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

THANK YOU