RNA/DNA ratios used to study growth in coastal nursery areas Comparison of methods and relation with environment Maarten Rutting, Richard Crooijmans , Ralf van Hal & Ingrid Tulp
Living below sea level... 2
Regular nourishments since 1991 3
Sand nourishments and nurseries
Impact on nursery function? § Knowledge on the impact: ● Benthic community restores within a year after sand nourishment ● Effects on fish community? ● Effects on the nursery function? =>fish growth?
June 2017: multidisciplinary survey § Wageningen Marine Research § Animal Breeding & Genetics § Multidisciplinary survey Richard Crooijmans MSC Maarten Rutting: Fish growth RNA/DNA 6
Locations § Location 1: Zuid-Holland § Location 2: Noord-Holland § Location 3: Texel § Location 4: Ameland =>4 consecutive weeks from South to North
Transects: fish sampling § Transects per location § Fish sampling: ● 0-1 m: walking push net ● 2-3 m: dinghy: 2 m beam ● 3-10 m: vessel 3 m beam § Stratification based on sediment § Continuous recording abiotics § Benthos sampling 8
Aim MSc project § compare methods to measure RNA:DNA ratio’s § Investigate growth juvenile flatfish in June in nurseries along the Dutch coast § Growth ~ related to abiotic factors? § Relation with sand nourishments: role of sediment?
Survey - Benthos
Survey - Sediment
Survey - Fish
Tissue collection § starting points ● For tissue collection: ● Directions from Benjamin Ciotti (thanks!) ● For isolation: ● Protocol and Guide for Estimating Nucleic Acids in Larval Fish Using a Fluorescence Microplate Reader (Caldarone, et al. , 2001)
RNA/DNA Quantification: two methods § Ethidium bromide § Qubit Fluorometer ● Already used before to analyse RNA:DNA ratio’s ● Using ● RNA High Sensitivity Assay Kit (Invitrogen™) ● dsDNA High Sensitivity Assay Kit (Invitrogen™)
RNA/DNA Quantification Add RNAse + Incubation 30 min Add Ethidium Calculate Measure #1 bromide fluorescence for DNA Measure #2 Quantify RNA and DNA Add Qubit DNA- and Measure Qubit Quantifies RNA dye RNA and DNA + Incubation 2 Correction for min sample input
Qubit range § Ideally ● maximum amount of sample Schematic overview of the Qubit range (20 µ l) 600 Fluorescence 500 ● measurements should 400 fall in the middle of the 300 range 200 § Possible for DNA 100 0 § Not possible for RNA 0 200 400 600 Concentration (ng/ml) Ø Dilution required
Dilution effect § Using less sample volume: 25 ● bias in the result Calculated Qubit RNA ng µ l -1 Dilution direction 20 § Correction needed for 15 diluting 10 § Dilution series to produce corrections for diluting 5 0 0 10 20 Sample in Qubit ( µ l)
Method comparison 8 7 6 RNA:DNA Qubit 6 RNA:DNA 5 4 4 2 3 2 0 1 0 2 4 6 8 RNA:DNA Ethidium Bromide Ethidium Bromide Qubit
Location differences 7 Plaice 7 Dab 7 Sole 6 6 6 5 5 5 RNA:DNA RNA:DNA RNA:DNA 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 1 2 3 4 1 2 3 4 1 2 3 4 Location Location Location N: 76 120 109 87 N: 48 103 74 77 N: 17 33 23 5
Factors considered ● Temperature ● Salinity ● Depth ● Tidal phase ● Location ● Sediment grain size <- No results yet ● Density of benthic prey <- No results yet ● Density of shore crab ● Density of large common shrimp (+30 mm) ● Density of flatfish (highly correlated with shore crab)
RNA/DNA~fish length 7 7 7 R 2 = 0.000231 R 2 = 0.232 R 2 = 0.333 Plaice Dab Sole p = 0.76 p = 5.6e-19 p = 2.6e-08 6 6 6 5 5 5 RNA:DNA RNA:DNA RNA:DNA 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Length (cm) Length (cm) Length (cm) 1 ( ο ) 3 ( + ) locations: 2 ( ∆ ) 4 ( × )
RNA/DNA~fish length R 2 = 0.199 7 Dab 0-group p = 1.8e-10 6 5 RNA:DNA 7 R 2 = 0.232 4 Dab p = 5.6e-19 6 3 5 RNA:DNA 4 2 3 1 2 1 0 0 0 1 2 3 4 5 6 0 5 10 15 20 Length (cm) Length (cm) 1 ( ο ) 3 ( + ) locations: 2 ( ∆ ) 4 ( × )
RNA/DNA~salinity 7 7 7 R 2 = 0.0148 R 2 = 0.239 R 2 = 0.015 Plaice Dab Sole p = 0.016 p = 1.5e-19 p = 0.28 6 6 6 5 5 5 RNA:DNA RNA:DNA RNA:DNA 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 28 30 32 34 36 28 30 32 34 36 28 30 32 34 36 Salinity (ppt) Salinity (ppt) Salinity (ppt) 1 ( ο ) 3 ( + ) locations: 2 ( ∆ ) 4 ( × )
Preliminary analysis dab 0 factor plaice dab sole group ns - + - fish length - - ns ns temperature - + + ns salinity - ns ns ns water visibility - ns ns ns density shore crab ns - ns ns density large brown shrimp ns s s ns Locations (factor)
Discussion § Qubit suitable to measure RNA/DNA § range RNA high sensitivity kit too limited to accurately quantify RNA in fastest growing juveniles =>Solution: Qubit™ RNA Broad Range Assay Kit § Seasonal effect ~ location effect § Variation in RNA/DNA related to several (a)biotic factors § Negative effect epibenthic predators § Location and salinity confounding § Relation with sediment: still too be included in analysis 25
Future work § Include sediment data § Refine Qubit method § Next step: collecting fish later in the year, when food becomes limiting and growth is reduced
Thanks for listening Questions (not too technical J ) 27
Discussion: Qubit vs Ethidium bromide Pros: Cons: ● Measure both DNA and RNA ● range RNA high sensitivity kit too limited to accurately ● Easy to use, less steps quantify RNA in fastest involved that influence growing juveniles outcome =>Solution: Qubit™ ● No enzymatic steps required RNA Broad Range ● safer to use and requires Assay Kit less training ● RNA quantification is ● possible to use the kits with sensitive to dilution a Fluorometric plate reader 28
Shore crab Density 7 7 7 R 2 = 0.052 R 2 = 0.00339 R 2 = 0.13 Plaice Dab Sole p = 5.1e-06 p = 0.31 p = 0.0012 6 6 6 5 5 5 RNA:DNA RNA:DNA RNA:DNA 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 0.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06 Shore crab m − 2 Shore crab m − 2 Shore crab m − 2 1 ( ο ) 3 ( + ) locations: 2 ( ∆ ) 4 ( × )
RNA/DNA~temperature 7 7 7 R 2 = 0.011 R 2 = 0.0532 R 2 = 0.0961 Plaice Dab Sole p = 0.038 p = 5.2e-05 p = 0.0054 6 6 6 5 5 5 RNA:DNA RNA:DNA RNA:DNA 4 4 4 3 3 3 2 2 2 1 1 1 0 0 0 16 17 18 19 20 21 22 16 17 18 19 20 21 22 16 17 18 19 20 21 22 Temperature ( ° C) Temperature ( ° C) Temperature ( ° C) 1 ( ο ) 3 ( + ) locations: 2 ( ∆ ) 4 ( × )
Recommend
More recommend
