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Diversity and relative abundance of the bacterial pathogen, Flavobacterium spp., infecting reproductive… Lemay, Matthew A; Russello, Michael A Nov 4, 2014

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RESEARCH ARTICLEnBackground Kokanee, the freshwater form of sockeye salmonLemay and Russello BMC Research Notes 2014, 7:778http://www.biomedcentral.com/1756-0500/7/778kokanee in four lakes across BC identified genes involvedwith pathogen resistance as being putatively underUniversity of British Columbia, Okanagan Campus, 3247 University Way,Kelowna, BC V1V 1V7, CanadaPathogens can play an important role in the evolution oftheir hosts [1-3]. This can occur when variation in patho-gen diversity over small spatial or temporal scales imposesdivergent selection on populations of their host species[1,4]. In salmonids, for example, genetic diversity associ-ated with the major histocompatability complex can varyat micro-geographical scales [1,5], reflecting local adapta-tion in response to heterogeneous pathogen regimes [2].(Oncorhynchus nerka), occurs as two reproductive eco-types, which differ in their choice of spawning habitat(streams vs. beaches) [6,7]. Previous research foundthat the abundance of cDNA from several pathogens(bacteria, fungi, and a parasitic flatworm) was greaterin pooled-transcriptome samples from the stream-spawning ecotype compared with the beach-spawningecotype in Okanagan Lake, British Columbia (BC),Canada [8]. Moreover, a subsequent genomic scan of* Correspondence: matt.lemay@ubc.caAbstractBackground: Understanding the distribution and abundance of pathogens can provide insight into the evolution andecology of their host species. Previous research in kokanee, the freshwater form of sockeye salmon (Oncorhynchusnerka), found evidence that populations spawning in streams may experience a greater pathogen load compared withpopulations that spawn on beaches. In this study we tested for differences in the abundance and diversity of thegram-negative bacteria, Flavobacterium spp., infecting tissues of kokanee in both of these spawning habitats (streamsand beaches). Molecular assays were carried out using primers designed to amplify a ~200 nucleotide region of thegene encoding the ATP synthase alpha subunit (AtpA) within the genus Flavobacterium. Using a combination of DNAsequencing and quantitative PCR (qPCR) we compared the diversity and relative abundance of Flavobacterium AtpAamplicons present in DNA extracted from tissue samples of kokanee collected from each spawning habitat.Results: We identified 10 Flavobacterium AtpA haplotypes among the tissues of stream-spawning kokanee and sevenhaplotypes among the tissues of beach-spawning kokanee, with only two haplotypes shared between spawninghabitats. Haplotypes occurring in the same clade as F. psychrophilum were the most prevalent (92% of all reads, 60% ofall haplotypes), and occurred in kokanee from both spawning habitats (streams and beaches). Subsequent qPCR assaysdid not find any significant difference in the relative abundance of Flavobacterium AtpA amplicons between samplesfrom the different spawning habitats.Conclusions: We confirmed the presence of Flavobacterium spp. in both spawning habitats and found weak evidencefor increased Flavobacterium diversity in kokanee sampled from stream-spawning sites. However, the quantity ofFlavobacterium DNA did not differ between spawning habitats. We recommend further study aimed at quantifyingpathogen diversity and abundance in population-level samples of kokanee combined with environmental sampling tobetter understand the ecology of pathogen infection in this species.Keywords: Bacteria, Bacterial coldwater disease, Flavobacterium, Pathogens, Kokanee, Sockeye salmon,Oncorhynchus nerkaDiversity and relative abupathogen, Flavobacteriumreproductive ecotypes ofMatthew A Lemay* and Michael A Russello© 2014 Lemay and Russello; licensee BioMedCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Accessdance of the bacterialspp., infectingkokanee salmonCentral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in thislowing the manufacturer’s recommended protocol.Flavobacterium DNA was isolated from each kokaneeDNA sample using primers designed to amplify a ~200 nu-cleotide region of the gene encoding the ATP synthasealpha subunit (AtpA) of all species within the genus Flavo-bacterium [Fspp1_F: 5′-TTRTTAAGAAGACCACCRGG-3′, Fspp1_R: 5′- GGRATATATGCAGAAACGTCACC-3′].This region was chosen because AtpA is part of a panel ofgenes used for strain identification in the species Flavobac-terium psychrophilum [16], allowing comparisons with pre-viously published sequence data.Each PCR contained 2 μl DNA, 2.5 μl 10X PCR buffer,2.5 μl dNTPs (2 mM), 1.0 μl forward primer (10 μM),1.0 μl reverse primer (10 μM), and AmpliTaq Gold poly-merase (0.5 units, Applied Biosystems) in a 25 μl totalvolume. Touchdown PCR protocol [17] was used withinitial denaturation of 94°C for 10 minutes, then 10 cyclesat 94°C for 30 seconds, 60°C for 30 seconds, 72°C for60 seconds, with the annealing step decreasing by 1°Cper cycle to 50°C. The annealing temperature was main-tained at 50°C for an additional 30 cycles, followed byextension at 72°C for 2 minutes. All PCR products werepurified using a Qiagen MinElute kit, diluted to 4 ng/μl,and ligated overnight at 4°C using the pGEM®-T EasyVector System (Promega). Transformed cells were addedLemay and Russello BMC Research Notes 2014, 7:778 Page 2 of 7http://www.biomedcentral.com/1756-0500/7/778divergent selection between stream and beach-spawningkokanee [9]. These data suggest that kokanee may experi-ence asymmetrical pathogen infection between spawningenvironments, however the lack of information on patho-gen diversity within each spawning habitat has precludeda direct test of this hypothesis.In this study, we measured the diversity and abun-dance of the gram-negative bacteria, Flavobacteriumsp., infecting Okanagan Lake kokanee from their twodivergent spawning habitats (streams and beaches).Flavobacterium was chosen for this study because itsabundance was highly correlated with ecotype inpooled transcriptome samples [8], and because severalFlavobacterium species are associated with high levelsof salmonid mortality [10], with devastating economicimpacts [11-13].MethodsThis study focused on Okanagan Lake, which is a long(135-km) and narrow (<5 km) post-glacial lake located inthe southern interior of British Columbia, Canada. Thelarge size of the lake (~350 km2) supports several spawningpopulations of both reproductive ecotypes [14,15].Current estimates suggest that there are ~200,000 spawn-ing adult kokanee in Okanagan Lake. Kokanee are semel-parous and philopatric, with the majority of adultsspawning at an age of three years [15]. Beach spawninghas been observed at most undeveloped areas of theshoreline. Stream-spawning is currently monitored at 18tributaries, of which 60% of stream spawning occurs at asingle location (Mission Creek) [15]. Stream and beach-spawning habitats experience differences in many abioticfactors such as seasonal temperature, turbidity, and rateof water-flow, which may affect the diversity of pathogenspresent in the two environments.Flavobacterium diversityIn order to infer the diversity of Flavobacterium speciesinfecting kokanee salmon, tissue samples from spawningadult kokanee were collected from four stream and threebeach-spawning sites in September 2010 (Table 1). Theseseven sites were chosen because they are annually moni-tored for kokanee abundance by the British ColumbiaMinistry of Forest, Lands and Natural Resource Opera-tions. All tissue samples were collected in partnershipwith the British Columbia Ministry of Forests, Lands andNatural Resource Operations (collection permit PE10-66394), and in accordance with animal care protocolA11-0127 as approved by the University of BritishColumbia’s Animal Care & Biosafety Committee, whichgoverns the ethical collection of specimens for research.Detailed collection information for these samples hasbeen previously reported by Lemay et al. [8]. GenomicDNA was extracted from the subcutaneous muscle tissueof one individual kokanee from each of these seven sitesusing a Macherey-Nagel single column extraction kit fol-Table 1 Samples used for each assaySpawninghabitatSamplelocation1. Flavobacteriumabundance:2. Flavobacteriumdiversity:Number of kokaneesampledNumber of coloniessequenced (numberof haplotypesobserved)Stream MissionCreek12 14 (7)PentictonCreek12 12 (2)PeachlandCreek12 12 (2)PowersCreek12 12 (4)Total 48 50Beach Northeast 8 15 (2)Northwest 20 15 (3)Southeast 20 20 (4)Total 48 50(1) The number of kokanee from each location used for the qPCR assay testingthe relative proportion of Flavobacterium sp. DNA infesting the host tissue; (2)The number of cloned Flavobacterium sp. amplicons that were sequencedfrom each sample location, and the number of different haplotypes observedat each location (in parentheses). Reads were cloned from genomic DNAextracted from a single kokanee at each sample location.to plates containing 100 μg/ml ampicillin, 0.5 mM IPTG,and 80 μg/ml X-Gal, and incubated for 16-20 hours atLemay and Russello BMC Research Notes 2014, 7:778 Page 3 of 7http://www.biomedcentral.com/1756-0500/7/77837°C. White colonies were then boiled at 100°C for 10 mi-nutes in 100 μl of TE buffer. We amplified cloned insertsfrom 100 colonies (50 from each ecotype) using T7 andSp6 primers (Promega). Each PCR contained 1 μl colonyboil, 1.25 μl 10X PCR buffer, 1.25 μl dNTPs (2 mM),0.5 μl each primer (10 μM), and KAPATaq polymerase(0.5 units; KAPA Biosystems) in a 13.5 μl total volume.Each PCR had an initial denaturation of 94°C for 2 mi-nutes, followed by 35 cycles at 94°C for 30 seconds, 50°Cfor 30 seconds, 72°C for 30 seconds, with a final exten-sion at 72°C for 2 minutes. Sequencing was carried outin one direction using Sp6 on an Applied Biosystems3130XL. Raw sequences were edited using Sequencher v.5; five sequences were discarded from further analysesdue to low quality and ambiguous bases.A phylogenetic approach was then used to quantifythe diversity of AtpA haplotypes among the remaining95 sequences (47 stream, 48 beach; Additional file 1).We retained all distinct haplotypes irrespective of thenumber of reads matching the sequence. Unique haplo-types from each spawning habitat (streams and bea-ches) were unambiguously aligned with the AtpAregion from the published genomes of nine Flavobacter-ium species (F. aquatile [GenBank: JX25684.1], F. bran-chiophilum [GenBank: NC016001.1], F. chungnamense[GenBank: JX256869.1], F. columnare [GenBank: CP003222.2], F. frigidarium [GenBank: HM443893.1], F. indi-cum [GenBank: NC017025.1], F. johnsoniae [GenBank;NC009441.1], F. koreense [GenBank: JX356867.1], F. psy-chrophilum [GenBank: NC009613.1]) using MUSCLEas implemented in Geneious v.6.1 (Biomatters). Thisalignment was then used to generate an unrootedneighbor-joining tree in Geneious v.6.1 (Biomatters);1000 Bootstrap replicates were performed with a 50%support threshold.Flavobacterium abundanceQuantitative PCR (qPCR) was used to measure the rela-tive abundance of Flavobacterium spp. present in koka-nee sampled from Okanagan Lake. This assay wascarried out using DNA extracted from operculum tissueof spawning adult kokanee collected in 2007 (n = 48)and 2010 (n = 48). All kokanee samples had been col-lected as part of previous research [7], and includedsamples from both ecotypes at seven different locationsin Okanagan Lake (Table 1).The total quantity of extracted DNA (fish and patho-gen) was first determined for each sample using theQuant-iT™ Pico Green ds DNA Assay Kit (Invitrogen)run on a ViiA7 real-time PCR machine (Life Technolo-gies). Quantitative PCR was carried out using the sameAtpA primers described above to quantify the pathogencomponent of each DNA sample using an absolute quan-tification protocol on a ViiA7 real-time PCR machine(Life Technologies). Using the same primers as the assayfor Flavobacterium diversity allows us to quantify theabundance of all documented haplotypes. To constructthe standard curve we used F. psychrophilum DNA ofknown strain and concentration [Strain: CIP103534(T),isolated from coho salmon, Oncorhynchus kisutch]. Forincreased precision, three replicates of each concentra-tion in the standard curve were used. Each PCR con-tained 1 μl of DNA template, 0.5 μl of 1 μM Fspp1_Fforward primer, 0.5 μl of 10 μM Fspp2_R reverse primer,and 5.0 μl of Fast SYBR® Green Master Mix (Applied Bio-systems) in a total volume of 10 μl. A two-step cyclingprotocol was carried out with an initial denaturation of94°C for 2 minutes, followed by 55 cycles at 94°C for30 seconds and 60°C for 30 seconds. A melt curve stagewas added to the end of the protocol beginning at 60°Cand increasing to a final temperature of 98°C.For each individual kokanee, the inferred quantity ofFlavobacterium spp. amplicons was normalized to thetotal DNA template concentration of the sample in orderto derive a measure of pathogen infection per unit ofkokanee DNA [18]. Deviations from normality in this re-sponse variable (ng of Flavobacterium DNA per ng of ko-kanee DNA) precluded the use of parametric statistics;instead we tested for differences in Flavobacterium abun-dance between ecotypes from each sampling year usingnon-parametric Kruskal-Wallis tests implemented in Rversion 3.0.1 [19]. In addition, results of the qPCR assaywere visualized using box plots generated in R using thedefault parameters for generating whisker lengths anddesignating outliers.Results and discussionWe tested for differences in the abundance and diversityof the salmonid pathogen, Flavobacterium spp., infectingkokanee salmon from two different spawning habitats(streams and beaches). Both of the molecular assays car-ried out in this study (DNA sequencing and qPCR)found evidence for the presence of Flavobacterium fromall study-sites and both spawning environments inOkanagan Lake.Using a phylogenetic approach, we identified 10 Flavo-bacterium AtpA haplotypes among the stream-spawningkokanee and seven haplotypes among beach-spawningkokanee, with only two haplotypes shared betweenspawning habitats (Figures 1 & 2, Additional file 1). Hap-lotypes occurring in the same clade as F. psychrophilumwere the most prevalent, occurring in kokanee from bothspawning habitats (streams and beaches) and accountingfor 60% of all haplotypes and 92% of all sequenced reads.The two most abundant haplotypes (haplotype 5 & 7)only differ from each other by a single nucleotide differ-ence and account for 82% of all sequenced reads; withHaplotype-7 identical to the reference sequence for F.Figure 1 Flavobacterium diversity. Unrooted neighbor-joining tree showing the relationship between the unique Flavobacterium AtpAhaplotypes amplified from each ecotype (n = 10 stream, n = 7 beach). Nine previously published reference sequences are included in thephylogram with Genbank accession numbers included next to species names. Branch labels are bootstrap support values (%). Pair wise nucleotidedistances between haplotypes are provided in Additional file 2.Figure 2 Haplotype frequency distribution. A frequency distribution showing the number of reads from each spawning habitat (n = 47 streamand n = 48 beach) that corresponds with each of the 15 haplotypes observed in this study. Haplotypes 4, 5, 7, 9, 10, 11, 13, 14, 15 occur within thesame clade as the F. psychrophilum reference sequence.Lemay and Russello BMC Research Notes 2014, 7:778 Page 4 of 7http://www.biomedcentral.com/1756-0500/7/778psychrophilum (Additional file 2). Haplotypes that fall onclades other than that of F. psychrophilum are only repre-sented by 1-2 reads per haplotype, and none of theseremaining haplotypes were shared between spawninghabitats.The results from the qPCR assay did not reveal anysignificant difference in the relative abundance of Flavo-bacterium AtpA amplicons between samples from stream-and beach-spawning sites (2010 p = 0.211; 2007 p = 0.204;Figure 3).The phylogenetic results provide preliminary evidencethat there may be differences in the composition of Fla-vobacteria species/strains between the two spawninghabitats, however the small sample size (3-4 kokanee perspawning habitat) severely limits our ability to draw de-finitive conclusions. For example the small sample sizesmay skew the observed distribution of rare haplotypes.Yet, the relatively high diversity of Flavobacterium AtpAhaplotypes observed in the tissue of only seven kokaneesystems including plants [21], animals [22,23], and soils[24], and provides an effective method to test for differ-ences in bacterial community structure. For example, inAtlantic salmon, Salmo salar, 16 s rRNA gene analysiswas used to identify bacterial communities putatively as-sociated with infectious amoebic gill disease [25].In order to better understand the ecological interactionsbetween kokanee and their bacterial pathogens, it wouldalso be useful for future research to examine Flavobac-terium diversity and abundance from environmental sam-ples in the two habitats. While F. psychrophilum is bothhorizontally and vertically transmitted to new hosts [26,27],it can also persist for long periods of time (300 days) with-out a host [28]. Therefore, the compliment of pathogensinfecting fish tissues may not be an accurate estimate of thetotal diversity and abundance present in each habitat.ConclusionsGenes associated with immune response are often identi-eriLemay and Russello BMC Research Notes 2014, 7:778 Page 5 of 7http://www.biomedcentral.com/1756-0500/7/778samples provides evidence that individuals may be in-fected by multiple strains and/or species of Flavobacter-ium, however the small size of the amplicon (~200 basepairs) precludes determination of species identity.It would be highly informative for future research to se-quence larger regions and additional genes in order to de-termine the identity of species present. Alternatively, theuse of high-throughput methods for quantifying microbialcommunity assemblages, such as 16 s rRNA gene sequen-cing [20], could be used to provide insight into the com-position of bacterial communities inhabiting kokaneefrom each spawning habitat. This approach has been usedto quantify microbial communities in a diversity of studyFigure 3 Flavobacterium abundance. Relative abundance of Flavobactsampled at stream and beach spawning sites in Okanagan Lake based on(B) 2010 (n = 48).fied as candidate regions for local adaptation in salmo-nids [29-33], including in kokanee [9], and it has beenshown that patterns of divergence at these genes may re-sult from localized differences in pathogen diversityacross fine spatial or temporal scales [34-37]. While thiscurrent study only found weak evidence for differences inpathogen diversity in kokanee collected from differentspawning habitats (streams and beaches), these resultswarrant further study. Future research aimed at quantify-ing pathogen diversity and abundance in population-levelsamples of kokanee combined with environmental sam-pling is needed to better understand the ecology ofpathogen infection in this system.um AtpA haplotypes amplified from the operculum tissue of kokaneeqPCR assays. Samples are separated by year: (A) 2007 (n = 48);Lemay and Russello BMC Research Notes 2014, 7:778 Page 6 of 7http://www.biomedcentral.com/1756-0500/7/778Availability of supporting dataThe data sets supporting the results of this article areincluded within the additional files.Additional filesAdditional file 1: Flavobacterium AtpA haplotype sequences. A listof the unique AtpA haplotypes used to generate Figure 1. Sections of theNCBI reference sequences from previously published Flavobacteriaspecies are also included.Additional file 2: Pair wise distance matrix. A pair wise matrixshowing the number of nucleotide differences between each AtpAhaplotype described in Figures 1 & 2.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsML and MR designed the study. ML carried out the laboratory assays anddata analyses. ML and MR drafted the manuscript. Both authors read andapproved the final manuscript.AcknowledgementsWe thank Eric Duchaud (French National Institute for Agricultural Research)for donating DNA standards and providing feedback on an earlier version ofthis manuscript. We also thank Katy Hind for assistance with the dataanalysis. This work was funded by Genome British Columbia (SOF130 to MR);ML was partially funded by an NSERC Postgraduate Fellowship.Received: 29 July 2014 Accepted: 24 October 2014Published: 4 November 2014References1. Fraser DJ, Weir LK, Bernatchez L, Hansen MM, Taylor EB: Extent and scale oflocal adaptation in salmonid fishes: review and meta-analysis. 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Dionne M, Miller KM, Dodson JJ, Bernatchez L: MHC standing geneticvariation and pathogen resistance in wild Atlantic salmon. Philos Trans RSoc Lond B Biol Sci 2009, 364(1523):1555–1565.36. Eizaguirre C, Lenz TL, Kalbe M, Milinski M: Divergent selection on locallyadapted major histocompatibility complex immune genesexperimentally proven in the field. Ecol Lett 2012, 15(7):723–731.37. Bernatchez L, Landry C: MHC studies in nonmodel vertebrates: what havewe learned about natural selection in 15 years? J Evol Biol 2003,16(3):363–377.doi:10.1186/1756-0500-7-778Cite this article as: Lemay and Russello: Diversity and relativeabundance of the bacterial pathogen, Flavobacterium spp., infectingreproductive ecotypes of kokanee salmon. BMC Research Notes2014 7:778.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionLemay and Russello BMC Research Notes 2014, 7:778 Page 7 of 7http://www.biomedcentral.com/1756-0500/7/778Submit your manuscript at www.biomedcentral.com/submit

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