Post on 02-Jan-2016
description
Bioscience – Aarhus Universitet
Jiayi Qin
Internship: Margarita Ustyuzhanina
Supervisor P.H. Krogh
The soil food web under stress by
application of ashes from biofuel
ASHBACK WP3 Ecotoxicology
Bioscience – Aarhus Universitet
Outline
Development of DNA metabarcoding method
• Total environmental DNA could represent the real biodiversity. • Minibarcode from mitochondrial DNA – amplification of sequence for
species identification
Biodiversity change in spruce forest under the stress of ash
Comparison of food webs affected by different ash application rates
Ecotoxicological effects on mesofauna by ash in a spruce forest soil and in agricultural soil
Bioscience – Aarhus Universitet
Develop DNA metabarcoding methodused for the investigating changes of the soil mesofauna biodiversity.
Bioscience – Aarhus Universitet
Morphospecies
• Take photo of each morphospecies.
• Sort morphospecies into different bottles.
• Use SoilBioStore to count the number of
animals.
• Identify species by barcoding and verify by
conventional taxonomic identification of
physical voucher subsequently archived
Bioscience – Aarhus Universitet
SoilBioStoreCounting webinterface and MySQL database storage
Trait database
Bioscience – Aarhus Universitet
Metabarcoding — DNA extraction
Environmental DNA (eDNA) extraction:
Total environmental
DNA
• 10 g soil• PowerMax®
Soil DNA isolation kit
Extracellular DNA
• 400 g soil• Dissolve soil
in PBS buffer
• Supernatant For DNA extraction
DNA soup
• All animals pooled togther
(Bienert et al. 2012; Taberlet et al. 2012, Yu et al. 2012, Yang et al. 2013 )
Bioscience – Aarhus Universitet
Metabarcoding — DNA extraction
Environmental DNA (eDNA) extraction:
Total environmental
DNA
• 10 g soil• PowerMax®
Soil DNA isolation kit
Extracellular DNA
• 400 g soil• Dissolve soil
in PBS buffer
• Supernatant For DNA extraction
DNA soup
• All animals pooled togther
Hypothesis 1: Total eDNA could represent the real biodiversity.
(Bienert et al. 2012; Taberlet et al. 2012, Yu et al. 2012, Yang et al. 2013 )
Bioscience – Aarhus Universitet
metabarcoding—PCR strategy
Primer:mt-COI12S/16S mt-rDNA18S/28S rDNA nuclear Internal Transcribed Spacer (ITS)
(Machida and Knowlton 2012)
Advantages and disadvantages of each gene region for metagenetic analyses.
Bioscience – Aarhus Universitet
metabarcoding—PCR strategy
Primer:mt-COI12S/16S mt-rDNA18S/28S rDNA nuclear Internal Transcribed Spacer (ITS)
(Machida and Knowlton 2012)
Advantages and disadvantages of each gene region for metagenetic analyses.
Hypothesis 2: Minibarcode from mitochondrial DNA could amplify sequence for specie identification.
Bioscience – Aarhus Universitet
Development of DNA metabarcodingfor the investigating of changes in the soil mesofauna biodiversity.
Bioscience – Aarhus Universitet
Biodiversity change in spruce forest under the stress of ash
• Ash treatment0, 3, 4.5, 6 t/ha
• Sampling occasions1) Pretreatment sampling, April 1, 20142) Spring 2014, 3) June 2-3 Autumn 2014, 4) (spring 2015) autumn 2015 5) spring 2016 6) autumn 2016 ?
• Analysis strategy– Conventional method - morphospecies– Metabarcoding
Bioscience – Aarhus Universitet
Comparison of food web changes when affected by different levels of ash
• Location: Agricultural field, Foulum• Ash treatments:
super phosphate at 30 and 300 kg ha-1 combined with either– wood pill ash– straw ash – Norway spruce, Picea abies, ash
• Sampling:Time: September, 2014
Content: soil cores for eDNA analysis• Analysis strategy: conventional method/metabarcoding
Ecotoxicology of ash exposed to mesofauna in a spruce forest soil and an agricultural soil
Test species Age for test(Days)
Test duration(Days)
Norminal concentration series(g kg-1)
Folsomia candida 9-12 28Foulum
soil 0, 5, 10, 12, 15, 17, 20Gedhus
soil 0, 5, 10, 15, 20
Onychiurus yodai 24-27 42 0, 5, 1, 15, 30, 35, 40, 50, 60, 75
Hypoaspis aculeifer 32-35 21 0, 5Enchytraeus crypticus Adult female 21 0, 5
Definitive test protocol in spruce forest soil and agricultural soil
(OECD 2009)
Bioscience – Aarhus Universitet
Result
0 5000 10000 15000 20000
0
200
400
600
800
1000
Reproduction of F.candida in Folumn Soil
Rep
rodu
ctio
n
Concentration of Ash (mg kg-1)
0 10000 20000 30000 40000 50000 60000 70000 800000
2
4
6
8
10
12
14
16
Reproduction of F.candida in Folumn Soil
Mot
ality
Concentration of Ash (mg kg-1)Motality of O.yodai in Folumn Soil
1E-51E-4100 1000 100000
50
100
150
200
250
300
350
Rep
rodu
ctio
n
Concentration of Ash (mg kg-1)
Reproduction of o.yodai in Folumn SoilSurvival
Mo
rta
lity
Bioscience – Aarhus Universitet
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