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DNA Metabarcoding to quantify the response of insect diversity to mountain forest die-offs in the French Pyrenees

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OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible

This is an author’s version published in: http://oatao.univ-toulouse.fr/21195

Any correspondence concerning this service should be sent

to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr

To cite this version:

Sire, Lucas and Rougerie, Rodolphe and Larrieu, Laurent and Bézier, Annie and Courtial, Béatrice and Bouget, Christophe and Herniou, Elisabeth and Lopez

Vaamonde, Carlos DNA Metabarcoding to quantify the response of insect diversity to

mountain forest die-offs in the French Pyrenees. (2018) In: International Conference

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response of insect diversity to

mountain forest die-offs in the

French Pyrenees

L. Sire1, R. Rougerie2, L. Larrieu3,4, A. Bézier1, B. Courtial5, C. Bouget6, E. Herniou1, C. Lopez-Vaamonde1,5

SFEcologie

Rennes – 23 Octobre 2018

1IRBI, UMR 7261 – Université de Tours – Tours, France; 2MNHN, UMR 7205 – Paris, France ; 3INRA, UMR 1201

DYNAFOR – Castanet Tolosan, France;4CRPFOcc – Auzeville Tolosane, France;5INRA, UR0633 Zoologie Forestière –

Orléans, France ;6IRSTEA, UR EFNO – Nogent-sur-Vernisson, France

lucas.sire@univ-tours.fr

Lucas Sire

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2

Allen, 2009

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• How to manage forest diebacks is a significant concern for forest stakeholders. • What should be done with the large volumes of dying trees and fresh deadwood

(salvage logging, sanitation salvage) ?

(5)

Global insects decline ?

https://2ser.com/rapid-insect-decline-caused-by-climate-change/ 4

(6)

Thesis aims

• Temporal turnover

• What is the species richness in arthropods ? • Develop biomonitoring pipelines for forest biodiversity

Quantify the ecological impact of climate-induced forest die-offs and

salvage logging on arthropod communities

• Test DAMe bioinformatic pipeline

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6 Badeau et al., 2005

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Situation of Silver fir in France

Proportion of dominant trees with more than 25% of dead branches

M o r e t h a n 3 5 % F rom 2 0 t o 3 5 % F rom 1 0 t o 2 0 % L e s s t h a n 1 0 % No tree in that case

Number of living and dominant trees

M o r e t h a n 1 0 0 F ro m 5 0 t o 1 0 0 F ro m 2 5 t o 5 0

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Experimental design

Healthy plots

|

Low / high level diebacks

|

No harvesting / Salvage logging

Vallée d’Aure Pays de Sault

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Ecological parameters

Micro-dendrohabitat assessment

Larrieu et al., 2018

Drénou et al., 2013

Stand structure, canopy openness, dead wood volume, large tree density…

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Biomonitoring using mass-trapping samples

• Sampling each month over 4 months

(from May to September) • 224 Malaise samples • 448 Polytrap samples

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Sample preparation

Drying and homogeneization Grinding Trap filtration DNA extraction

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12

Twin-tagging dual indexing PCRs

Leray et al., 2016 (Chap 14) Leray et al., 2013

• Highly degenerated primers

Geller et al., 2013

• 313bp COI barcode fragment

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Twin-tagging dual indexing PCRs

Leray et al., 2016 (Chap 14) Leray et al., 2013

• Highly degenerated primers

Geller et al., 2013

• 313bp COI barcode fragment

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14

Twin-tagging dual indexing PCRs

Leray et al., 2016 (Chap 14) Leray et al., 2013

• Highly degenerated primers

Geller et al., 2013

• 313bp COI barcode fragment

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(17)

16

(18)
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18

Bio-informatic pipeline

3756 OTUs

after Arthropod 80% filtering and sample selection

Midori 4296 Vsearch 4615 Sumaclust 5065

Minimum 4 reads in each of the 3 PCR replicates

OTU 97% clustering

RDPClassifier

Taxonomic assignment Chimeras removal

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Species richness for Malaise traps

3756 OTUs found 18 Insecta orders represented

Arachnida 66 Chilopoda 2 Collembola12 Diplopoda 5 Diplura 1 Insecta Malacostraca 1 Diptera 1534 Hymenoptera 1087 Lepidoptera 512 Coleoptera 332 Hemiptera 116 Others 88

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Species richness for Malaise traps

3756 OTUs found 18 Insecta orders represented

Arachnida 66 Chilopoda 2 Collembola12 Diplopoda 5 Diplura 1 Insecta 3669 Malacostraca 1 Diptera 1534 Hymenoptera 1087 Lepidoptera 512 Coleoptera 332 Hemiptera 116 Others 88

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Trap composition

20 40 60 80 100 120 140 160

Number of bulk samples per OTU

0 50 100 150 200 250

Number of OTUs per bulk sample

Min = 18 | Max = 223 | Mean ~110

Vespula vulgaris Bombus lucorum Bombus pascuorum

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22

Trap composition

0 20 40 60 80 100 120 140 160

Number of bulk samples per OTU

0 50 100 150 200 250

Number of OTUs per bulk sample

Min = 18 | Max = 223 | Mean ~110

Vespula vulgaris Bombus lucorum Bombus pascuorum

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442 482 452 491 286 225 282 57 36 74 361 228 51 58 231

High temporal turnover from May to August

May 1519 OTUs June 1995 OTUs July 1894 OTUs August 1527 OTUs 3756 OTUs

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24

Future work

• Expand reference libraries (saproxylic beetles)

• Improve taxonomic assignment into the bioinformatic pipeline

• Reanalize with new version DAMe 2.0 incoming

• Compare with other bioinformatic methods (mBRAVE, CROP…)

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Doug Yu’s team

Paul Schmidt Susan Mbedi

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26

0.5mm

1.0mm

Hylesinus toranio Xylosandrus germanus

Detection of pests and invasive species

Vespa vetulina

1.0cm

(28)

Pre-PCR migration and rincing test

for MPG removal

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T a g 1 T a g 1 0 T a g 1 0 0 T a g 1 1 T a g 1 2 T a g 1 3 T a g 1 4 T a g 1 5 T a g 1 6 T a g 1 7 T a g 1 8 T a g 1 9 T a g 2 T a g 2 0 T a g 2 1 T a g 2 2 T a g 2 3 T a g 2 4 T a g 2 5 T a g 2 6 T a g 2 7 T a g 2 8 T a g 2 9 T a g 3 T a g 3 0 T a g 3 1 T a g 3 2 T a g 3 3 T a g 3 4 T a g 3 5 T a g 3 6 T a g 3 7 T a g 3 8 T a g 3 9 T a g 4 T a g 4 0 T a g 4 1 T a g 4 2 T a g 4 3 T a g 4 4 T a g 4 5 T a g 4 6 T a g 4 7 T a g 4 8 T a g 4 9 T a g 5 T a g 5 0 T a g 5 1 T a g 5 2 T a g 5 3 T a g 5 4 T a g 5 5 T a g 5 6 T a g 5 7 T a g 5 8 T a g 5 9 T a g 6 T a g 6 0 T a g 6 1 T a g 6 2 T a g 6 3 T a g 6 4 T a g 6 5 T a g 6 6 T a g 6 7 T a g 6 8 T a g 6 9 T a g 7 T a g 7 0 T a g 7 1 T a g 7 2 T a g 7 3 T a g 7 4 T a g 7 5 T a g 7 8 T a g 7 9 T a g 8 T a g 8 0 T a g 8 1 T a g 8 2 T a g 8 3 T a g 8 4 T a g 8 5 T a g 8 6 T a g 8 7 T a g 8 8 T a g 8 9 T a g 9 T a g 9 0 T a g 9 1 T a g 9 2 T a g 9 3 T a g 9 4 T a g 9 5 T a g 9 6 T a g 9 7 T a g 9 8 T a g 9 9 Tag99 Tag98 Tag97 Tag96 Tag95 Tag94 Tag93 Tag92 Tag91 Tag90 Tag9 Tag89 Tag88 Tag87 Tag86 Tag85 Tag84 Tag83 Tag82 Tag81 Tag80 Tag8 Tag79 Tag78 Tag77 Tag76 Tag75 Tag74 Tag73 Tag72 Tag71 Tag70 Tag7 Tag69 Tag68 Tag67 Tag66 Tag65 Tag64 Tag63 Tag62 Tag61 Tag60 Tag6 Tag59 Tag58 Tag57 Tag56 Tag55 Tag54 Tag53 Tag52 Tag51 Tag50 Tag5 Tag49 Tag48 Tag47 Tag46 Tag45 Tag44 Tag43 Tag42 Tag41 Tag40 Tag4 Tag39 Tag38 Tag37 Tag36 Tag35 Tag34 Tag33 Tag32 Tag31 Tag30 Tag3 Tag29 Tag28 Tag27 Tag26 Tag25 Tag24 Tag23 Tag22 Tag21 Tag20 Tag2 Tag19 Tag18 Tag17 Tag16 Tag15 Tag14 Tag13 Tag12 Tag11 Tag10 Tag1

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975

604

1571

vouchers

861

species

Sire et al., in prep. Rougerie et al., 2015

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