 JQ772726 Sphingobacteriaceae (Bacteroidetes) 93 90 539 542 10 JQ772740 Oxalobacteraceae (Betaproteobacteria) 565 566 487 494

Published by NRC Research Press Winters et al. 77

Fig. 3. Distance tree of 353 16S rRNA gene sequences detected in Fig. 4. Neighbor-joining consensus phylogeny of Bacteroidetes Diporeia spp. collected from 5 waterbodies. The tree was generated 16S rRNA gene sequences of clones from Diporeia and closely related with the neighbor-joining algorithm and the Jukes–Cantor reference sequences. Bootstrap values (1000 replicates) greater than correction. Lineage-specific significance (*) was determined using 70% are indicated at the nodes. Taxa observed in Diporeia samples UniFrac (blue, Lake Superior; red, Lake Michigan; light blue, Lake collected from lakes Superior, Michigan, Superior, and Ontario Huron; green, Lake Ontario; yellow, Cayuga Lake). “Others” refers to (Great Lakes), and Cayuga Lake (inland lake) are in bold. Triangle Actinobacteria, Chloroflexi, Deltaproteobacteria, Firmicutes, Planctomycetes, indicates a collapsed branch containing multiple taxa. Value to the and Verrucomicrobia. Note: to readers of the print issue, please see right of triangle indicates the number of taxa in the collapsed branch. the Web site at http://www.nrcresearchpress.com/doi/abs/10.1139/ cjm-2014-0434 to view a coloured version of the figure. For personal use only.

Virtual digestion and phylogenetic analysis RDP’s “classifier” revealed the presence of Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, and Verrucomicrobia in Diporeia 16S rRNA clone libraries. The in silico digestion of the representative clones provided for the putative identification of several of T-RFs. Despite evidence of “T-RF drift” (T-RF length differing from true length; Kaplan and Kitts 2003), patterns were observed in that a number of T-RFs can be matched to cloned sequences (Table 3). For For the phylum Bacteroidetes (Fig. 4), 2 significant clusters were example, T-RFs at 86 HhaI/80 MspI are likely to match Flavobacterium; observed. One large cluster contained 121 sequences from all T-RFs at 205 HhaI/489 MspI are likely to match Pseudomonas; and 5 clone libraries, where the vast majority of sequences were from T-RFs at 366 HhaI/493 MspI are likely to match Methylotenera. the Lake Ontario library. In the second smaller Bacteroidetes cluster As displayed in Fig. 3, phylogenetic analysis of 16S rRNA se- containing 11 sequences, the majority of sequences were from the

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 quences using UniFrac revealed significant clustering among Lake Michigan library. Phylogenetic analysis of these sequences Diporeia clone libraries, indicating a unique bacterial structure for showed that, while a greater genetic diversity was observed for Diporeia from each lake. Further analysis of the sequences in com- the Lake Ontario Diporeia sequences, both clusters were repre- parison with similar reference sequences derived from RDP re- sented by Flavobacterium spp. Additionally, although not signifi- vealed the presence of diverse bacterial groups present in Diporeia cant (P > 0.05) based on the specified distance from the root clone libraries. The phylum Actinobacteria, the class Betaproteobacteria, (11.3333), a third Flavobacterium cluster characterized by an abun- the order Pseudomonadales (Gammaproteobacteria), and the genus dance of sequences from the Lake Superior library was observed. Flavobacterium (Bacteroidetes) were present in all 5 libraries. For the For Alphaproteobacteria (Fig. 5a), sequences belonging to the order composite Diporeia clone library (353 16S rRNA sequences), a cov- Rhodobacteriales were from both the Lake Michigan and Cayuga erage level of 81.1%, 89.1%, 96.1%, and 100% was obtained for a Lake libraries. Sequences belonging to the order Sphingomonadales genetic distances of 0.02, 0.05, 0.10, and 0.20, respectively. At a were only detected in the Lake Ontario library. Interestingly, a genetic distance of 0.02, a total of 126 OTUs were observed. bacterium belonging to the order Rickettsiales was only detected in

Published by NRC Research Press 78 Can. J. Microbiol. Vol. 61, 2015

Fig. 5. Neighbor-joining consensus phylogeny of Alphaproteobacteria (a) and Betaproteobacteria (b) 16S rRNA gene sequences of clones from Diporeia and closely related reference sequences. Bootstrap values (1000 replicates) greater than 70% are indicated at the nodes. Taxa observed in Diporeia samples collected from lakes Superior, Michigan, Superior, and Ontario (Great Lakes), and Cayuga Lake (inland lake) are in bold. Triangle indicates a collapsed branch containing multiple taxa. Value to the right of triangle indicates the number of taxa in the collapsed branch. For personal use only.

the Lake Ontario library. However, the corresponding T-RF (450 MspI) ously reported for both freshwater and marine crustacean species was only detected in Diporeia samples collected from lakes Mich- (Møller et al. 2007; Peter and Sommaruga 2008; Grossart et al. igan and Superior, with a prevalence of 0.03% and an mean 2009; Tang et al. 2009). Recent studies using culture-independent relative abundance of 1.73% ± 0.56%. For the class Betaproteobacteria methods have identified as many as 6 major bacterial groups (Fig. 5b), a significant clustering of sequences similar to Rhodoferax associated with freshwater crustacean species (Actinobacteria, spp. and Albiferax spp. was observed, with the higher proportion Firmicutes, Bacteroidetes, Alpha-, Beta-, and Gammaproteobacteria). In the of sequences from the Lake Michigan Diporeia library having the current study, we identified 10 major bacterial groups associated highest relative contribution to the overall differences between with Great Lakes Diporeia (Actinobacteria, Bacteroidetes, Chloroflexi, environments. For the class Gammaproteobacteria (Fig. 6a), a signif- Firmicutes, Planctomycetes, Verrucomicrobia, Alpha-, Beta-, Gamma-, icant clustering of sequences similar to Perlucidibaca piscinae was and Deltaproteobacteria). observed, with the higher proportion of sequences from the Lake It is unknown how the composition of the bacterial community Ontario Diporeia library having the highest relative contribu- in the surrounding sediment influences that of Diporeia. However, tion to the overall differences between environments. For Gram- positive bacteria belonging to the phylum Firmicutes (Fig. 6b), with comparison of the 16S rDNA genes associated with Diporeia ob- the exception of the Lakes Huron library, bacteria belonging to served in the current study with those reported to be associated the orders Actinomycetales and Acidimicrobiales (Actinobacteria) were with the surrounding Great Lakes sediment in a recent study Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 detected in all libraries. A bacterium belonging to the order (Winters et al. 2014) show that, while the sediment is largely Bacilliales (Firmicutes) was only detected in the Lake Michigan dominated by Actinobacteria followed by Acidobacteria, Beta-, and library. Gammaproteobacteria, Diporeia is largely dominated by Bacteroidetes followed by Beta- and Gammaproteobacteria. This finding suggests Discussion that a number of the observed bacteria are intimately associated This study investigated the bacterial communities associated with Diporeia. While we cannot be sure which bacteria came from with Diporeia, an ecologically important organism in the Great the surface or gut of Diporeia or how the fullness of individual guts Lakes, which has declined considerably in abundance across affected the overall bacterial community structure, results of much of the lakes (Barbiero et al. 2011). Results of this study show 16S rRNA sequence and T-RFLP analysis demonstrate that the bac- that the diversity of the bacterial communities associated with terial communities of Diporeia were dominated by Flavobacterium Diporeia is relatively high compared with what has been previ- spp. (Bacteroidetes) and Pseudomonas spp. (Gammaproteobacteria).

Published by NRC Research Press Winters et al. 79

Fig. 6. Neighbor-joining consensus phylogeny of Gammaproteobacteria (a) and Firmicutes (b) 16S rRNA gene sequences of clones from Diporeia and closely related reference sequences. Bootstrap values (1000 replicates) greater than 70% are indicated at the nodes. Taxa observed in Diporeia samples collected from lakes Superior, Michigan, Superior, and Ontario (Great Lakes), and Cayuga Lake (inland lake) are in bold. Triangle indicates a collapsed branch containing multiple taxa. Value to the right of triangle indicates the number of taxa in the collapsed branch. For personal use only.

Analysis of the phylogenetic tree using UniFrac revealed that A number of differences were observed among the profiles for a number of bacterial species were common among different Diporeia samples collected from multiple sites in Lake Michigan in Diporeia populations, indicating the presence of a core micro- 2007 (MI-18M, MI-27M, MI-40, and MI-47). The finding that the biome. Additionally, analysis of the phylogenetic tree using Chao similarity index revealed a significant difference in ␤ diver- UniFrac revealed that a number of significant clusters were present sity among Lake Michigan profiles (MspI data set) suggests that the within the composite clone library, indicating the bacterial com- observed difference is due to low relative abundances of multiple munities associated with the surrounding sediment influenced bacterial species. This is further supported by the findings that the structure the Diporeia bacterial communities. The extent of MI-18M had higher species richness compared with other Lake how sediment bacterial communities influence Diporeia bacterial Michigan profiles and that a high number of T-RFs with low communities is unknown. Additionally, it is possible that amount mean relative abundances were unique to the MI-18M profiles. Al- of gut contents contained within individual Diporeia within each together, these findings suggest the bacterial communities asso- sample had varying effects on the observed bacterial community ciated with Diporeia at site MI-18M are distinctly different from structure. others in Lake Michigan. Interestingly, in a survey of trends in Diporeia abundances Temporal shifts were observed for Diporeia samples collected throughout the Great Lakes between 1997 and 2009, Barbiero et al. from Lake Superior (SU-23B). Whether the observed temporal (2011) showed that, although Diporeia abundances had signifi- shifts are due to the relatively shallow depth of this site, it’s cantly declined throughout Lake Michigan, significant declines unique location in Whitefish Bay near the Saint Mary’s River

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 were not observed at the 2 most southerly deep sites (MI-11 and through which Lake Superior drains into Lake Huron, or some MI-18M), which exhibited a negative (–0.60) albeit nonsignificant unknown limnological feature remains to be determined. The (P = 0.06) trend. Results of the current study showed that MI-18M cause(s) and potential biological significance of the shifts are un- profiles contained 20 (HhaI data set) and 27 (MspI data set) unique known and warrant further investigation. T-RFs compared with other Lake Michigan samples. Additionally, A highly significant correlation between depth and the diver- of the significantly different relative T-RF abundances for the Lake sity of Diporeia bacterial communities was observed for the com- Michigan Diporeia profiles, all were enriched in samples collected posite data set, suggesting depth may play a role in regulating the from site MI-18M. Furthermore, of all the samples analyzed in this bacterial communities associated with Diporeia. However, the study, on average, samples from MI-18M had relatively high spe- same correlation was not observed when only Lake Michigan data cies diversity. Collectively, the findings of this study together with were considered. It is possible that a significant correlation be- those of Barbiero et al. (2011) point to the possibility that the tween depth and diversity would have been observed if a larger profiles of MI-18M Diporeia represent a healthy bacterial community. range of depths were sampled for Lake Michigan. Further research

Published by NRC Research Press 80 Can. J. Microbiol. Vol. 61, 2015

is required to determine the relationship between depth and the Barbiero, R.P., and Tuchman, M.L. 2002. Results from GLNPOS’s biological open diversity of Diporeia bacterial communities. water surveillance program of the Laurentian Great Lakes. U.S. Environmen- tal Protection Agency, Great Lakes National Program Office, 2002. While the exact cause for the decline in Diporeia populations Barbiero, R.P., Schmude, K., Lesht, B.M., Riseng, C.M., Warren, G.J., and remains unexplained, bacterial pathogens could be a contribut- Tuchman, M.L. 2011. Trends in Diporeia populations across the Laurentian ing factor. In the current study, we detected some bacterial groups Great Lakes, 1997-2009. J. Great Lakes Res. 37: 9–17. doi:10.1016/j.jglr.2010.11. that contain members known to be pathogens of aquatic organ- 009. Briée, C., Moreira, D., and López-García, P. 2007. Archaeal and bacterial commu- isms. In terms of obligate pathogens, a bacterium belonging to nity composition of sediment and plankton from a suboxic freshwater pond. Rickettsiales, an order of Bacteria containing known pathogens for Res. Microbiol. 158: 213–227. doi:10.1016/j.resmic.2006.12.012. PMID:17346937. Diporeia and other freshwater amphipods (Federici et al. 1974; Chao, A., Chazdon, R.L., Colwell, R.K., and Shen, T.J. 2005. A new statistical Larsson 1982; Graf 1984; Messick et al. 2004), was detected in the approach for assessing similarity of species composition with incidence and abundance data. Ecol. Lett. 8: 148–159. doi:10.1111/j.1461-0248.2004.00707.x. Lake Ontario Diporeia clone library. One T-RF representing the Cole, J.R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D.M., Sun, Y., et al. 2014. order Rickettsiales (449 M) had a prevalence of 0.03% and an mean Ribosomal Database Project: data and tools for high throughput rRNA anal- relative abundance of 1.73% ± 0.56%. This would be considered a ysis. Nucleic Acids Res. 41: D633–D642. doi:10.1093/nar/gkt1244. PMID: relatively high abundance for a parasite in a free-ranging organ- 24288368. Dermott, R. 2001. Sudden disappearance of the amphipod Diporeia from eastern ism like Diporeia and can contribute, either alone or combined Lake Ontario, 1993-1995. J. Great Lakes Res. 27: 423–433. doi:10.1016/S0380- with other opportunistic bacteria, to Diporeia declines (Anderson 1330(01)70657-0. and May 1981). Additionally, Flavobacterium spp. and Pseudomonas Dermott, R., and Kerec, D. 1997. Changes to the deepwater benthos of eastern spp. genera that are known to contain pathogens of aquatic ani- Lake Erie since the invasion of Dreissena: 1979–1993. Can. J. Fish. Aquat. Sci. mals were detected in Diporeia samples. 54(4): 922–930. doi:10.1139/f96-332. Edmonson, W.T. 1959. Fresh-water Biology. 2nd ed. John Wiley and Sons, New Despite the constraints that can be associated with T-RFLP anal- York, USA. ysis of complex bacterial communities, such as multiple taxa shar- Egert, M., and Friedrich, M.W. 2003. Formation of pseudo-terminal restriction ing similar T-RFs and the possibility of pseudo T-RF formation fragments, a PCR-related bias affecting terminal restriction fragment length (Egert and Friedrich 2003), the combination of analysis of multi- polymorphism analysis of microbial community structure. Appl. Environ. Microbiol. 69: 2555–2562. doi:10.1128/AEM.69.5.2555-2562.2003. PMID:12732521. ple restriction enzyme data sets (HhaI and MspI) with T-RF pattern Federici, B.A., Hazard, E.I., and Anthony, D.W. 1974. Rickettsia-like organism confirmation by in silico digestion of cloned 16S rRNA gene se- causing disease in a crangonid amphipod from Florida. Appl. Microbiol. 28: quences allowed for considerable elucidation of Diporeia bacterial 885–886. PMID:4441067. communities. Furthermore, the reasonably high estimate of cov- Graf, F. 1984. Présence de bactéries dans les caecums postérieurs de l’intestin moyen du Crustacé hypogé Niphargus virei (Gammaridae : Amphipoda). Can. J. erage obtained for the composite Great Lakes Diporeia 16S rRNA Zool. 62: 829–833. doi:10.1139/z84-266. library suggests a large portion of the bacteria associated with Grossart, H.-P., Dziallas, C., and Tang, K.W. 2009. Bacterial diversity associated Diporeia in the Great Lakes was observed. Even though we did not with freshwater zooplankton. Environ. Microbiol. Rep. 1(1): 50–55. doi:10.1111/ sample to saturation (100% coverage), after 16S rRNA gene clones j.1758-2229.2008.00003.x. Hugenholtz, P., Goebel, B.M., and Pace, N.R. 1998. Impact of culture-independent were analyzed with T-RFLP, a few corresponding T-RFs were not studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. found in the community profiles. Similar findings were observed 180: 4765–4774. PMID:9733676. in an interlaboratory comparison for the microbial communities Hughes, J.B., Hellmann, J.J., Ricketts, T.H., and Bohannan, B.J.M. 2001. Counting of seafloor basalts (Orcutt et al. 2009). With this in mind, it is likely the uncountable: statistical approaches to estimating microbial diversity. Appl. Environ. Microbiol. 67: 4399–4406. doi:10.1128/AEM.67.10.4399-4406. these detected bacteria represent a rather small fraction of bacte- 2001. PMID:11571135. rial communities associated with the Diporeia. For personal use only. Inniss, W.E., and Mayfield, C.I. 1978. Psychrotrophic bacteria in sediments from In conclusion, the combination of T-RFLP analysis and 16S rRNA the Great Lakes. Water Res. 12: 237–241. doi:10.1016/0043-1354(78)90092-1. gene sequencing proved to be a powerful tool for investigating the Ishii, S., Yan, T., Shively, D.A., Byappanahalli, M.N., Whitman, R.L., and Sadowsky, M.J. 2006. Cladophora (Chlorophyta) spp. harbor human bacterial bacterial diversity of Diporeia. Since a similar study has never been pathogens in nearshore water of Lake Michigan. Appl. Environ. Microbiol. conducted on Diporeia, this study represents the most extensive 72: 4545–4553. doi:10.1128/AEM.00131-06. PMID:16820442. list of bacteria associated with this amphipod in relation to its Jukes, T.H., and Cantor, C.R. 1969. Evolution of protein molecules. In Mamma- surrounding environment and provides useful insights on the lian protein metabolism. Vol. 3. Edited by H.N. Munro. Academic Press, Massachesetts, USA. pp. 21–123. microbiota of Diporeia. Our hope is that the knowledge gained in Kaplan, C., and Kitts, C.L. 2003. Variation between observed and true terminal this study will shed light on the potential causes of the decline of restriction fragment length is dependent on true TRF length and purine Diporeia populations and will foster the development of effica- content. J. Microbiol. Methods, 54: 121–125. doi:10.1016/S0167-7012(03)00003-4. cious management strategies for the restoration and conservation PMID:12732430. Larsson, R. 1982. A rickettsial pathogen of the amphipod Rivulogammarus pulex. of both Diporeia and other Great Lakes organisms that rely on Diporeia. J. Invertebr. Pathol. 40: 28–35. doi:10.1016/0022-2011(82)90033-7. Legendre, P., and Legendre, L. 1998. Numerical ecology. 2nd English ed. Elsevier, Acknowledgements Amsterdam, Netherlands. We are thankful to the crew and staff of the R/V Lake Guardian Liu, W., Marsh, T.L., Cheng, H., and Forney, L.J. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymor- for helping in sample collection. This study was partially funded phisms of genes encoding 16S RNA. Appl. Environ. Microbiol. 63: 4516–4522. by the United States Environmental Protection Agency – Great PMID:9361437. Lakes National Protection Office (grant No. GL00E36101) and the Lozano, S.J., Scharold, J.V., and Nalepa, T.F. 2001. Recent declines in benthic Great Lakes Fisheries Trust (grant No. 2009.1058). macroinvertebrate densities in Lake Ontario. Can. J. Fish. Aquat. Sci. 58(3): 518–529. doi:10.1139/f01-002. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18 Lozupone, C., Hamady, M., and Knight, R. 2006. UniFrac — an online tool for References comparing microbial community diversity in a phylogenetic context. BMC Abdo, Z., Schütte, U., Bent, S., Williams, C., Forney, J., and Joyce, P. 2006. Statis- Bioinformatics, 7: 371. doi:10.1186/1471-2105-7-371. PMID:16893466. tical methods for characterizing diversity in microbial communities by anal- Maki, J.S., and Remsen, C.C. 1981. Comparison of two direct-count methods for ysis of terminal restriction fragment length polymorphism of 16S rRNA. determining metabolizing bacteria in freshwater. Appl. Environ. Microbiol. Environ. Microbiol. 8: 929–938. doi:10.1111/j.1462-2920.2005.00959.x. PMID: 41: 1132–1138. PMID:16345767. 16623749. Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., et al. Anderson, R.M., and May, R.M. 1981. The population dynamics of microparasites 1998. Design and evaluation of useful bacterium-specific PCR primers that and their invertebrate hosts. Phil. Trans. R. Soc. Lond. B Biol. Sci. 291: 451– amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64: 424. doi:10.1098/rstb.1981.0005. 795–799. PMID:9464425. Ashelford, K.E., Chuzhanova, N.A., Fry, J.C., Jones, A.J., and Weightman, A.J. Messick, G.A., Overstreet, R.M., Nalepa, T.F., and Tyler, S. 2004. Prevalence of 2005. At least 1 in 20 16S rRNA sequence records currently held in public parasites in amphipod Diporeia spp. from Lakes Michigan and Huron, U.S.A. repositories is estimated to contain substantial anomalies. Appl. Environ. Dis. Aquat. Organ. 59: 159–170. doi:10.3354/dao059159. PMID:15212283. Microbiol. 71: 7724–7736. doi:10.1128/AEM.71.12.7724-7736.2005. PMID:16332745. Møller, E.F., Riemann, L., and Søndergaard, M. 2007. Bacteria associated with

Published by NRC Research Press Winters et al. 81

copepods: abundance, activity and community composition. Aquat. Microb. Rinta-Kanto, J.M., Konopko, E.A., DeBruyn, J.M., Bourbonniere, R.A., Boyer, G.L., Ecol. 47(1): 99–106. doi:10.3354/ame047099. and Wilhelm, S.W. 2009. Lake Erie Microcystis: relationship between micro- Nalepa, T.F. 1987. Long-term changes in the macrobenthos of southern Lake cystin production, dynamics of genotypes and environmental parameters in Michigan. Can. J. Fish. Aquat. Sci. 44(3): 515–524. doi:10.1139/f87-064. a large lake. Harmful Algae, 8: 665–673. doi:10.1016/j.hal.2008.12.004. Nalepa, T.F. 1989. Estimates of macroinvertebrate biomass in Lake Michigan. Sait, M., Hugenholtz, P., and Janssen, P.H. 2002. Cultivation of globally distrib- J. Great Lakes Res. 15(3): 437–443. doi:10.1016/S0380-1330(89)71499-4. uted soil bacteria from phylogenetic lineages previously only detected in Nalepa, T.F., Hartson, D.J., Fanslow, D.L., Lang, G.A., and Lozano, S.J. 1998. De- cultivation-independent surveys. Environ. Microbiol. 4: 654–666. doi:10.1046/ clines in benthic macroinvertebrate populations in southern Lake Michigan, j.1462-2920.2002.00352.x. PMID:12460273. 1980–1993. Can. J. Fish. Aquat. Sci. 55(11): 2402–2413. doi:10.1139/f98-112. Saitou, N., and Nei, M. 1987. The neighbor-joining method: a new method for Nalepa, T.F., Fanslow, D.L., and Messick, G. 2005. Characteristics and potential reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. PMID:3447015. causes of declining Diporeia spp. populations in southern Lake Michigan and Smith, C.J., Danilowicz, B.S., Clear, A.K., Costello, F.J., and Wilson, B. 2005. T-Align, a web-based tool for comparison of multiple terminal restriction Saginaw Bay, Lake Huron. Great Lakes Fishery Commission Technical Report 66. fragment length polymorphism profiles. FEMS Microbiol. Ecol. 54: 375–380. Nalepa, T.F., Fanslow, D.L., Pothoven, S.A., Foley, A.J., and Lang, G.A. 2007. Long- doi:10.1016/j.femsec.2005.05.002. PMID:16332335. term trends in benthic macroinvertebrate populations in Lake Huron over Stackebrandt, E., and Goebel, B.M. 1994. A place for DNA–DNA reassociation and the past four decades. J. Great Lakes Res. 33(2): 421–436. doi:10.3394/0380- 16S rRNA sequence-analysis in the present species definition in bacteriology. 1330(2007)33[421:LTIBMP]2.0.CO;2. Int. J. Syst. Bacteriol. 44: 846–849. doi:10.1099/00207713-44-4-846. Newton, R.J., Jones, S.E., Eiler, A., McMahon, K.D., and Bertilsson, S. 2011. A guide Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. to the natural history of freshwater lake bacteria. Microbiol. Mol. Biol. Rev. MEGA5: molecular evolutionary genetics analysis using maximum likeli- 75: 14–49. doi:10.1128/MMBR.00028-10. hood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Oksanen, J., Blanchet, F.G., Kindt, R., Legendre, P., Minchin, P.R., O’Hara, R.B., Evol. 28(10): 2731–2739. doi:10.1093/molbev/msr121. PMID:21546353. et al. 2013. Package ‘vegan’. Community ecology package. Version 2.0-0. Tang, K.W., Turk, V., and Grossart, H.P. 2010. Linkage between crustacean zoo- Available from http://CRAN.R-project.org/package=vegan. plankton and aquatic bacteria. Aquat. Microb. Ecol. 61: 261–277. doi:10.3354/ Orcutt, B., Bailey, B., Staudigel, H., Tebo, B.M., and Edwards, K.J. 2009. An inter- ame01424. laboratory comparison of 16S rRNA gene-based terminal restriction fragment Tang, X., Gao, G., Qin, B., Zhu, L., Chao, J., Wang, J., et al. 2009. Characterization length polymorphism and sequencing methods for assessing microbial di- of bacterial communities associated with organic aggregates in a large, shal- versity of seafloor basalts. Environ. Microbiol. 11: 1728–1735. doi:10.1111/j.1462- low, eutrophic freshwater lake (Lake Taihu, China). Microb. Ecol. 58: 307– 2920.2009.01899.x. PMID:19508561. 322. doi:10.1007/s00248-008-9482-8. PMID:19169740. Peter, H., and Sommaruga, R. 2008. An evaluation of methods to study the gut Wang, Q., Garrity, G.M., Tiedje, J.M., and Cole, J.R. 2007. Naïve Bayesian classifier bacterial community composition of freshwater zooplankton. J. Plankton for rapid assignment of rRNA sequences into the new bacterial taxonomy. Res. 30(9): 997–1006. doi:10.1093/plankt/fbn061. Appl. Environ. Microbiol. 73: 5261–5267. doi:10.1128/AEM.00062-07. PMID: Pilloni, G., Granitsiotis, M.S., Engel, M., and Lueders, T. 2012. Testing the limits of 17586664. 454 pyrotag sequencing: reproducibility, quantitative assessment and com- Winters, A.D., Marsh, T.L., Brenden, T.O., and Faisal, M. 2014. Molecular charac- parison to T-RFLP fingerprinting of aquifer microbes. PLoS One, 7: e40467. terization of bacterial communities associated with sediments in the Lauren- doi:10.1371/journal.pone.0040467. PMID:22808168. tian Great Lakes. J. Great Lakes Res. 40: 640–645. doi:10.1016/j.jglr.2014.04. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., and R Development Core Team. 008. 2010. nlme: linear and nonlinear mixed effects models. R package version Zwart, G., Crump, B.C., Kamst-van Agterveld, M.P., Hagen, F., and Han, S.K. 2002. 3.1-97. R Foundation for Statistical Computing, Vienna, Austria. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences Ramette, A. 2007. Multivariate analyses in microbial ecology. FEMS Microbiol. from plankton of lakes and rivers. Aquat. Microb. Ecol. 28: 141–155. doi:10. Ecol. 62: 142–160. doi:10.1111/j.1574-6941.2007.00375.x. PMID:17892477. 3354/ame028141. For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIVERSITY on 04/12/18

Published by NRC Research Press