Development of q-PCR approaches to assess water quality: Effects of cadmium and pesticides on gene expression of diatoms

(1)

HAL Id: hal-02599228

https://hal.inrae.fr/hal-02599228

Submitted on 16 May 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Development of q-PCR approaches to assess water quality: Effects of cadmium and pesticides on gene

expression of diatoms

S. Kim Tiam, S. Moisset, A. Feurtet Mazel, Nicolas Mazzella, A. Arini, François Delmas, Soizic Morin, G. Daffe, P. Gonzalez

To cite this version:

S. Kim Tiam, S. Moisset, A. Feurtet Mazel, Nicolas Mazzella, A. Arini, et al.. Development of q-PCR approaches to assess water quality: Effects of cadmium and pesticides on gene expression of diatoms. SETAC North America, Nov 2013, Nashville, United States. pp.1, 2013. �hal-02599228�

(2)

Development of q-PCR approaches to assess water quality: Effects of cadmium and pesticides on gene

expression of diatoms

.

Kim Tiam S.

1,2

, Moisset S.

1

, Feurtet-Mazel A.

1

, Mazzella N.

2

, Arini A

1

., Delmas F.

2

, Morin S.

2

, Daffe G.

1

and Gonzalez P.

1

_{University of Bordeaux, EPOC, CNRS UMR 5805, Aquatic Ecotoxicology Team, Place du Dr Peyneau, 33120 Arcachon, France}

2

_{IRSTEA, UR REBX, Anthropic Contaminants and Responses of Aquatic Systems Team, 50 avenue de Verdun, F-33612 Cestas cedex, France}

SETAC North America34

TH

Annual Meeting – Nashville, TN, USA, 17-21 November 2013

e-mail: p.gonzalez@epoc.u-bordeaux1.fr

Figure 1. Experimental units (I) and species studied (II), A : Eolimna minima ;

B : Gomphonema parvulum ; C : Nitzschia palea ; D : Planothidium lanceolatum).

Objective of the study

Methodology

Results

Conclusion

Periphytic diatoms are valuable tools to determine freshwater rivers quality

based on diatoms identifications and distribution and constitute key indices in international standards. Nevertheless, indices currently used for water quality assessment do not take in consideration the diverse nature of contaminants, are time consuming and require important taxonomic knowledge. In this context development of q-PCR approaches have been developed to enhance a better reactivity in the diagnosis of modifications due to pollution conditions and to understand the genetic impact in relation with diatoms species. Through different laboratory experiments (14 days exposure) concerning various contamination (metallic with Cd: 10 and 100 µg/L) or pesticides with diuron: 1 and 10 µg/L), several target genes involved in mitochondrial metabolism (coxI, nad4, 12S), oxidative stress response (sod Mn), detoxification (cyp1A1) and photosynthesis (psaA, d1) have been characterized and their expression levels have been determined.

[KAuCl

₄

]=

50µg/mL à 7

jours

PsaA D1 CoxI Nad5 12S Sod Mn  Actine Rpl7 A B D C

E. minima was exposed to Cd (10 and 100 µg/L) and E. minima, G. parvulum, N. palea and P. lanceolatum were exposed to diuron (1 and 10 µg/L)

Parameters:

- Growth (cellular enumerations)

- Photosynthetic efficiency (fluorimeter Phyto-PAM)

- Gene expression by real time qPCR

Figure 2. Target genes

I

II

Figure 3. E. minima growth during Cd

exposure

Cd exposure

a a a a b _b 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 12 14 D e n s ity (1 0 ^3 C e ll s /mL )

Exposure time (days)

0 μg Cd/L 10 μg Cd/L 100 μg Cd/L

Results are expressed as induction factors (>1) or repression factors (<1) compared to control E.minima. / : identical to control expression level.

Tableau 1. Differential expression observed after exposure of E. minima to Cd after 1, 2, 7

and 14 days.

Diuron exposure

Functions Genes EOMI

C1 (1µg/L) C2 (10 µg/L) 6h D2 D7 D14 6h D2 D7 D14 Photosynthesis psaA 0.4 / / 13.1 0.3 4.4 / 4.2 d1 0.1 / 0.3 16 / 3.8 / 8.1 Mitochondrial metabolism cox1 0.3 / / 5.3 0.2 3.4 / 2.3 nad5 0.1 / / / / 4.6 / / 12s 0.5 / 0.4 2.3 0.4 4.6 / / 0,0 20,0 40,0 60,0 80,0 100,0 120,0 C0 C1 C2 C0 C1 C2 C0 C1 C2 C0 C1 C2 P h o to sy n th eti c ef fi ci en cy (% c o m p ar ed to c o n tr o l c o n d iti o n C 0) Time a a c a b c a b d a a d 0 20 40 60 80 100 120 C0 C1 C2 C0 C1 C2 C0 C1 C2 C0 C1 C2 P h o to sy n th et ic ef fi ci en cy ( % c o m p a re d to c o n tr o l c o n d it io n C 0 ) Time 6h D2 D7 D14 a a b a _a b a _a b a a b

Tableau 2. Differential expression observed after exposure of E. minima and P. lanceolatum

to diuron after 6h, 2, 7 and 14 days.

Figure 4. Optimal photosynthetic quantum yield

E. minima

(similar behavior for P. lanceolatum)

P. Lanceolatum

(similar behavior for G. parvulum)

Results showed that both compounds impacted the mitochondrial and the photosynthetic metabolisms. The observed effects are closely related to the growth rate efficiency of the cultures used and the photosynthetic quantum yield but appear earlier. Thus, molecular biomarkers could be used to evidence early response and sensitiveness of diatoms during environmental pollutions (or xenobiotic exposures).These first innovative results constitute a first step towards the study of periphytic diatoms through molecular biology.

Results are expressed as induction factors (>1) or repression factors (<1) compared to control. / : identical to control expression level.

Functions Genes PTLA

C1 (1µg/L) C2 (10 µg/L) 6h D2 D7 D14 6h D2 D7 D14 Photosynthesis psaA 0.3 39.3 / / 15.8 22.4 / 0.1 d1 / 51.5 / / 6.8 42 / 0.1 Mitochondrial metabolism cox1 / 8.3 / / 109.3 5.1 / / nad5 / / / / 3.7 / / / 12s 0.3 54.8 / / 22.4 21.8 / 0.08

-Slight significant difference between growth of C₀ and 10 µg/L

-Great significant difference between growth of C₀ and 100µg/L from day 7

-No population growth along the 14 days exposure

Tolerance of E. minima towards Cd - Fv/Fm at C1 (1 µg/L) ≈ Fv/Fm for C₀(Controls ) - Fv/Fm at C2 (10 µg/L) > > Fv/Fm for C₀and C1

-strong impacts on P. Lanceolatum -Early response of E. minima

E. minima more resistant to diuron

Functions Genes Exposure conditions

C1 (10 µg/l) C2 (100 μg/l) D1 D2 D7 D14 D1 D2 D7 D14 Mitochondrial metabolism cox1 / / / / / / 9.5 / nad5 / / / 2.5 / / / 9.5 12s / / / / / / 15 /

Oxidative stress sodMn / / / / / / / /

Photosynthesis d1 / / / 2 / / 5.5 24

psaA / / / 2.5 / / 7.5 48