Les interactions hôte-parasite en écotoxicologie : l’exemple des

parasites de la dreissène en tant que facteur confondant

Laëtitia MINGUEZ, Laure GIAMBERINI

LIEBE – Laboratoire Interactions Ecotoxicologie Biodiversité Ecosystèmes

CNRS UMR 7146

Journées ZAM10 et 11 Mai 2010

Context of the studyContext of the study

Ecotoxicology / Aquatic Ecology Parasitology

Environmental Parasitology

Important to determine combined effects of pollution and parasitism in ecotoxicological studies

Two questions: (1) Could parasites be indicators of environmental quality? (2) Could parasites modify biological responses of their hosts and interfere with bioindication procedures?

Could parasites modify biological responses of their hosts and interfere with bioindication procedures?

Test organism: The zebra mussel (Dreissena polymorpha)

Sentinel organism – more than 30 symbionts described

3 streams in France :

3 cellular biomarkers:

- the structural changes of the digestive lysosomal system

- the accumulation of neutral lipids

- the accumulation of lipofuscin granules

Context of the study

+ Condition and Gonadal indexes

Villaine

Moselle

Meuse

THE LYSOSOMAL SYSTEM

= Group of organelles (acid hydrolases)

- Intracellular digestion, reproduction, immune response, embryogenesis, cellular turnover, …

- Sequestration / accumulation of xenobiotics

Unspecific response to pollution

Context of the study

UNSATURATED NEUTRAL LIPIDS

Energetic metabolism

- long term alimentary use

- reproduction

- thermic isolation

= mostly triglycerides

Context of the study

Context of the study

LIPOFUSCIN GRANULES

= Intracellular granulous pigments(in secondary lysosomes)

Fast accumulation : pathogenic

Oxydative catabolism of : - lipids- proteins- ascorbic acid- polyenic compounds

MethodsMethods

Remaining tissuesDigestive gland excision

Cryosections -27°C (8µm)

Histochemical staining

Image analysis (5 fields / ind.)

Stereological parameters

Lysosomal β-glucuronidase (Cajaraville et al., 1991)

Unsaturated Neutral Lipids (Moore, 1988)

Lipofuscin granules (Moore, 1988)

Lysosomal: VvL, SvL, NvL, S/VL

Neutral lipids: SvNL

Lipofuscines: SvLF

Biomarker revelation

sections (5µm)

Hematoxylin/Eosin staining

Inventory of parasites + Gonadal index

Prevalence, Mean Intensity

Observation (30-40 sections / ind.)

Results and DiscussionResults and Discussion

Focus on bacteria and ciliates:

Rickettsiale-like organisms (RLO)

Ophryoglena spp.

-Intracellular bacteria-Digestive gland

-Ciliates-Digestive gland

Could it be sources of distortion in ecotoxicological studies ?

Previous study : Meuse vs Moselle (Sierck-les-Bains) (Minguez et al., 2009)

Study on the Moselle River: up- and downstream WSTP of Metz

Results - Discussion

Infection status = infected or not

Only station discrimination

Parasite species (Downstream)

Lysosomal system

Experimental groups: at least 5 organisms, gender

Ratio BM response infected / non-infected

-Differences between ♂ and ♀

-Coinfection: synergism between parasite species

♂ : synergism –

♀ : synergism +

The primary parasite in the study site turns biological responses (i.e. RLOs)

* : significant

RLOs and Ophryoglena

-80%

-60%

-40%

-20%

0%

20%

40%

60%

80%

oph M oph F rlo M rlo F oph-rlo M oph-rlo F

Lyso

som

al v

olum

e de

nsity

/con

trol

* *

*

*CoinfectionBacteriaCiliates

Phyllodistomum folium

Focus on two pathogenic trematodes:

Could it be sources of distortion in ecotoxicological studies ?

Bucephalus polymorphus

Molloy et al., 19975 mm

Results - Discussion

- Digenea

- ZM = the only intermediate host

- Gills Deformation

- Digenea

- ZM = 1st intermediate host

- Gonade Castration

Sporocyst

Vv L

(10

-4 µ

m3/µ

m3)

2

3

4

5

6

7

8

9

NI M NI F P. folium M P. folium F

a a

b

ab

Phyllodistomum folium

LYSOSOMAL SYSTEM and NEUTRAL LIPIDS

-♂ : no significant differences (trends)

-♀ : infection more developped lysosomal system and more lipid reserves

LIPOFUSCIN GRANULES

- Differences between ♂ and ♀

Results - Discussion

Sampling : Meuse river Prevalence rate = 4.8% (564 dissected mussels)-Males = 2 times more infected

1.5

2.5

3.5

4.5

5.5

6.5

7.5

8.5

NI M NI F P. folium M P. folium F

Sv L

F (

10-4 µ

m2 /

µm

3 )

a a

bb

♂ : oxidative stress

Vv L

(10

-4 µ

m3/µ

m3)

0

1

2

3

4

5

6

7

8

9

0 + ++ +++

Results - Discussion

Infection:

- Delay in gonad development (gonadal index: NI > I)

- Condition index: NI > I

and the infection intensity ??

+ : ≤ 5 sporocysts ++ : 5 < sporocysts ≤ 10 +++ : gills covered by sporocysts

Only for males

LYSOSOMAL SYSTEM

Tendency : Negative correlation between infection intensity and lysosomal system responses (lysosomes more and more numerous and smaller)

5 mm

Confounding factor in ecotoxicological monitoring

sensibility to environmental

conditions

Results – Discussion

Males:Females:

lysosome system,

lipidic reserves,

lipofuscin granules

lysosome system lipidic reserves lipofuscin granules

Better defenses More sensitive

( infection prevalence)

Parasite development or

Host’s eating behaviour modification( food intake)

Oxidative stress:Inflammatory processes

and/or antioxidant enzyme activities

vs

Bucephalus polymorphus

Sampling : Villaine river Prevalence rate = 1.9% (905 dissected mussels)

Studied Compartments : Lysosomes, Lipids, Lipofuscins

No significant differences between infected and non-infected organisms

Condition index: FW/(L*H*W)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

NI B. polymorphus

Con

ditio

n in

dex

a

b

CI: Non-infected < Infected

Infection :

speed weight intake

Parasite = beneficial ??

Results – Discussion

FW: Fresh Weight L: shell Length

H: shell Height W: shell Width

Reproduction Reproduction

P. FoliumIn males

B. polymorphus

Results - Discussion

ReproductionParasite

DevelopmentGrowth MaintenanceGrowth MaintenanceGrowth Maintenance

Modification of the host’s energetic metabolism

Energy for reproduction used for parasite development

STRESS NO STRESS

Confounding factor Confounding factor

ConclusionConclusion

Parasitism = confounding factor ? Yes

But it depends of :

- parasite species

- infection intensity

- host gender

AcknowledgementsAcknowledgements

Studies supported by an EC2CO program and the CPER Lorraine- ZAM

ReferencesReferencesCajaraville, M.P., Marigómez, J.A., Angulo, E., 1991. Arch. Environ. Contam. Toxicol. 21, 395-400.Minguez, L., Meyer, A., Molloy, D.P., Giambérini, L., 2009. Environ. Res. 109, 843-850.Moore, M.N., 1988. Mar. Ecol. – Prog. Ser. 46, 81-89.