RNA/DNA ratios used to study growth in coastal nursery areas - - PowerPoint PPT Presentation

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...

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Regular nourishments since 1991

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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 § Multidisciplinary survey § Animal Breeding & Genetics

Richard Crooijmans

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MSC Maarten Rutting: Fish growth RNA/DNA

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

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§ 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

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 bromide Measure #1 Add Qubit DNA- and RNA dye Measure Measure #2 Incubation 2 min Calculate fluorescence for DNA Qubit Quantifies RNA and DNA + Correction for sample input Quantify RNA and DNA

Qubit range

§ Ideally

• maximum amount of sample

(20 µl)

• measurements should

fall in the middle of the range

§ Possible for DNA § Not possible for RNA

Ø

Dilution required

200 400 600

Fluorescence Concentration (ng/ml)

Schematic overview of the Qubit range

5 10 15 20 25 10 20

Calculated Qubit RNA ng µl -1

Sample in Qubit (µl)

Dilution effect

§ Using less sample volume:

• bias in the result

§ Correction needed for

diluting

§ Dilution series to produce

corrections for diluting

Dilution direction

Method comparison

Ethidium Bromide Qubit 1 2 3 4 5 6 7 RNA:DNA

2 4 6 8 2 4 6 8 RNA:DNA Ethidium Bromide RNA:DNA Qubit

Location differences

1 2 3 4 1 2 3 4 5 6 7 Location RNA:DNA

Sole

1 2 3 4 1 2 3 4 5 6 7 Location RNA:DNA

Dab

1 2 3 4 1 2 3 4 5 6 7 Location RNA:DNA

Plaice

N: 48 103 74 77 N: 17 33 23 5 N: 76 120 109 87

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

5 10 15 20 1 2 3 4 5 6 7 Length (cm) RNA:DNA

R 2 = 0.333 p = 2.6e-08

Sole

5 10 15 20 1 2 3 4 5 6 7 Length (cm) RNA:DNA

R 2 = 0.232 p = 5.6e-19

Dab

5 10 15 20 1 2 3 4 5 6 7 Length (cm) RNA:DNA

R 2 = 0.000231 p = 0.76

Plaice

locations: 1 (ο) 2 (∆) 3 (+) 4 (×)

RNA/DNA~fish length

5 10 15 20 1 2 3 4 5 6 7 Length (cm) RNA:DNA

R 2 = 0.232 p = 5.6e-19

Dab

1 2 3 4 5 6 1 2 3 4 5 6 7 Length (cm) RNA:DNA

R 2 = 0.199 p = 1.8e-10

Dab 0-group

locations: 1 (ο) 2 (∆) 3 (+) 4 (×)

RNA/DNA~salinity

28 30 32 34 36 1 2 3 4 5 6 7 Salinity (ppt) RNA:DNA

R 2 = 0.015 p = 0.28

Sole

28 30 32 34 36 1 2 3 4 5 6 7 Salinity (ppt) RNA:DNA

R 2 = 0.239 p = 1.5e-19

Dab

28 30 32 34 36 1 2 3 4 5 6 7 Salinity (ppt) RNA:DNA

R 2 = 0.0148 p = 0.016

Plaice

locations: 1 (ο) 2 (∆) 3 (+) 4 (×)

Preliminary analysis

factor plaice dab dab 0 group sole

fish length

ns

• +
• temperature
• ns

ns

salinity

• +

+ ns

water visibility

• ns

ns ns

density shore crab

• ns

ns ns

density large brown shrimp

ns

• ns

ns

Locations (factor)

ns s s ns

§ 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

Discussion

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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)

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Discussion: Qubit vs Ethidium bromide

Pros:

• Measure both DNA and RNA
• Easy to use, less steps

involved that influence

• utcome
• No enzymatic steps required
• safer to use and requires

less training

• possible to use the kits with

a Fluorometric plate reader

Cons:

• range RNA high sensitivity

kit too limited to accurately quantify RNA in fastest growing juveniles =>Solution: Qubit™ RNA Broad Range Assay Kit

• RNA quantification is

sensitive to dilution

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Shore crab Density

0.00 0.02 0.04 0.06 1 2 3 4 5 6 7 Shore crab m−2 RNA:DNA R 2 = 0.13 p = 0.0012

Sole

0.00 0.02 0.04 0.06 1 2 3 4 5 6 7 Shore crab m−2 RNA:DNA R 2 = 0.00339 p = 0.31

Dab

0.00 0.02 0.04 0.06 1 2 3 4 5 6 7 Shore crab m−2 RNA:DNA R 2 = 0.052 p = 5.1e-06

Plaice

locations: 1 (ο) 2 (∆) 3 (+) 4 (×)

RNA/DNA~temperature

16 17 18 19 20 21 22 1 2 3 4 5 6 7 Temperature ( ° C) RNA:DNA

R 2 = 0.0961 p = 0.0054

Sole

16 17 18 19 20 21 22 1 2 3 4 5 6 7 Temperature ( ° C) RNA:DNA

R 2 = 0.0532 p = 5.2e-05

Dab

16 17 18 19 20 21 22 1 2 3 4 5 6 7 Temperature ( ° C) RNA:DNA

R 2 = 0.011 p = 0.038

Plaice

locations: 1 (ο) 2 (∆) 3 (+) 4 (×)