DOI: 10.5061/dryad.w9ghx3fw7
title: Effects of environmental translocation and host characteristics on skin microbiomes of sun-basking fish

description: Variation in the composition of skin-associated microbiomes has been
 attributed to host species, geographic location, and habitat, but the role
 of intraspecific phenotypic variation among host individuals remains
 elusive. We explored if and how host environment and different phenotypic
 traits were associated with microbiome composition. We conducted repeated
 sampling of dorsal and ventral skin microbiomes of carp individuals
 (Cyprinus carpio) before and after translocation from laboratory
 conditions to a semi-natural environment. Both alpha and beta diversity of
 skin-associated microbiomes increased substantially within and among
 individuals following translocation, particularly on dorsal body sites.
 The variation in microbiome composition among hosts was significantly
 associated with body site, sun-basking, habitat switch, and growth, but
 not temperature gain while basking, sex, personality, or colour morph. We
 suggest that the overall increase in the alpha and beta diversity
 estimates among hosts were induced by individuals expressing greater
 variation in behaviours and thus exposure to potential colonizers in the
 pond environment compared to the laboratory. Our results exemplify how
 biological diversity at one level of organization (phenotypic variation
 among and within fish host individuals) together with the external
 environment impacts biological diversity at a higher hierarchical level of
 organisation (richness and composition of fish-associated microbial
 communities).
description: Estimation of alpha and beta diversity All
 statistical analyses were performed in Rstudio v1.3.1093 (53) with R
 v3.6.0. We included three alpha diversity estimates. Observed number of
 ASVs (100% identical ‘amplicon sequence variants’) was used to illustrate
 the partitioning of ASVs according to sample type and environment.
 Statistical analysis of richness was based on estimates generated with
 default settings in the breakaway function (package breakaway v4.6.11). To
 incorporate an alpha diversity measurement that takes abundance and
 evenness into account we used Shannon-Weaver diversity index estimated
 from data subsampled to the smallest sample size (3775 reads per sample)
 using the diversity function in the vegan package (v2.5-6).
 Apart from the subsampling prior to the Shannon index, raw data
 was used throughout the analyses. However, to explore the contribution of
 rare ASVs, we conducted a filtering step for comparison with the results
 based on raw data. To this end, we followed the method described in
 Bokulich, Subramanian: ASVs with a total count &lt;10 within each
 sample and total abundance &lt;0.01% across all samples were
 considered “rare”. This step decreased the total number of ASVs found in
 fish skin microbiomes from 16,881 to 1883 representing 11% of the total
 number of ASVs. The results based on data subsets are reported in Table S2
 and S3. For beta diversity, the data was transformed by
 centred log ratio (clr) allowing it to be used as input for linear
 regressions. Distances to group centroid for samples from each of the two
 environments were estimated from Euclidean distance matrix on clr-values,
 using the function betadisper (type = centroid) in the vegan
 package.
description: This README file was generated on 2023-11-13 by Hanna Berggren. GENERAL
 INFORMATION 1. Title of Dataset: "Effects of environmental
 translocation and host characteristics on skin microbiomes of sun-basking
 fish" 2. Author Information A. Principal Investigator Contact
 Information Name: Anders Forsman Institution: Linnaeus University Address:
 Kalmar, Sweden Email: B. Associate or
 Co-investigator Contact Information Name: Hanna Berggren Institution:
 Linnaeus University Address: Kalmar, Sweden Email:
 We explored if and how host environment
 and different phenotypic traits were associated with microbiome
 composition. We conducted repeated sampling of dorsal and ventral skin
 microbiomes of carp individuals (*Cyprinus carpio*) before and after
 translocation from laboratory conditions to a semi-natural environment. We
 collected microbiome samples from dorsal and ventral body sites for each
 individual (*n* = 27) and environment (laboratory versus pond)
 representing 108 samples in total. Sequencing libraries were prepared by
 using the primer pair 341F and 805R that target the V3-V4 hypervariable
 regions of the bacterial 16S gene complex and sequenced on the Illumina
 MiSeq platform (Illumina, USA) using MiSeg Reagent Kit v3 with 600 cycles
 (2×300 bp). SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on
 the data: CC0 1.0 Universal (CC0 1.0) Public Domain 2. Links to
 publications that cite or use the data: Berggren H, Nordahl O, Yildirim Y,
 Larsson P, Tibblin P, Forsman A. (2023). Effects of environmental
 translocation and host characteristics on skin microbiomes of sun-basking
 fish. Proceedings of the Royal Society B. 1. Links to other publicly
 accessible locations of the data (raw sequence data):
  
   2.
 Links/relationships to ancillary data sets: None 3. Was data derived from
 another source? No A. If yes, list source(s): NA 4. Recommended citation
 for this dataset: Berggren H, Nordahl O, Yildirim Y, Larsson P, Tibblin P,
 Forsman A. 2023 Data from: Effects of environmental translocation and host
 characteristics on skin microbiomes of sun-basking fish. Dryad Digital
 Repository (doi: ). DATA
 & FILE OVERVIEW The data is divided in two steps; Rawdata processing
 (raw data originated from qiime2 v2018.8 i.e., feature-tables, taxonomy
 files) for instance removing sequences present in negative controls and
 separating sequences from different libraries into correct files for
 further analysis. For this workflow, please see raw_data_processing.Rmd.
 The resulting files from this first part are provided on dryad and thus it
 is not a necessary step for going through the statistical analyses and
 data transformations described in and Sunbasking_carp_2022.Rmd. For
 statistical analyses and data transformation, rawdata are provided that
 can be used for all the data handling described in
 Sunbasking_carp_2022.Rmd. However, some variables such as estimated
 richness, shannon and distance to centroid are included in the metadata
 file. Some of the transformed data i.e., centered-log-ratio are not
 provided due to very large file size. Please note that since these data
 transformations are based on probability estimates, the exact file cannot
 be generated, which might lead to slightly different test-statistics. 1.
 File List: Step 1: A) raw_data_processing.Rmd B)
 P11965-feature-table.tsv.zip C) P11965_q2_taxonomy.zip D)
 P12860-feature-table.tsv.zip E) P12860_q2_taxonomy.zip F)
 P13155-feature-table.tsv.zip G) P13155_q2_taxonomy.zip H)
 P13156-feature-table.tsv.zip I) P13156_q2_taxonomy.zip J) metadata-merged
 Step 2: K) Sunbasking_carp_2022.Rmd L) metadata_carp_2021_03_14.tsv M)
 carp_counts.tsv.zip N) literature_search.txt O) taxonomy_filtered.tsv.zip
 LEfSe Results: P) Supplementary Data File 1.txt Q) Supplementary Datafile
 2.txt 1. Relationship between files, if important: None 2. Additional
 related data collected that was not included in the current data package:
 None 3. Are there multiple versions of the dataset? No A. If yes, name of
 file(s) that was updated: NA i. Why was the file updated? NA ii. When was
 the file updated? NA
 \#########################################################################
 The data is divided in two steps; DATA-SPECIFIC INFORMATION FOR STEP 1:
 A-J Rawdata processing = preprocessing the raw data and annotated taxonomy
 originated from qiime2 v2018.8 (i.e., feature-tables) such as removing
 sequences present in negative controls,taxonomic filtering, and splitting
 samples into right files for further analysis. Please see
 raw_data_processing.Rmd. The resulting files from this part are provided
 and thus not a neccessary step for going through the statistical analysis
 in the file described in and Sunbasking_carp_2022.Rmd. DATA-SPECIFIC
 INFORMATION FOR: raw_data_processing.Rmd R.markdown containing code for
 removing sequences present in negative controls,taxonomic filtering, and
 splitting samples into right files for further analysis.
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: P11965-feature-table.tsv 1. Number of
 variables: 3 2. Number of cases/rows: 26988 3. Variable List: * seqid:
 unique ASV id originating from qiime2 * sample: sample id containing
 library information and sample number * count: number of times the
 specific ASV occurred in the specific sample 4. Missing data codes: None
 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: P11965_q2_taxonomy.zip 1. Number of
 variables: 9 2. Number of cases/rows: 9634 3. Variable List: * asv: unique
 ASV id originating from qiime2 * domain: taxonomic rank * phylum:
 taxonomic rank * class: taxonomic rank * order: taxonomic rank * family:
 taxonomic rank * genus: taxonomic rank * species: taxonomic rank *
 Confidence: confidence level for assigned genus 4. Missing data codes: NA
 (data not available) 5. Specialized formats or other abbreviations used:
 None \############################################################
 DATA-SPECIFIC INFORMATION FOR: P12860-feature-table.tsv 1. Number of
 variables: 3 2. Number of cases/rows: 26076 3. Variable List: * seqid:
 unique ASV id originating from qiime2 * sample: sample id containing
 library information and sample number * count: number of times the
 specific ASV occurred in the specific sample 4. Missing data codes: None
 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: P12860_q2_taxonomy.zip 1. Number of
 variables: 9 2. Number of cases/rows: 9675 3. Variable List: * asv: unique
 ASV id originating from qiime2 * domain: taxonomic rank * phylum:
 taxonomic rank * class: taxonomic rank * order: taxonomic rank * family:
 taxonomic rank * genus: taxonomic rank * species: taxonomic rank *
 Confidence: confidence level for assigned genus 4. Missing data codes: NA
 (data not available) 5. Specialized formats or other abbreviations used:
 None \##########################################################
 DATA-SPECIFIC INFORMATION FOR: P13155-feature-table.tsv 1. Number of
 variables: 3 2. Number of cases/rows: 52846 3. Variable List: * seqid:
 unique ASV id originating from qiime2 * sample: sample id containing
 library information and sample number * count: number of times the
 specific ASV occurred in the specific sample 4. Missing data codes: None
 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: P13155_q2_taxonomy.zip 1. Number of
 variables: 9 2. Number of cases/rows: 13170 3. Variable List: * asv:
 unique ASV id originating from qiime2 * domain: taxonomic rank * phylum:
 taxonomic rank * class: taxonomic rank * order: taxonomic rank * family:
 taxonomic rank * genus: taxonomic rank * species: taxonomic rank *
 Confidence: confidence level for assigned genus 4. Missing data codes: NA
 (data not available) 5. Specialized formats or other abbreviations used:
 None \#########################################################
 DATA-SPECIFIC INFORMATION FOR: P13156-feature-table.tsv 1. Number of
 variables: 3 2. Number of cases/rows: 44418 3. Variable List: * seqid:
 unique ASV id originating from qiime2 * sample: sample id containing
 library information and sample number * count: number of times the
 specific ASV occurred in the specific sample 4. Missing data codes: None
 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: P13156_q2_taxonomy.zip 1. Number of
 variables: 9 2. Number of cases/rows: 15486 3. Variable List: * asv:
 unique ASV id originating from qiime2 * domain: taxonomic rank * phylum:
 taxonomic rank * class: taxonomic rank * order: taxonomic rank * family:
 taxonomic rank * genus: taxonomic rank * species: taxonomic rank *
 Confidence: confidence level for assigned genus 4. Missing data codes: NA
 (data not available) 5. Specialized formats or other abbreviations used:
 None \###########################################################
 DATA-SPECIFIC INFORMATION FOR: metadata-merged.txt 1. Number of variables:
 6 2. Number of cases/rows: 384 3. Variable List: * sample: sample id
 containing library information and sample number * flowcell: library name
 * sample_type: sample originated from water och fish * study: indicates
 which study species that was used * date_extraction: Date of DNA
 extraction * kit_used: samples with same number were extracted using the
 same kit-package. 4. Missing data codes: None 5. Specialized formats or
 other abbreviations used: None
 \########################################################### DATA-SPECIFIC
 INFORMATION FOR STEP 2: K-O K) Sunbasking_carp_2022.Rmd L)
 metadata_carp_2021_03_14.tsv M) carp_counts.tsv.zip N)
 literature_search.txt O) taxonomy_filtered.tsv.zip DATA-SPECIFIC
 INFORMATION FOR: Sunbasking_carp_2022.Rmd R-markdown with the code for
 statistical analysis.
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: metadata_carp_2021_03_14.tsv 1. Number of
 variables: 36 2. Number of cases/rows: 116 3. Variable List: * sample:
 sample id containing library information and sample number * est:
 estimated richness from breakaway package * flowcell: sequence library
 name * samplename: original sample name * sample_type: sample originated
 from water or fish * study: indicates which study species that was used *
 sampling_location: whether sample was collected when fish were in
 laboratory or filed (pond) * Individ: The individual ID number that was
 also used in Nordahl O, Tibblin P, Koch-Schmidt P, Berggren H, Larsson P,
 Forsman A. Sun-basking fish benefit from body temperatures that are higher
 than ambient water ambient water. Proceedings of the Royal Society
 B-Biological Sciences. 2018;285(1879). * occasion: experimental period
 (pre or post translocation) * body_cite.c: abbreviated exact body
 position: VL= ventral left, VR = Ventral right, DL= dorsal left, DR=
 dorsal right * body_site: whether sample was taken on dorsal or ventral
 body sites * tube_number: number of the tube the sample was stored in *
 date_extraction: Date of DNA extraction * kit_used: samples with same
 number were extracted using the same kit-package. * cm_release: length of
 fish in cm at time of release into pond * cm_recap: length of fish in cm
 at time of recapture from pond * gr_recap: weight of fish in gram at time
 of recapture from pond * gr_release: weight of fish in gram at time of
 release into pond * Date of release into the pond: as described in
 variable name * Date of recapture: as described in variable name *
 PIT_tag_number: ID number on the individual fish PIT-tag * DST_number: ID
 number on the individual fish DST-tag * Colour: which colour morph the
 fish belong to * Sex: which gender * Weight: growth in gram during period
 in pond (i.e., weight gain calculated from gr_recap - gr_release) *
 Bold_Shy: value of bold-shy continuum * Thermal_switch: We extracted the
 maximum and minimum in body temperature of each individual during each
 hour of the last week in the pond and calculated a delta value (max-min)
 in °C that was summed up for the whole period. * Basking: accumulated time
 of dormancy in the surface layer (measured in minutes) during periods of
 time categorized as basking * Mean_basktemp: mean of the temperature
 excess acquired during periods of sun-basking for each individual compared
 to the water measured in °C * Vertical_switch: We extracted the maximum
 and minimum in water depth of each individual during each hour of the last
 week in the pond and calculated a delta value (max-min) in cm that was
 summed up for the whole period. * groups: grouping sample according to
 experimental period (occasion) and body site (dorsal/ventral) *
 distance_to_centroid_occasion: from the Euclidean distance matrix, this
 the distance to centroid for each individual sample that belonged to
 either the groups of samples taken pre- or post-translocation,
 respectively. * distance_to_centroid_occasion_abundant_ASVs: same as above
 but for abundant ASVs only * shannon: shannon diversity index for each
 sample. * distance_to_centroid_occasion_rare: same as above but for rare
 ASVs only * color_morph: which colour morph the fish belong to 4. Missing
 data codes: None 5. Specialized formats or other abbreviations used: F =
 female, M = male
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: carp_counts.tsv.zip 1. Number of variables:
 3 2. Number of cases/rows: 42671 3. Variable List: * sample: sample id
 containing library information and sample number * seqid: unique ASV id
 originating from qiime2 * count: number of times the specific ASV occurred
 in the specific sample 4. Missing data codes: None 5. Specialized formats
 or other abbreviations used: None
 \################################################################
 DATA-SPECIFIC INFORMATION FOR: literature_search.txt 1. Number of
 variables: 5 2. Number of cases/rows: 287 3. Variable List: * Year: year
 of publication * records: number of publications * body_site: whether
 publication stydy skin or gut microbiome * where: aquatic or terrestrial
 habitat * groups: variables "body_site" and "where"
 combined 4. Missing data codes: None 5. Specialized formats or other
 abbreviations used: None
 \####################################################################
 DATA-SPECIFIC INFORMATION FOR: taxonomy_filtered.tsv.zip 1. Number of
 variables: 9 2. Number of cases/rows: 40 175 3. Variable List: * seqid:
 unique ASV id originating from qiime2 * domain: taxonomic rank * phylum:
 taxonomic rank * class: taxonomic rank * order: taxonomic rank * family:
 taxonomic rank * genus: taxonomic rank * species: taxonomic rank *
 Confidence: confidence level for assigned genus 4. Missing data codes:
 None 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 LEfSe Results: DATA-SPECIFIC INFORMATION FOR: Supplementary Data File
 1.txt 1. Number of variables: 3 2. Number of cases/rows: 58 3. Variable
 List: * taxonomy: taxonomic annotation * total_score: total LDA score for
 the specific biomarker * occasion: group of comparison (pre or post
 translocation) * enriched_score: enriched LDA score 4. Missing data codes:
 None 5. Specialized formats or other abbreviations used: None
 \########################################## DATA-SPECIFIC INFORMATION FOR:
 Supplementary Data File 2.txt 1. Number of variables: 4 2. Number of
 cases/rows: 58 3. Variable List: * taxonomy: taxonomic annotation *
 LDA_score_total: total LDA score for the specific biomarker * body_site:
 group of comparison (dorsal or ventral) * score_body_site: enriched LDA
 score 4. Missing data codes: None 5. Specialized formats or other
 abbreviations used: None