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Doctoral Thesis

Dirty water and diseasepesticide mediated interactions between Daphnia and theirparasites

Author(s): Buser, Claudia Carolina

Publication Date: 2011

Permanent Link: https://doi.org/10.3929/ethz-a-006713645

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DISS. ETH NO. 19964

Dirty water and disease: Pesticide mediated interactions between Daphnia

and their parasites

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by

CLAUDIA CAROLINA BUSER Master of Science in Biology, University of Zürich

born 05.05.1982

citizen of Basel (BS)

accepted on the recommendation of

PD Dr. Piet Spaak

Prof. Dr. Paul Schmid-Hempel

Dr. Christoph Haag

2011

Contents

Summary ..................................................................................................................................... 1

Zusammenfassung ...................................................................................................................... 3

Chapter 1 .................................................................................................................................... 5

Introduction and outline of the thesis ..................................................................................... 5

Chapter 2 .................................................................................................................................. 19

Variation within and between species cannot be ignored in toxicity tests ........................... 19

Chapter 3 .................................................................................................................................. 31

Combined exposure to parasites and pesticides causes increased mortality in the water

flea Daphnia ......................................................................................................................... 31

Chapter 4 .................................................................................................................................. 43

Disease and pollution alter Daphnia community structure .................................................. 43

Chapter 5 .................................................................................................................................. 63

Evolutionary and environmental effects of pesticide exposure on parasite fitness and

virulence ................................................................................................................................ 63

Chapter 6 .................................................................................................................................. 77

Environment-dependent virulence affects disease spread in host populations ..................... 77

Chapter 7 .................................................................................................................................. 87

Concluding remarks and recommendations for future research ........................................... 87

Curriculum vitae ....................................................................................................................... 97

Acknowledgments .................................................................................................................. 103

1

Summary

Communities in the wild are subject to a multitude of abiotic and biotic stressors. In turn,

environmental stressors acting on the individual level can further influence population and

community structure. In my doctoral research, I studied the effect of parasites and pesticides

on host individuals as well as on hosts in experimental communities. I used several Daphnia

(water flea) taxa and their endoparasites, as well as the pesticide diazinon. Chapter 1

introduces these animals and the pesticide, and additionally includes a short review about

host-parasite-environment interactions.

Pesticides are one of the most ubiquitous pollutants in water bodies. Although the effects of

chemical contamination can permeate whole ecosystems, many toxicological studies are

conducted using a single clone of one species. Chapter 2 shows variation in mortality rates

towards the exposure of the common pesticide diazinon within and between three different

Daphnia species (D. magna, D. pulicaria and D. galeata). Not taking that variation into

account by using only one clone for assessing the hazardous potential of a chemical might

lead to an underestimation of risk towards other, more sensitive clones in the community.

In Chapter 3, I investigated the combined influence of disease and chemical pollution on host

fitness. I exposed D. magna clones to the parasite Pasteuria ramosa and the pesticide

diazinon. The clones were hatched from different sediment layers from a pond in Belgium

where they co-occur with different densities of this parasite. Although I did not observe

visible signs of infection, D. magna clones had reduced survival when simultaneously

exposed to both parasites and chemical pollution. I did not find that host clones were adapted

to pesticides or parasites over time.

Effects of parasites and/or pesticides on single individuals may shape the structure and

dynamic of the whole community. However, extrapolation of experimental results from tests

on single individuals to a community level may not always be accurate given that, for

example, competition between individuals is not considered. In Chapter 4, I followed

changes in experimental Daphnia communities after exposure to the fungal parasite

Metschnikowia sp. and/or the pesticide diazinon. On a community level, including

competition within and among taxa, dynamics were altered by parasite and pesticide

exposure. One taxon went extinct under parasite and combined stress treatments, and I also

show clonal/taxon sensitivity towards the pesticide. Furthermore, the density of all adult

Summary

2

females was significantly reduced in the parasite treatment, but not in the pesticide treatment.

In general, the parasite exposure inserted stronger selection pressure than the pesticides.

Not only hosts are affected by pollutants. Parasites can be affected as well. Parasite virulence

can be strongly dependent on host condition and can be mediated by environmental variation.

In Chapter 5 the environmental and evolutionary effects of diazinon on parasite fitness and

virulence were tested. Parasites exposed for several generations to the pesticide evolved

towards higher fitness and lower virulence, a finding that could have a significant impact on

disease dynamics.

Using epidemiological models, I examined the impact of environment-dependent virulence on

parasite virulence and disease spread in Chapter 6. The results suggest that the underlying

relationship between virulence and host environment can drastically affect whether disease

spreads as well as the intensity of the epidemic.

In summary, the studies I conducted for my doctoral thesis show that two forms of

environmental stress, parasites and pesticides, can mediate the fitness of single individuals,

affect the structure and evolution of communities, and determine whether disease can spread.

Parasites act together with ecologically-relevant levels of pesticide pollution to impact host

individuals and communities, but the strength of the combine effects highly depends on host

genotype and the organismal level (i.e., individual, community) considered. Moreover,

pesticides not only reduce host fitness and alter community structure, but also shape the

evolution of parasites towards higher fitness and lower virulence. These outcomes may affect

the theoretical spread of disease in environments of varying quality. I discuss the implications

of these findings, as well as future research directions in Chapter 7.

3

Zusammenfassung

Populationen in natürlichen Habitaten sind häufig einer Vielzahl von abiotischen und

biotischen Stressoren ausgesetzt. Diese Umweltstressoren, agierend auf einzelne Individuen,

können Auswirkungen auf die Struktur und Dynamik von Populationen und Gemeinschaften

haben. Während meinem Doktorat habe ich den Effekt solcher Umweltstressoren, in Form

von Parasiten und Pestiziden, auf einzelne Wirte als auch auf experimentelle

Wirtsgemeinschaften untersucht. Als Modelsystem habe ich verschiedene Daphnien Arten

und ihren Endoparasiten sowie das Pestizid Diazinon benutzt. Dieses Modelsystem wird

zusammen mit Grundlagentheorie zum Thema Wirt-Parasiten Interaktion und deren Effekt

auf die Umwelt in Kapitel 1 beleuchtet.

Pestizide sind häufig vorkommende Chemikalien, welche unsere Gewässer verschmutzen.

Auch wenn Giftstoffe ganze Ökosysteme verändern können, werden toxikologische Studien

häufig nur mit einem Klon einer Art durchgeführt. Kapitel 2 zeigt, dass Variation in der

durch das Pestizid Diazinon verursachten Mortalität zwischen und innerhalb verschiedener

Daphnien Arten vorhanden ist. Wird diese Variation nicht berücksichtigt, kann dies zu einer

Fehleinschätzung der Gefährlichkeit des Schadstoffes für natürliche Gemeinschaften führen.

Kapitel 3 beschreibt meine Studie über den kombinierten Effekt von Parasiten und Pestizid

auf die Fitness des Wirtes. Dafür habe ich D. magna Klone dem Parasit Pasteuria ramosa und

dem Pestizid Diazinon ausgesetzt. Die D. magna Klone stammen aus verschiedenen

Sedimentschichten eines Weihers in Belgien. Diese Schichten weisen eine unterschiedliche

Dichte der Parasitensporen auf. Eine visuelle Infektion konnte anhand des Experimentes nicht

festgestellt werden. D. magna Klone zeigten aber bei simultaner Exponiertheit von Krankheit

und Umweltstressor eine Reduktion der Überlebensrate. Keine Adaption, weder an den

Parasit noch an das Pestizid, konnte über die Zeit festgestellt werden.

Parasitäre Effekte und/oder solche von Pestiziden auf einzelne Individuen können die Struktur

und Dynamik einer ganzen Gemeinschaft formen. Eine Hochrechnung von Resultaten, welche

auf Tests mit einzelnen Individuen basieren, auf Populationsebene reflektiert die reale

Situation in einer Gemeinschaft nicht ganzheitlich. Unter anderem wird zum Beispiel die

Konkurrenz innerhalb und zwischen den Individuen nicht in Betracht gezogen. In dem im

Kapitel 4 beschriebenen Experiment habe ich die Änderung von experimentellen Daphnien-

Gemeinschaften verfolgt, nachdem sie dem Pilzparasit Metschnikowia sp. und/oder dem

Zusammenfassung

4

Pestizid Diazinon exponiert worden waren. In einer Gemeinschaft und somit unter Einbezug

von inner- und zwischenartlicher Konkurrenz wurde die Dynamik durch den Parasit sowie

durch das Pestizid geändert. Dabei verschwand eine Art bei Behandlung mit Parasiten sowie

auch mit Parasiten/Pestizid komplett. Ebenfalls konnte ich bei einer Art eine erhöhte

Sensitivität gegenüber dem Pestizid feststellen. Ausserdem reduzierte der Parasit, jedoch nicht

das Pestizid, die Dichte von allen erwachsenen Weibchen signifikant. Unter diesen

experimentellen Bedingungen hatte der Parasit einen stärkeren Selektionsdruck auf die

Daphnien-Gemeinschaft ausgeübt als das Pestizid.

Nicht nur die Wirte, auch deren Parasiten können von chemischen Verschmutzungen

beeinträchtigt sein. Die Virulenz des Parasiten ist von der Wirtkondition abhängig, kann aber

auch von Umweltveränderungen beeinflusst werden. In Kapitel 5 wurden die evolutionären

und ökologischen Effekte von Diazinon auf die Fitness und Virulenz des Parasiten getestet.

Parasiten, welche für einige Generationen dem Pestizid ausgesetzt waren, entwickelten eine

höhere Fitness und eine niedrigere Virulenz. Eine Entdeckung, welche eine signifikante

Auswirkung auf die Krankheitsdynamik haben kann.

Mit Hilfe eines epidemiologischen Modells habe ich in Kapitel 6 die Auswirkung von

verschiedenen Umwelt-Virulenz Beziehungen auf die Krankheitsverbreitung untersucht. Die

Resultate deuten darauf hin, dass die Verbreitung der Krankheit in unterschiedlichen

Umweltqualitäten drastisch von der zugrunde liegenden Umwelt-Virulenz-Beziehung

beeinflusst wird.

Zusammenfassend zeigen die Studien in meiner Dissertation, dass zwei Formen von

Umweltstressoren - Parasiten und Pestizid - die Fitness von einzelnen Individuen sowie die

Struktur und Evolution von Gemeinschaften beeinflussen und auch bestimmen, ob sich die

Krankheit verbreiten kann. Die Kombination von Parasiten und ökologisch-relevanten

Pestizidkonzentrationen haben einen Einfluss auf Wirt-Individuen und -Gemeinschaften. Die

Stärke des kombinierten Effektes hängt stark vom Wirtsgenotyp und dem Organismuslevel

(einzelne Individuen oder Gemeinschaften) ab. Desweiteres hat das Pestizid nicht nur die

Wirtfitness und die Gemeinschaftsstrukturen verändert, sondern formt auch die Evolution von

Parasiten in Richtung höhere Fitness und schwächere Virulenz. Diese Ergebnisse können die

potentielle Verbreitung von Krankheiten in verschiedenen Umweltqualitäten beeinflussen. Ich

diskutiere die Auswirkungen dieser Resultate sowie auch mögliche zukünftige

Forschungsprojekte in Kapitel 7.

5

Chapter 1

Introduction and outline of the thesis

Claudia C. Buser

Introduction Chapter 1

6

Parasites in ecosystems

Parasites are ubiquitous and play an important role in host ecology and evolution. For

example, parasites are a driving factor for the maintenance of sexual reproduction (Jaenike

1978; Hamilton 1980; Bell 1982; Hamilton et al. 1990), and they can alter the genetic

structure of their host populations (Sasaki 2000) and structure of host communities (Anderson

& May 1979, 1982; Hudson & Greenman 1998; Boots & Sasaki 2003). Host-parasite

interactions may result in continuous coevolutionary changes (Hamilton 1980), in which

parasites evolve new adaptations to infect and hosts evolve new adaptations to resist parasites,

also known as the Red Queen hypothesis (van Valen 1977; Jaenike 1978; Bell 1982). This

type of coevolution can result in host and parasite genotypes oscillating in frequency over

time (Jaenike 1978; Hamilton 1980). Such oscillatory dynamics were observed in for example

snails or Daphnia (Dybdahl & Lively 1998; Decaestecker et al. 2007; Jokela et al. 2009;

Koskella & Lively 2009). Although host and parasites genotypes are major determinants of

infection success (Lambrechts et al. 2006), these host-parasite interactions can be influenced

by environmental conditions (Lazzaro & Little 2009; Wolinska & King 2009), having impact

on populations and even whole ecosystems (Fig. 1).

Environment and host-parasite interactions

As most parasites cannot live outside their host for extended periods, parasite success is

strongly linked with host ecology and evolution. Consequently, if environmental factors affect

host fitness, they likely also highly impact on parasite fitness. Host × parasite × environment

interactions can mediate parasite prevalence, disease dynamics and, ultimately affect

populations and whole ecosystems (Fig. 1).

Environmental stress can make hosts more susceptible to parasites. Abiotic and biotic factors

can affect immune defence. In general, the immune system is energetically costly to maintain

and therefore, environmental stress, such as poor feeding conditions (Schmid-Hempel 2005)

or pollution (McDowell et al. 1999), can lead to a weaker immune system. This enables

parasites to infect more easily. Increased infectivity when hosts experience environmental

stress has been observed in several host-parasite systems: pesticide exposures increases

infectivity in oysters (Chu & Hale 1994) and amphibians (Rohr et al. 2008) predation

increases infectivity in crustaceans (Yin et al. 2011).

Chapter 1 Introduction

7

Fig. 1: Schematic representation of host-parasite interactions (black arrows), its influence (thinner black arrows)

on disease dynamic and consequently the effects on populations and whole ecosystems (wide grey arrows). The

black dashed arrows denote the feedback of the different levels on host and/or parasite fitness. The environment

can have an influence on everything.

Thus, parasite fitness can increase in a poor host environment due to reduced host immune

function (Sheldon & Verhulst 1996; Moret & Schmid-Hempel 2000). Conversely, not only

hosts, but also parasites can be negatively affected when the host is exposed to environmental

stress, for example, due to reduced resources available for the parasite growth (Pulkkinen &

Ebert 2004; Tschirren et al. 2007; Seppälä et al. 2008; Hall et al. 2009b). Furthermore,

parasites themselves can be killed by the environmental stressor itself due to higher sensitivity

to the stressor (Pietrock & Marcogliese 2003). Also, parasite virulence has been repeatedly

shown to be influenced by environmental variation in a variety of animal-parasite systems

(empirical examples in Lazzaro & Little 2009; Wolinska & King 2009). The relationship

between virulence and environmental quality can take many forms (see examples in Thomas

& Blanford 2003). Often, mortality of infected hosts is increased in bad environments, for

example by starvation (Jokela et al. 1999; Ferguson & Read 2002; Brown et al. 2003; Tseng

2004; Jokela et al. 2005) or exposure to pesticides (Coors et al. 2008; Coors & De Meester

2011), microorganism-enriched water (Cornet & Sorci 2010) and high temperatures (Mitchell


8

& Read 2005). Less frequently reported, parasites can induce higher host mortality when

environmental conditions are good (e.g., Vale et al. 2011).

Parasite transmission can be affected by environmental stressors too. Environmental stress

can increase (e.g., Murray et al. 1998) or reduce (e.g., Coors & De Meester 2011) the

production of parasite transmission stages, or transmission stages could be sensitive towards

direct exposure (Pietrock & Marcogliese 2003). Furthermore, transmission rate is linked with

host density (e.g., Ebert 1995; Bittner et al. 2002), and therefore environmental stressors

reducing host density could affect parasite transmission.

Transmission rate, host and parasite fitness, host immunocompetence and parasite virulence

all can influence disease occurrence, the timing of epidemics and epidemic intensity (Fig. 1).

Environmental parameters can also spark epidemics (Johnson et al. 2006; Johnson et al.

2009). Temperature and predators, for example, can jointly drive the timing of epidemics

(Hall et al. 2006).

The emergence of disease in fresh water can be imputed to anthropogenic changes (Daszak et

al. 2001). Epidemiological models are increasingly integrating the environment to determine

how local conditions affect spread of diseases (Lafferty & Holt 2003; Hall et al. 2007; Hall et

al. 2009a). As infection disease models suggest that the likelihood and impact of an epidemic

increases with host density (Anderson & May 1978), stressors depressing population density

should reduce the chance of an epidemic. Lafferty and Holt (2003) suggest that stress reduces

density and/or host quality, and therefore transmission rate is lowered. On the other hand, the

spread of disease can be enhanced by improved environmental conditions (Hall et al. 2007).

Over the last few years, studies including the effect of environmental factor on host × parasite

interactions have become more frequent (e.g., Wolinska & King 2009). Nevertheless, there is

still a general need for more empirical studies testing predictions of how environmental stress

influence the different steps in Fig. 1. The study system (host-parasite-environmental stress)

used to answer the aims (see below) of this thesis is presented below.

Host-parasite system and pesticides as environmental stressors

The host Daphnia

The genus Daphnia comprises planktonic crustaceans belonging to the Cladocera (crustacean)

which inhabit freshwater habitats (water bodies from small temporary pools to huge lakes)

throughout the world. Over 100 species are included in the genus. Some species can form

hybrid complexes which co-exist with the parental species in Daphnia communities (e.g.,

Schwenk & Spaak 1995; Keller et al. 2008; Petrusek et al. 2008; Yin et al. 2010).


9

Many Daphnia species have a cyclic parthenogenetic reproduction mode (Fig. 2). Under good

conditions, Daphnia produce clones inside their brood pouch which are released as fully-

developed juveniles. When conditions are deteriorating, most Daphnia clones are able to

switch to sexual reproduction. The sexual eggs must be fertilized by asexually-produced

males. These sexual eggs are encapsulated and protected by an ephippium. They enter into

diapause until environmental clues (e.g., light, warmer temperature) induce hatching.

Otherwise, dormant eggs can accumulate in the sediment. A new field of resurrection ecology

is using dormant eggs as a tool to address ecological, evolutionary and toxicological based

questions.

Fig. 2: Life cycle of a cyclic parthenogenic Daphnia. Drawing by Dita B. Vizoso, Fribourg University (Ebert

2005)

Daphnia is a model organism in ecology, evolutionary biology, and toxicological and

biomedical research. The animal has a cyclic parthenogenic reproduction mode, short

generation time, central role in ecosystems, worldwide distribution, small size, and quickly

responds to environmental changes.


10

Daphnia and their parasites used in this thesis

Daphnia species are host of different parasites, carrying a large number of epi- and

endobionts (Green 1974). Most parasites of Daphnia are microparasites, belonging to

bacteria, fungi and microsporidians (Ebert 2005). In my thesis, I worked with two different

Daphnia taxa-parasite systems: a) Daphnia magna and Pasteuria ramosa, b) Daphnia of the

D. longispina hybrid complex (specifically, D. galeata and its hybids

D. galeata × longispina) and Metschnikowia sp..

a) D. magna and P. ramosa

D. magna is found in temporary and permanent ponds. P. ramosa is a bacteria parasitizing

Daphnia (Ebert 2005) and co-occurring with D. magna in the field (e.g., Little & Ebert 2000;

Jansen et al. 2010). This Gram-positive bacterium, belonging to the family of the

Alicyclobacillaceae (e.g., Ebert et al. 1996), is an extracellular parasite infecting the

hemolymph (Metchnikoff 1888). Infected hosts grow large, the body becomes darkish and

non-transparent (Fig. 3), and infected hosts become infertile. P. ramosa is transmitted

horizontally through spores released from dead infected hosts. P. ramosa spores are found in

pond sediments and remain infective for decades (Decaestecker et al. 2004).

Fig. 3: D. magna uninfected (left) and infected (right) with P. ramosa (photo and copyright by Ebert (2005)).

b) D. galeata, the hybrid D. galeata × longispina and Metschnikowia sp.

D. galeata is one of the three parental species (D. cucullata G. O. Sars, 1862, D. galeata

G. O. Sars, 1863 and D. longispina O. F. Müller, 1776) belonging to the D. longispina hybrid

complex (Petrusek et al. 2008). D. galeata × longispina hybrids can co-occur with parental


11

species and dominate Daphnia communities of European lakes (e.g., Keller et al. 2008;

Petrusek et al. 2008; Yin et al. 2010). The yeast parasite Metchnikowia sp. (family

Hemiascomycetes, Wolinska et al. 2009) commonly infects Daphnia populations in Europe

and the United States (Hall 2005; Caceres et al. 2006; Yin et al. 2010; Wolinska et al. 2011).

Needle-like ascospores accumulate in the body cavity and are released and horizontally

transmitted after host death (Green 1974)(Fig. 4). Infected Daphnia have fewer offspring and

a reduced lifespan (Duffy & Hall 2008; Lohr et al. 2010).

Fig. 4: D. galeata uninfected (left) and infected (right) with Metschnikowia sp. (photo and copyright by Yin et

al. (2011)).

The pesticide diazinon and the interaction with Daphnia

Many chemicals find their way into the water (e.g., through surface run-offs from agricultural

fields and urban areas after rain events). Among anthropogenic chemicals, pesticides are

harmful because they were originally designed to kill a variety of target species. Pesticides are

known to affect fitness of individual organisms, as well as to change community and

ecosystem structure from non-target individuals (reviewed in Hanazato 2001; Rohr &

Crumrine 2005). Organophosphosphate pesticides can be regularly detected in the aquatic

environment (Giddings et al. 2000; Strom et al. 2003; Pedersen et al. 2006; Gaworecki et al.

2009; Wittmer et al. 2010). They were introduced as replacements for the persistent

organochlorine pesticides (e.g., DDT), which caused adverse health effects in non-target

species (Woodwell et al. 1967). Organophosphosphate pesticides can have toxic effects on the

immune systems and immune functions of invertebrates, fish and higher vertebrate wildlife

(reviewed in Galloway & Handy 2003). Because of its wide use in agriculture and


12

households, the organophosphate diazinon (O,O-diethyl O-[2-isopropyl-6-methyl-4-

pyrimidinyl] phosphothionate; Fig. 5; initially produced in 1952 by Ciba-Geigy, later

Novarits and then Syngenta) is among the most used pesticides and is still frequently

measured in aquatic environments. For example, diazinon is found in Switzerland around

Lake Greifensee (e.g., Singer et al. 2010; http://www.umweltschutz.zh.ch; Wittmer et al.

2010) or Greece (Konstantinou et al. 2006). It can be detected in surface waters mainly in the

ng/L range, but also acute concentrations (μg/L range) in short pulses have been recorded

(Wittmer et al. 2010). Exposure to a sub-lethal diazinon concentration causes a delay in

reproduction, a decrease in fecundity and offspring size and higher adult mortality in

Daphnia magna (Sanchez et al. 1998).

Fig. 5: Chemical structure of Diazinon (Structure retrieved from toxipedia.org/display/ toxipedia/Diazinon)

Daphnia, their parasites and pesticide interactions

Pesticides and biotic stressors, like parasites, can interact in the wild. Coors found increased

parasite virulence and/or increased host susceptibility to infections, when exposing D. magna

to the pesticide carbaryl (Coors & De Meester 2008; Coors et al. 2008; Coors et al. 2009;

Coors & De Meester 2011). Moreover, Jansen found that natural Daphnia populations are

able to rapidly evolve resistance towards the pesticide carbaryl, but at the cost of a greater

susceptibility towards the parasite (Jansen et al. 2011a; Jansen et al. 2011b).

Aim of the thesis

This thesis aimed to examine the influence of pesticide pollution on host and parasite fitness,

as well as parasite virulence. It additionally focuses on the effect of multiple stressors on host-

parasite interactions and the consequences on population dynamics and disease spread.


13

Specifically, aims of the chapters are:

Chapter 2: to highlight the importance of testing a wide variety of genotypes in

toxicity tests, and to show that there is Daphnia genetic and species variation in

sensitivity to the pesticide diazinon.

Chapter 3: to test whether the combined exposure to parasites and pesticides affect

the fitness of Daphnia magna and whether these fitness consequences change through

time as a population evolves.

Chapter 4: to follow the taxa and clonal dynamics in experimental Daphnia

communities after parasite and pesticide exposure.

Chapter 5: to investigate the environmental and evolutionary effects of pesticide

exposure on parasite fitness and virulence.

Chapter 6: to explore how eight relationships between parasite virulence and

environmental quality alter the ability of parasites to spread across an environmental

gradient using epidemiological models.

Chapter 7: to summarize the above findings and discuss the importance of multiple

stressors, as well as G×E×E interactions, in an ecological and evolutionary context,

and to highlight future research questions.

Outline of the thesis

There are many studies showing the harmful effects of the pesticide diazinon on Daphnia.

Although it is common to conduct toxicology studies experiments on single clones,

Chapter 2 highlights the importance of considering the genetic diversity of test organisms. I

further show that acute toxicity of diazinon affects Daphnia species and conspecific clones

differently. In ecosystems, organisms are exposed to natural stressors, in addition to

anthropogenic inputs. Therefore, in Chapter 3 I tested the combined influence of disease, a

natural stressor, and chemical pollution on host fitness by exposing Daphnia magna clones

hatched from different sediment layers to the parasite Pasteuria ramosa and the pesticide

diazinon. Effects observed in life-history experiments on single individuals are useful to test

the effect on changes in susceptibility and life history traits for the specific combination of

abiotic and biotic stressors. However, extrapolating these results to predict the effect on

communities is difficult given that interactions between and within taxon are not taken into

account. In Chapter 4 I followed changes in experimental Daphnia communities (consisting

of two taxa and three clones per taxon), after exposure to the fungal parasite

Metschnikowia sp. and/or the pesticide diazinon. I tracked changes in density, prevalence,

genetic composition, fecundity and size of Daphnia over more than a 3 month period. In

addition to the hosts, parasites exposed to pesticide could also be affected, yet the

evolutionary consequences of long-lasting exposure to pesticides on parasites have not been

studied in great detail. In Chapter 5, I tested whether there were environmental and


14

evolutionary effects of diazinon on parasite fitness and virulence. If an environmental stress

factor has high impact on parasite virulence, this might significantly impact disease dynamics

in aquatic systems. Although theoretical studies have found that the environment can affect

host-parasite population dynamics in nature, little is known of the influence of the interaction

between environment and virulence on the spread of disease through a host population.

Assuming different virulence relationships with the environment, I used an epidemiological

model in Chapter 6 to explore how condition-dependent virulence and environmental quality

alter disease dynamics.

Final discussion, overall conclusions and future perspectives are presented in Chapter 7.

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19

Chapter 2

Variation within and between species cannot be ignored in toxicity

tests

Claudia C. Buser, Jennifer Fox, Andreas Kretschmann & Piet Spaak

(In preparation, target journal Aquatic Toxicology)

Abstract

Although chemical contamination can affect whole ecosystems, many toxicological studies of

aquatic pollutants are still conducted on only a single clone of a single Daphnia species.

Variation in sensitivity towards a toxicant within and between species is not taken into

account, which could lead to inaccurate estimates of the harm of the toxicant. In this study we

show that there is substantial variation within and between three different Daphnia species in

their responses to the common pesticide. To estimate the effect on a population or even on

ecosystems level we therefore suggest conducting toxicological experiments with more than

one clone.

Keywords

Acute toxicity test, Daphnia clonal variation, diazinon, ecology, fitness

LC50 and species variation Chapter 2

20

Introduction

Chemical contaminants are very widespread in the environment. Aquatic ecosystems in

particular encounter many chemical stressors because runoff into rivers and lakes concentrates

chemicals from across a watershed. Watersheds are important and for humans widely used as

drinking water and food sources, though we are also affected (directly or indirectly) by

pollutants in ponds and lakes.

Although scientists from different fields try to assess the toxic effect of chemical

contaminants on individual organisms in order to predict potential impacts of chemicals in the

environment, the fields of toxicology and ecology are still quite separate (Chapman 2002).

While in ecology genetic variation is taken into account when testing for effects of stressors,

in toxicology it is still common to test the toxic potential of a potential toxicant on only one

clone of a single species. For example, many toxicology studies on aquatic organisms focus

on a single Daphnia magna reference clone (Adema 1978). This provides toxicologists with a

good estimate of the toxic potency of a chemical and leads, for example, to fast decisions

about whether and in which concentrations a pesticide can be used in agriculture. But natural

populations normally harbour a genetically diverse set of individuals, all potentially adapted

to quite different environments, and thus the harm of a chemical compound can vary between

and also within different taxa. This variation in response can be due to variation in traits, such

as size, mode of respiration, feeding type and habit and life-cycle duration (Baird & Van den

Brink 2007; Rubach et al. 2010), variation in biotransformation processes and in sensitivity of

the target site (e.g., a certain enzyme) inside an organism (Keizer et al. 1995; Barata et al.

2001; Damasio et al. 2007), or because of adaptation of a species towards the chemical

compound (Klerks & Weis 1987).

Acute toxicity tests are one common method for assessing the hazardous potential of

chemicals (OECD/OCDE 2004). They focus on acute toxic effects like immobilization and

mortality of organisms after short-term exposure. Therefore they are appropriate for the

estimation of effects of chemicals present in the environment in rather high concentrations

and short pulses e.g., due to an accidental spill or a fast degradation. These acute effects

observed on an individual level (e.g., mortality after exposure to short pulses of acutely toxic

concentrations) might have a long-term effect at the population level.

Organophosphorous insecticides are regularly detected in aquatic environments, e.g., in

surface waters due to surface run-off from agricultural fields after rain events (Strom et al.

2003; Pedersen et al. 2006; Wittmer et al. 2010). The organophosphate diazinon (O,O-diethyl

O-[2-isopropyl-6-methyl-4-pyrimidinyl] phosphothionate) is an insecticide widely applied in

Chapter 2 LC50 and species variation

21

agricultural systems (e.g., Konstantinou et al. 2006; Wittmer et al. 2010) and has been shown

to affect and to be highly toxic to aquatic animals (Ankley et al. 1991; Ceron et al. 1996; van

der Geest et al. 2000; Marcial et al. 2005; Gaworecki et al. 2009). Acute toxic effects

(immobilization, mortality) of diazinon are related to inhibition of acetylcholinesterase

(AChE), an enzyme essential for proper functioning of the nervous system (Chambers 1992;

Maxwell et al. 2006). Diazinon is often found in aquatic environments (Miles & Harris 1978;

Konstantinou et al. 2006; Singer et al. 2010). It can be detected in surface waters mainly in

the ng/L range, but also acute concentrations (μg/L range) in short pulses have been recorded

(Wittmer et al. 2010).

Plankton crustaceans are very abundant and sensitive to environmental conditions, for

example to organophosphorous insecticides (Vaal et al. 1997). They are near the bottom of

aquatic food webs and are ecologically important as both grazers of algae and food sources

for fish and other invertebrates. Daphnia, belonging to the Cladocera, are one genus of

planktonic crustaceans often used as model organism in fields like ecology and toxicology.

There are a dozens of Daphnia species differing in size and life history traits (Koivisto 1995),

distributed all over the world. For example Daphnia pulicaria is found in permanent, usually

clear-water, ponds and lakes. Daphnia galeata Sars., 1863 is found in small to very large

permanent lakes, and Daphnia magna is typically found in temporary and permanent ponds

(Hebert 1978). Since D. magna is easy to culture in the lab (Adema 1978; Baudo 1988), this

species is commonly used for toxicity tests (Baudo 1988; ISO_10706 2000).

Many studies have shown the negative effects of diazinon on Daphnia (Fernandez-

Casalderrey et al. 1994; Sanchez et al. 1998; Jemec et al. 2007). Most of these studies test

acute toxicity using only one clone and do not consider the variation within and between taxa.

In nature we rarely find Daphnia populations consisting of only a single clone, so it makes

sense to investigate how results obtained from “one clone studies” relate to the variation that

can be obtained by doing experiments with several clones. Ideally the “one clone” would be

somewhere in the middle of the distribution of the many clones, and would not be an extreme

genotype that shows a behaviour quite different from clones chosen from natural

environments.

In this study, we highlight the importance of taking the genetic diversity of organisms into

account when performing toxicity tests. We show that the toxic response to acutely toxic

diazinon concentrations affect Daphnia species and even clones within a species differently.

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Nervous_system


22

Material and Methods

Acute toxicity test

Acute toxicity tests were performed according to the OECD Guideline 202 (OECD/OCDE

2004). Diazinon (CAS 333-41-5, 99.5 % purity) was obtained from Ultra Scientific Analytical

Solutions (N. Kingstown RI, USA). For exposure experiments diazinon was dissolved in

acetone and spiked into filtered lake water. We tested 6 different concentrations of diazinon

(0.00, 0.13, 0.25, 0.50, 1.00, and 2.00 µg/L), with lake water containing the same amount of

acetone as in the treatments as a control. Nominal exposure concentrations of diazinon were

verified with LC-MS/MS as described in (Kretschmann et al. 2011b). As measured diazinon

concentrations were within ± 20 % of the nominal concentrations, for data analysis nominal

concentrations were used.

Six clones of Daphnia magna, four clones of Daphnia pulicaria, and four clones of

Daphnia galeata were used. The clones were selected from different lakes from several

countries as listed in Table 1, to increase genetic variation. All clones had been cultured in the

laboratory for at least 1 year. Prior to the experiment, clones were raised in multiple source

jars for two generations under standard laboratory conditions. From the second or later clutch

of the third generation we put 5 juveniles (<24 h old) in 10 mL of medium in 50-mL jars, with

3 replicates for each clone in each of the 7 treatments (6 diazinon concentrations and acetone

control). Animals were assessed for survival after 48 h. Daphnia were not fed during the

experiment. Experiments were performed at a temperature of 20 ± 1 °C and a photoperiod of

16 h light and 8 h dark.

Data analysis

Acute toxicity data were evaluated with the program SigmaPlot version 10.0. LC50 values

(concentration at which 50 % of the tested organisms died) and the hill slope (steepness of the

dose-response curve) were obtained by fitting a sigmoidal dose-response curve (four-

parameter logistic equation) to the plot of the fraction of surviving animals (%) vs. log-

transformed diazinon concentrations.

To test if mortality differed within and among taxa according to diazinon concentrations we

used a generalized linear model in SPSS 15.0 with proportion of animals surviving after 48h

as the dependent variable, the concentrations and taxa as fixed factors, and the clones nested

within taxa as a random factor.


23

Table 1: Overview and distribution of the clones used in the acute toxicity test

Taxa Clone Origin

D. galeata ENDI Lago di Endine, Italy

SEGR Lago del Segrino, Italy

ZUG Lake Zug, Switzerland

ZURI Lake Zurich, Switzerland

D. magna CN2-4 Czech Republic

EK1-1 United Kingdom

HO-1 Hungary

Klon 5 Germany

M10 Belgium

M12 Germany

D. pulicaria A16 Czech Republic

PU3 Netherlands

PUL-ARE Oregon, USA

REZ Czech Republic

Results

Acute toxicity test on three Daphnia species

A comparison of the LC50 values reveals that D. pulicaria and D. galeata had a lower LC50

than D. magna (Table 2, Fig. 1). The LC50 of D. pulicaria was almost 50 % lower than that of

D. magna (0.878 µg/L versus 1.512 µg/L).

Table 2: Summary of LC50 values and hill slope for D. galeata, D. magna and D. pulicaria.

Species R Best fit hill slope value ± SE Best fit LC50 (µg/L) value ± SE

D. galeata 0.9960 -0.099 ± 0.025 1.112 ± 0.022

D. magna 0.9997 -0.122 ± 0.005 1.512 ± 0.006

D. pulicaria 0.9995 -0.070 ± 0.014 0.878 ± 0.013

The generalized linear model revealed highly significant main effects of diazinon

concentration (χ2= 1955.187, df= 6 , p< 0.001), species (χ

2= 81.264, df= 2, p< 0.001, Fig. 1),

and clone (χ2= 146.691, df= 12, p< 0.001, Fig. 2) on survival. The higher the diazinon

concentration, the lower the proportion of animals that survived (Fig. 1 & Fig. 2). The three

species differed in their response, with D. magna showing higher survival than the other two

species.


24

Fig. 1: Fitted dose-response curves show the variation in survival (mean ± SE) after 48 h of diazinon exposure

among the three Daphnia species. The LC50 values for each species are back transformed.

There was also a significant interaction for species × diazinon concentration (χ2= 121.448,

df= 12 , p< 0.001). For example, in the absence of diazinon or at low diazinon concentrations

fewer animals of D. pulicaria died compared to D. galeata, but at higher diazinon

concentrations D. pulicaria was the most sensitive species (Fig. 1). We also observed similar

interactions within the species on a clonal level (clone × diazinon concentration: χ2= 428.965,

df= 66 , p< 0.001, Fig. 2). Within different clones of the species D. magna, the clones CN2-4

and M10 were found to be the most resistant ones (see Fig. 2B). Survival of the CN2-4 clone

was not affected by any diazinon concentration (in all concentrations 100% survival).

Survival of the M10 clone was affected only at the highest diazinon concentration applied

(proportion of M10 clones surviving at 2 µg/L: 0.65 ± 0.236 (mean ± SE); proportion of CN2-

4 clones surviving at 2 µg/L: 1.0 ± 0).


25

Fig. 2: Proportion (mean ± SE) of clones

surviving after 48 hours at different

concentrations of diazinon within the (A)

D. galeata, (B) D. magna, (C) D. pulicaria.

Discussion

The species and even clones within a single species reacted significantly differently from each

other when exposed to diazinon for 48 h. In our acute test D. magna was the species least

sensitive to diazinon, whereas D. galeata and D. pulicaria have a higher mortality rate when

exposed to the pesticide. The difference in sensitivity could be due to several (not mutually

exclusive) reasons. First, larger animals often have a lower sensitivity towards toxicants. For

example, a positive relationship between EC50 (concentration at which 50 % of the tested

organism show an effect) values and body size was found by Vesela and Vijverberg (2007)

when four different Daphnia species were exposed to zinc. In other interspecies comparisons

large-sized Daphnia tended to be more tolerant to toxic substances than smaller ones (Winner


26

& Farrell 1976; Koivisto et al. 1992). A correlation between sensitivity and size would

explain why the larger D. magna is less sensitive than the two smaller species in our

experiments. This hypothesis does not hold true for the finding that D. pulicaria is more

sensitive than D. galeata in our study; however the size difference between these two species

is not as great as with D. magna. Also this size-hypothesis does not explain the high variation

within taxa, as the size differences within a taxa are small. Second, the difference in

sensitivity could be due to differences in the habitats the species live in. D. magna lives more

in shallow ponds, which are unpredictable habitats with large temporal and spatial variability

in abiotic factors; whereas the other two species live in a larger water habitats that are more

stable. Adaptation to natural abiotic stress may increase pollution tolerance (Fisher 1977;

Leblanc 1985). For example, Koivisto et al. (1992) show that Daphnia species living in ponds

were less sensitive to copper than lake-living cladocerans. Finally, the observed difference in

sensitivity towards diazinon might also be due to different intrinsic properties, like different

reactivities/sensitivities of biotransforming enzymes as well as of the target site AChE. Keizer

et al. (1995) explained differences in the toxicity of diazinon towards different fish species by

an observed variation in AChE sensitivity toward inhibition by diazoxon, the active

metabolite of diazinon, in combination with the ability to biotransform diazinon. A correlation

between the sensitivity towards diazinon and the sensitivity of AChE has also been shown for

D. magna (Kretschmann et al. 2011a).

In our study we not only showed variation between species; we also demonstrated that clones

within a species show very different levels of sensitivity to the pesticide. An ideal model

clone should be in the middle of the distribution to provide a good mean estimate for the harm

within the species. In our example, if only the CN2-4 or M10 clone of the species D. magna

was used for assessing the hazardous potential of a chemical this might lead to an

underestimation of risk towards other, more sensitive clones.

When assessing the effects of chemicals on aquatic organisms, it makes sense to choose an

ecologically significant species, such as Daphnia, that may be near the base of the food web.

Concentrations affecting this species could have an impact on species higher up in the food

chain. Therefore we recommend the use of more than one clone in toxicology studies in order

to take into account the variation within and between species and to minimize the risk of

underestimating the effects of a chemical (Cairns 1984; Chapman 2000). If (for whatever

reason) it is possible to perform an experiment with only a single clone, we recommend using

a clone known to be in the middle of the sensitivity distribution.


27

Acknowledgment

We thank Nora Brede for her help setting up the experiment and Christoph Haag for

comments on the manuscript. The study was financially supported by the Swiss National

Science Foundation (SNF) and the ETH Board (CCES-GEDIHAP).

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29

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31

Chapter 3

Combined exposure to parasites and pesticides causes increased

mortality in the water flea Daphnia

Claudia C. Buser, Mieke Jansen, Kevin Pauwels, Luc De Meester & Piet Spaak

(In preparation, target journal Freshwater Biology)

Abstract

Organisms are exposed to multiple biotic and abiotic environmental stressors, which can

influence dynamics of individual populations and communities. Species may also genetically

adapt to both natural (e.g., disease) and anthropogenic (e.g., chemical pollution) stress. We set

out to study fitness consequences of exposure to both parasites and pesticides in the water flea

Daphnia and to quantify whether these fitness consequences change through time as a

population evolves. We exposed Daphnia magna clones hatched from dormant eggs isolated

from different time layers of a natural dormant egg bank to the parasite Pasteuria ramosa and

the insecticide diazinon. While our experimental treatments for unknown reasons failed to

induce disease in the Daphnia we did observe a reduced survival of D. magna when exposed

to both parasites and pesticides. No increased mortality upon exposure to individual stressors

was observed. As also no induction of phenoloxidase activity was found, we cannot confirm if

the difference in survival was related to immune suppression. We did not observe an

evolutionary change in fitness response of the Daphnia clones hatched from different time

horizons upon exposure to stressors.

Keywords

Daphnia magna, diazinon, fitness, multiple stressors, Pasteuria ramosa, parasites,

resurrection ecology, dormant eggs

Multiple stress on Daphnia hatchlings Chapter 3

32

Introduction

In recent years, the importance of multiple stressors on the health of organisms has received

increased attention (e.g., Marcogliese & Pietrock 2011). It is well known that responding to

additional disturbances leads to higher costs, which can result in a reduction of fitness of

exposed individuals (reviewed in Relyea 2003; Sih et al. 2004). Anthropogenic stress is

expected to increase diseases in natural populations (e.g., Daszak 2000). Pesticides, for

example, are used worldwide in agriculture, and have been shown to increase susceptibility to

parasite infections (e.g., Chu & Hale 1994; Gendron et al. 2003; Coors & De Meester 2008;

Rohr et al. 2008; King et al. 2010; Kreutz et al. 2010).

Studies considering abiotic and biotic stressors are often conducted with genotypes sampled

over a spatial gradient (King et al. 2010; Schoebel et al. 2010; Bryner & Rigling 2011; Jansen

et al. 2011). Although this approach does not directly track changes over time (De Meester et

al. 2007), it provides valuable information patterns of genetic adaption to local environmental

conditions (Kawecki & Ebert 2004). In organisms that produce dormant eggs, however,

“resurrection ecology” can be a powerful tool for studying microevolutionary responses to

environmental change (Hairston et al. 1999; Jeppesen et al. 2001; Kerfoot & Weider 2004). In

lake and pond sediments, dormant stages of different organisms can remain viable for decades

or longer (Hairston 1996). Thus it is possible to hatch dormant eggs from a dated sediment

core in the laboratory and compare these “old” genotypes in experiments with current

genotypes from the same pond or lake (Kerfoot et al. 1999). Dormant eggs from different time

periods can therefore be used to examine evolutionary responses to environmental changes.

Several resurrection ecology studies have provided evidence for microevolutionary responses

over only a few generations (e.g., Hairston et al. 1999; Cousyn et al. 2001; Hairston et al.

2001). For example, Hairston et al. (1999) showed that Daphnia hatched from a period when

toxic cyanobacteria were present in Lake Constance were more resistant to a diet with

cyanobacteria. Cousyn et al. (2001) documented microevolutionary changes in phototactic

behaviour in a natural Daphnia population in response to changes in fish predation pressure

and Pauwels et al. (2010) showed that the same population also evolved with respect to

phenoloxidase activity, a component of the invertebrate innate immunity. Decaestecker et al.

(2007) using both Daphnia and its microparasites hatched from different sediment layers,

showed very strong parasite-host co-adaptive responses.

To our knowledge, there are no studies observing the effect of multiple stressors over a

timescale of several decades. In the present study we set out to test whether the combined

exposure to a parasite and a pesticide affected the fitness of Daphnia magna (Straus, 1820)

hatched from different sediment layers.

Chapter 3 Multiple stress on Daphnia hatchlings

33


Origin and culture conditions of the host-parasite system and the pesticide

Daphnia magna host clones were used from a previous study (Decaestecker et al. 2007). The

hatched Daphnia came from seven different sediment layers of the Belgian pond OM 2

situated in Heverlee (50°50‟22‟‟N, 4°39‟18‟‟E), covering a time period from about 17 - 28

years (Decaestecker et al. 2007). This pond is characterized by epidemics of Pasteuria

ramosa (Metchnikoff, 1888), a parasite of Daphnia magna (Decaestecker et al. 2007), as well

as intense agricultural activity in the catchment (Coors et al. 2009). In total, we used 13

genetically different clones, two from each sediment layer, except from the oldest, where only

one clone could be used.

P. ramosa spores used for infection were obtained by exposing different individuals of a

single D. magna clone (M10, see Cousyn et al. 2001) originating from the pond Oud Heverlee

to the first few centimetres of the sediment core from the pond Knokke Nat (Belgium

51°21‟25‟‟N, 3°19‟50‟‟E), as described in Jansen et al. (2010). The parasites were collected

from a different pond than the host clones used in the experiment to avoid possible effects of

co-adaption of hosts and parasites (Decaestecker et al. 2007). P. ramosa from Knokke Nat

have been shown to heavily infect D. magna clones from OM 2 (Jansen et al. 2010). To

increase the amount of P. ramosa spores, infected M10 clones were grown for 21 days and

used to infect Daphnia juveniles of the same clone for another generation, before using them

in the experiment as described in Coors et al. (2008). Infected Daphnia become sterilized, are

darkish-red and become larger in body size (Ebert 2005) and are therefore easily recognizable

by eyes. Grounded up, non-infected Daphnia from the M10 clones were used as placebo in

the treatments without parasites.

The organophosphate diazinon (O,O-diethyl O-[2-isopropyl-6-methyl-4-pyrimidinyl]

phosphothionate, CAS 333-41-5, 99.5 % purity, Ultra Scientific Analytical Solutions, N.

Kingstwon RI, USA) is a pesticide developed in 1952 that was heavily used between 1970

and the early 1980s in many parts of the world. Nowadays, it is still frequently used in

agriculture and households, and can thus be detected in aquatic environments (Konstantinou

et al. 2006; Singer et al. 2010). Acute toxic effects of diazinon (e.g., immobilization and

death) are related to the inhibition of acetylcholinesterase, an enzyme essential for proper

function of the nervous system (Chambers 1992; Maxwell et al. 2006). Exposure of D. magna

to sub-lethal concentrations of diazinon leads to a delay in reproduction, a decrease in the

number and size of offspring, and higher adult mortality (Sanchez et al. 1998).


34

Experimental set-up and procedures

Thirteen clones (from seven sediment layers) were exposed to P. ramosa and diazinon in a

full factorial design, and replicated six times, resulting in 312 experimental units. To

minimize maternal effects at the start of the experiment, the clones were kept under standard

experimental laboratory conditions [1:5 diluted Aachener Daphnien Medium (ADaM,

Klüttgen et al. 1994), 20 ± 2 °C, 16 h:8 h light/dark photoperiod, fed daily with 2 * 105

Scenedesmus obliquus cells/mL] for at least two generations. To start the experiment ten

third-clutch juveniles (less than 24 h old) were put together in 250 mL glasses containing 20

mL medium. The pesticide treatment consisted of a concentration of diazinon (0.25 μg/L),

which is sub-lethal for Daphnia, whereas the pesticide-free control treatment included the

addition of the same amount of the solvent (acetone 0.008 %). Concentrations of diazinon

were verified with spot test with LC-MS/MS as described in Kretschmann et al. (2011b) and

were all within ± 20 % of the nominal concentrations. Individuals belonging to the parasite

treatment were exposed on day 0 and day 2 to 375 × 102

mature spores of P. ramosa per mL

(Jansen et al. 2011). All glasses belonging to the control treatment were treated with ground

up, non-infected, Daphnia of the M10 clone. On day five all Daphnia were transferred into

new glasses (500 mL) containing 400 mL diluted ADaM medium, which was renewed every

third day thereafter. Animals were checked for infections, the number of offspring was

counted and the survival rate was checked daily (and the dead individuals removed) till the

end of the experiment. On day 21, 1 μL haemolymph was taken from two animals per glass

and frozen at -21 °C in 100 μL phosphate buffered saline (PBS) buffer. Phenoloxidase (one

component of the invertebrate innate immune reponse, Söderhäll & Cerenius 1998) was

activated with chymotrypsin (Sigma aldrichchemie GmbH, Buchs, Switzerland) and

quantified on a spectrophotometer (absorbance 475 nm) following Mucklow et al. (2004). All

traits were quantified as a mean or proportion per set of 10.

Statistical analyses

Statistical analyses were performed using SPSS 19.0. In total, seven experimental units were

lost due to handling errors and excluded from the analysis. Differences in survival rate,

reproductive output as well as phenoloxidase activity were analysed using linear mixed

effects models, with exposure to parasites (yes/no), exposure to diazinon (yes/no) and

sediment layer as fixed factors and clone nested in sediment layer as random factor. We did

not include the interactions between the fixed factors and the random factor clone, as our aims

were not related to clonal variation. The individual reproductive output was calculated (total

amount of offspring born on day x divided by the total amount of living females on day x, for

x going from day 4 to 21, Van Doorslaer et al. 2009). All residuals were distributed normally.


35

Results

In this experiment no visible signs of infections could be observed at day 21. Nevertheless,

significant differences in survival rate were observed when Daphnia were exposed to both

stressors: The proportion of surviving individuals was significantly reduced when Daphnia

were exposed to parasite spores and pesticides simultaneously, but not when they were

exposed to a single stressors (Table 1; Fig. 1). There was neither a significant effect of these

factors or their interactions on the individual reproductive output nor on phenoloxidase

activity (see Table 1). Furthermore, there were no interaction effects with sediment layer, nor

a main effect of sediment layer (all p> 0.1, see Table 1). All variables were highly variable

among clones (Table S1).

Table 1: Results of linear mixed effects models describing the influence of exposure to parasite spores, exposure

to diazinon, sediment layer and interactions among these factors on survival, reproduction and phenoloxidase

activity (PO).

Dependent variable Source Numerator df Denominator df F p

Survival rate parasite (P) 1 271.034 3.008 0.084

diazinon (DZ) 1 271.034 2.876 0.091

sediment layer (S) 6 6.036 1.936 0.220

P × DZ 1 271.087 5.099 0.025

P × S 6 271.037 0.689 0.659

DZ × S 6 271.037 1.601 0.147

P × DZ × S 6 271.093 0.982 0.438

reproductive output parasite (P) 1 267.060 0.873 0.351

diazinon (DZ) 1 267.139 0.280 0.597

sediment layer (S) 6 6.034 2.192 0.180

P × DZ 1 267.147 0.234 0.629

P × S 6 267.067 1.042 0.398

DZ × S 6 267.154 1.642 0.136

P × DZ × S 6 267.162 1.321 0.248

PO parasite (P) 1 227.054 0.339 0.561

diazinon (DZ) 1 227.527 0.526 0.469

sediment layer (S) 6 5.898 1.521 0.313

P × DZ 1 227.576 0.341 0.560

P × S 6 227.071 1.225 0.294

DZ × S 6 227.597 1.603 0.147

P × DZ × S 6 227.608 0.803 0.569


36

Fig. 1: Reaction norms of mean proportion of surviving individuals per glass (± SE) as a function of exposure to

the pesticide diazinon(no/yes) and parasite spores (no= open circles/yes= closed circles).

Discussion

Our key result is an increased mortality upon exposure to both parasites and a pesticide, even

though no parasite infections could be detected visually (see further). Many studies showed

hosts in poor condition have higher parasite-induced mortality than hosts in good condition

(e.g., Braune & Rolff 2001; Krist et al. 2004; Jokela et al. 2005). The negative effect of two

stressors on survival is much stronger compared to individual effects. Immune suppression

under direct exposure to pollutants is a well-known phenomenon (e.g., McDowell et al. 1999;

Aggarwal et al. 2008) and also diazinon is known to have immunomodulatory effects

(Galloway & Handy 2003; Oostingh et al. 2009; Holmstrup et al. 2010). In this study we did

not observe a decrease in phenoloxidase activity in animals exposed to the pesticide diazinon.

This could either be because phenoloxidase activity is not affected by diazinon, which stays in

contrast to other pesticides (Campero et al. 2008), or due to the fact that phenoloxidase was

measured 17 days after pesticide exposure of the Daphnia. The Daphnia which did not die

might have recovered from pesticide stress over the day 21 period, resulting in identical

phenoloxidase activity between pesticide treatments. Kretschmann et al. (2011a) observed a

slow recovery from diazinon exposure after transferring D. magna from media containing

diazinon into clean water. According to their model, a recovery from diazinon exposure

would take place within approximately 17 days. Nevertheless, our results indicate that the

defence against parasite spores, even if they do not cause a visible infection, is associated with

costs for D. magna. Yin et al. (2011) showed that defence against predators induced stronger

infectivity of parasites, while Coors et al. (2008) showed that exposure to pesticides increased

disease progression in Daphnia. Here, we show the reverse, that the enhanced costs of


37

defending against parasites spores, even if they do not cause visible infections, increases

mortality upon exposure to a pesticide.

There are several potential reasons why D. magna clones in our study did not get infected by

P. ramosa. First of all, it is possible that either during parasite isolation from the sediment or

during enrichment of parasite spores, M10 clones were infected by parasite spores that were

not infective for OM2 clones, even though P. ramosa from Knokke Nat have been shown to

heavily infect D. magna clones from OM 2 (Jansen et al. 2010). It is possible that our

additional round of infection of clone M10 resulted in a bottleneck with respect to the number

of parasite lineages, and that a strain was selected which by chance did not infect OM2

clones. Alternatively, it may be that, for unknown reasons, the number of viable P. ramosa

spores decreased to below threshold levels in the time between estimating their density and

using them in the experiment. Low spore concentrations result in failure to induce disease

(Ben-Ami et al. 2008). The observed increased mortality in our combined parasites ×

pesticides exposure treatment may thus be associated with either exposure to a below-

threshold concentration of parasite spores or exposure to spores that were not infective.

We did not find any effect of sediment layer on any measured trait, which is in contrast to

Decaestecker et al. (2007), who found a correlation between parasite density and years as well

as with the study of Pauwels et al. (2010), who found differences in levels of Daphnia

phenoloxidase activity over time. The fact that our spore solution did not cause visible

infections in this study as well as having few clones per sediment layer could be the reason

why we do not find any sediment layer effect. In case of the diazinon treatment alone, we

never expected to find a sediment layer effect. First of all, OM 2 is surrounded by farm land,

but the historical use of diazinon is not well documented. Therefore we do not know if

diazinon was ever present in this lake. Additionally, it might be diazinon degraded too fast not

allowing enough time for the hosts to adapt. Nevertheless, we encourage scientist to study

evolutionary responses to environmental changes using dormant eggs, as this approach is

straightforward to document microevolutionary changes in natural populations. Also

multiples stressors should be included, since this is a more realistic representation of natural

situations.

Acknowledgment

We thank several members of the Luc De Meester‟s group for their help during the

experiment and Andreas Kretschmann for helping verifying the diazinon concentration. The

manuscript improved by comments from Andreas Bruder, Christoph Tellenbach and Mat


38

Seymour. This research was funded by the ETH Board (CCES-GEDIHAP), the Mobility

Support from Eawag and the IWT, Flanders.

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41

Web appendix

Table S1: Descriptive statistic (mean, standard deviation (SD), number of vials (N)) for survival, reproductive

output and phenoloxidase activity for each clone exposed to the four treatments.

Survival Reproductive output Phenoloxidase activity Clone Sediment layer Treatment mean SD N mean SD N mean SD N

D2-2 1 Control 0.24 0.18 5 6.39 2.03 5 63.97 28.63 3

Diazinon (D) 0.35 0.27 6 4.32 2.87 6 86.52 65.65 5

Parasite (P) 0.30 0.30 6 2.95 1.96 6 132.10 23.21 4

P & D 0.18 0.21 6 3.28 2.76 6 25.85 21.81 3

D2-3 1 Control 0.47 0.32 6 3.52 0.57 6 19.83 27.18 6

Diazinon (D) 0.38 0.35 6 2.60 1.64 6 4.52 7.02 4

Parasite (P) 0.38 0.12 6 4.51 0.60 6 8.57 10.06 6

P & D 0.30 0.32 6 3.12 1.09 6 59.31 41.63 4

D3-1 2 Control 0.56 0.46 6 3.41 1.35 6 47.32 91.88 4

Diazinon (D) 0.83 0.19 4 3.92 2.22 4 85.39 148.79 4

Parasite (P) 0.65 0.34 5 3.08 1.92 5 43.60 24.28 3

P & D 0.75 0.42 6 3.62 1.59 6 69.69 35.49 5

D3-2 2 Control 0.64 0.25 6 4.03 1.54 6 8.46 10.65 5

Diazinon (D) 0.63 0.33 6 4.02 1.11 6 30.52 51.71 5

Parasite (P) 0.65 0.25 6 4.58 1.51 6 9.35 13.65 6

P & D 0.66 0.11 6 4.85 1.27 6 60.74 126.64 6

D4-1 3 Control 0.43 0.22 6 2.73 1.72 6 104.07 116.95 6

Diazinon (D) 0.41 0.36 6 4.30 2.15 5 154.63 149.09 3

Parasite (P) 0.43 0.30 6 3.40 1.61 6 49.01 46.13 4

P & D 0.27 0.12 6 3.52 0.83 6 39.16 62.68 6

D4-2 3 Control 0.90 0.20 6 1.77 0.37 6 35.83 27.22 6

Diazinon (D) 0.68 0.29 6 2.16 0.61 6 150.15 62.94 6

Parasite (P) 0.70 0.31 6 2.47 0.42 6 73.04 45.78 5

P & D 0.30 0.31 6 3.13 1.85 6 92.18 77.72 5

D5-1 4 Control 0.83 0.26 6 2.96 0.63 6 177.30 135.14 5

Diazinon (D) 0.85 0.17 6 3.09 0.29 6 66.83 48.92 3

Parasite (P) 0.52 0.30 6 4.22 1.24 6 62.75 42.63 5

P & D 0.63 0.22 6 4.25 1.21 6 100.93 78.69 5

D5-2 4 Control 0.39 0.14 6 3.11 0.87 6 16.31 19.13 6

Diazinon (D) 0.55 0.25 6 3.31 0.49 6 38.03 49.39 6

Parasite (P) 0.63 0.22 6 3.40 0.79 6 60.49 45.41 6

P & D 0.59 0.11 6 2.71 0.98 6 33.76 28.40 6

D6-1 5 Control 0.58 0.20 6 3.58 0.79 6 54.73 81.01 6

Diazinon (D) 0.28 0.30 6 4.91 2.37 5 13.03 16.18 4

Parasite (P) 0.53 0.19 6 5.56 1.62 6 57.87 52.30 6

P & D 0.23 0.17 6 4.81 1.74 6 26.69 31.50 5

D6-2 5 Control 0.22 0.13 6 3.92 1.01 5 32.45 18.87 5

Diazinon (D) 0.43 0.14 6 5.06 1.11 6 34.50 36.75 6

Parasite (P) 0.33 0.36 6 4.20 0.51 5 58.93 72.87 5

P & D 0.40 0.20 6 4.61 0.87 6 58.29 70.43 6

D7-1 6 Control 0.91 0.12 6 3.23 0.61 6 13.81 15.59 6

Diazinon (D) 0.77 0.14 6 3.89 0.71 6 24.82 16.68 6

Parasite (P) 0.75 0.08 6 3.00 1.10 6 7.48 9.01 6

P & D 0.76 0.25 6 3.44 0.72 6 27.18 40.70 6

D7-2 6 Control 0.52 0.28 6 3.38 1.44 6 15.21 30.71 6

Diazinon (D) 0.50 0.29 6 4.35 1.45 6 30.24 45.06 6

Parasite (P) 0.50 0.25 6 5.40 2.09 6 7.92 8.97 5

P & D 0.41 0.30 5 4.18 1.32 5 5.63 5.33 5

D8-1 7 Control 0.39 0.22 6 3.19 1.06 6 27.11 27.87 6

Diazinon (D) 0.56 0.32 6 2.42 0.48 6 30.59 34.24 5

Parasite (P) 0.66 0.33 5 2.28 0.47 5 53.94 39.48 5

P & D 0.25 0.24 5 3.06 1.42 5 20.54 10.26 4

43

Chapter 4

Disease and pollution alter Daphnia community structure

Claudia C. Buser, Piet Spaak & Justyna Wolinska

(submitted to Freshwater Biology)

Abstract

Environmental stressors can influence population and community structure. But a majority of

experimental work on multiple environmental stressors have been tested on an individual

level only. In the present work we followed changes in experimental Daphnia (waterflea)

communities (consisting of two taxa and three clones per taxon) after exposure to the fungal

parasite Metschnikowia sp. and/or the pesticide diazinon. We found a significant shift in taxon

and clonal composition under both stressors. Strikingly, one taxon went extinct in the parasite

and parasite + pesticide treatments. While the pesticide had no effect on total parasite

prevalence, one taxon was more infected when additionally exposed to the pesticide.

Furthermore, the density of all adult females was significantly reduced in the parasite

treatment, but not in the pesticide treatment. Our results demonstrate that dynamics in

Daphnia communities are altered by parasites and pesticide exposure. Given Daphnia‟s key-

role in aquatic food webs, converting primary production to fish food, this may lead to a

higher stress on aquatic ecosystems.

Key words

Competition, Daphnia galeata, frequency-dependent selection, hybrids, Metschnikowia sp.,

parasite, pesticide

Competing taxa exposed to multiple stressors Chapter 4

44

Introduction

Communities are subject to a multitude of abiotic and biotic stressors. One approach to

understand the effect of these multiple stressors on communities is to study the effect of

stressors on single individuals and extrapolate these findings to the higher community level.

The effect of multiple stressors on the individual level has been investigated for several taxa

across the plant and animal kingdom. It has been shown that a combination of abiotic and

biotic stressors is often more harmful than either stressor alone (e.g., Hanazato & Dodson

1995; Folt et al. 1999; Relyea 2003; Sih et al. 2004).

Abiotic stressors (e.g., temperature change, eutrophication, acidification) are very abundant

and can affect the functioning and structure of ecosystems (e.g., Bronmark & Hansson 2002;

Brede et al. 2009). Pesticide pollution is one frequently occurring stressor in aquatic habitats

as pesticides are washed out from agricultural fields after rain events (Pedersen et al. 2006;

Wittmer et al. 2010). For example, zooplankton exposed to a chronic concentration of

pesticides often have fewer and smaller offspring and suffer increased mortality (reviewed in

Hanazato 2001). Among biotic stressors, parasites seem to play an especially important role.

They affect the fitness of host individuals and thus can alter the structure and the diversity of

host populations as well as competition among species (e.g., Combes 1996; Hudson &

Greenman 1998; Jokela et al. 2003; Duncan & Little 2007; Wolinska & Spaak 2009).

Parasite-mediated selection can be increased or decreased by additional biotic or abiotic

stressors in the host environment, like extreme temperature or food shortage (reviewed in

Wolinska & King 2009).

Parasites and pollution may interact to enhance the damage to and selection on host

populations. For example, exposure to pesticides can increase host susceptibility to infection,

as well as enhance parasite virulence, as shown for amphibians (Gendron et al. 2003; Rohr et

al. 2008; King et al. 2010), fish (Kreutz et al. 2010), oysters (Chu & Hale 1994) and

planktonic crustaceans (Coors & De Meester 2008; Coors et al. 2008; Coors et al. 2009;

Coors & De Meester 2011). Many of these studies have been conducted on the individual and

population level. But, the effects of pesticide exposure on the relative genotypes frequencies

is rarely investigated. Hanazato & Yasuno (1987) observed changes in zooplankton species

composition after exposure to insecticides, and Relyea & Hovernan (2008) demonstrated that

pesticide exposure lowered zooplankton diversity and abundance. However, the extent to

which the combined effect of parasites and pesticides alter host composition on a community

or population level remains largely unexplored.

Chapter 4 Competing taxa exposed to multiple stressors

45

In this study, we examined how Daphnia (waterflea) communities are affected by exposure to

the parasite Metschnikowia sp. and pesticide diazinon. More specifically, we followed taxa

and genotype dynamics in experimental communities when exposed to both stressors in a full

factorial design. As Daphnia reproduce by cyclical parthenogenesis, we could easily follow

the frequencies of the two taxa and three genotypes (clones) per taxon. We predicted that

parasite and pesticide exposure would additively decrease host density and increase parasite

prevalence because effects of stressors are worse in combination than alone (Hanazato &

Dodson 1995; Folt et al. 1999). However, predictions for the effects of stressors on clonal and

taxa composition are difficult to form given the conflicting results on whether

Metschnikowia sp. infects different taxa and clones to the same extent (Stirnadel & Ebert

1997; Wolinska et al. 2009; Yin et al. 2011) or not (Duffy & Sivars-Becker 2007; Duffy et al.

2011). If the latter is true, the parasite would infect various taxa and clones to different

extents, possible altering the competition among taxa and clones.

Materials and Methods

Host-parasite system

As a model host, we used Daphnia (waterflea) belonging to the D. longispina hybrid

complex. This complex consists of three parental species (D. cucullata G. O. Sars, 1862,

D. galeata G. O. Sars, 1863 and D. longispina O. F. Müller, 1776, as revised in Petrusek et al.

2008) and their interspecific hybrids, and dominates Daphnia communities of European lakes

(e.g., Keller et al. 2008; Petrusek et al. 2008; Yin et al. 2010). The yeast parasite

Metchnikowia sp. (family Hemiascomycetes, Wolinska et al. 2009) commonly infects

Daphnia populations in Europe and the United States (Hall et al. 2005; Caceres et al. 2006;

Wolinska et al. 2011). Needle-like ascospores accumulate in the body cavity and are released

only after host death (Green 1974). Infected Daphnia have fewer offspring and a reduced

lifespan (e.g., Duffy & Hall 2008; Lohr et al. 2010).

Pesticide exposure

The organophosphate diazinon (O,O-diethyl O-[2-isopropyl-6-methyl-4-pyrimidinyl]

phosphothionate) is one of the most used and applied pesticide in agriculture and households,

and is thus detected in several aquatic environments (Miles & Harris 1978; Singer et al.

2010). Acute toxic effects of diazinon (like immobilization, mortality) are related to the

inhibition of acetylcholinesterase, an enzyme essential for proper function of the nervous

system. Exposure of Daphnia magna to a sub-lethal concentration leads to a delay in

reproduction, a decrease in the number of offspring and offspring size and higher adult

mortality (see Sanchez et al. 1998). Based on the results of sub-lethal concentrations of


46

diazinon (CAS: 333-41-5, 99.5 % purity, Ultra Scientific Analytical Solution, N. Kingstown

RI, USA) on D. galeata (see supplementary material, S1) we set our pesticide concentration

to 0.1 μg/L. Similar diazinon concentrations as used in this study have been measured in

natural systems (e.g., Konstantinou et al. 2006; Wittmer et al. 2010).

Origin and care of host and parasite

Both host and parasite were isolated from Ammersee, a natural lake in Germany (surface area:

46.6 km2; coordinates: 48°00‟34‟‟N, 11°07‟02‟‟E), two years prior to the experiment (in

autumn 2008). The Daphnia community of this lake is dominated by D. galeata and

D. galeata × D. longispina hybrids (Yin et al. 2010). Three D. galeata and three

D. galeata × longispina hybrid clones were selected from a larger collection of sampled

clones (taxon and multilocus genotype assignment based on 15 microsatellite loci, for

methods see Yin et al. 2010). Metschnikowia was maintained on a single

D. galeata × longispina clone (AMM_12) by adding uninfected juveniles into the infected

culture every second week (Lohr et al. 2010). Uninfected and infected Daphnia cultures were

kept under standardized conditions (20 °C, 16:8 light-dark photoperiod, fed three times a

week with 1.0 mg C/L green algae, Scenedesmus obliquus). Before the experiment, the

uninfected Daphnia clones were raised in 10 L buckets filled with lake water (Greifensee,

Switzerland; 0.45-μl-filtered and UV-light sterilized), to obtain enough individuals.


We tested whether the presence of parasite and/or pesticide alters the competition between

Daphnia taxa (hereafter we use the term "taxon" for each of the two Daphnia forms tested:

parental D. galeata and D. galeata × longispina hybrids) and within taxa (three clones per

taxon tested). Experimental communities were exposed to four treatments: control (lake water

only), parasite, pesticide and parasite + pesticide. Each treatment was replicated five times.

The diazinon, dissolved in acetone, was added at a final concentration 0.1 μg/L; the respective

amount of acetone was also added to non-pesticide treatments. To obtain parasite spores for

experimental infection, 24 juveniles (four per clone; 3-6 days old) were placed together in a

200 mL jar (one-hundred jars were prepared). Then, 300 heavily infected/uninfected Daphnia

of clone AMM_12 were crushed and equally distributed to the corresponding jars (i.e., fifty

infected and fifty uninfected jars were prepared). The water was stirred daily to re-suspend the

spores and maximise Daphnia/spore contact. Daphnia were daily fed with S. obliquus at an

amount of 1.0 mg C/L. Four days later (i.e., on experimental day 1), the contents of the jars

were added into the respective aquarium (see below).


47

On day 1, twenty aquaria (4 treatments × 5 replicates) were prepared, containing 4 L of lake

water. Each aquarium was set up with three D. galeata and three D. galeata × longispina

clones (40 adult females of each clone). In addition, the contents of five spore/control jars was

added into each aquarium, resulting in a total of 360 Daphnia in each aquarium. Every day

200 mL water was added (with or without diazinon), reaching a final volume of 8 L after 22

days. Daphnia were daily fed with 0.3 mg C L-1

S. obliquus. Magnetic stirrers were turned

one minute per day to provide a uniform distribution of parasite spores, pesticide and algae.

Under same experimental conditions, the diazinon degrades at a rate of 2.8 % per day

(supplemental information S2). To compensate for that loss, a corresponding amount of

diazinon/acetone was added to the aquaria every 2nd

week. The experiment lasted 99 days. On

day 57, all animals of one control aquarium died because of a handling error.

Data collection

Daphnia were first sampled on day 29 and then again every second week (i.e., day 43, 57, 71,

85 and 99). From each aquarium three replicate samples were collected after stirring the water

using a Plexiglas tube (diameter: 3 cm) and Daphnia were preserved in 100 % EtOH

(infection estimates from the ethanol preserved samples are similar to those estimated from

live samples; J.W. pers. observation, see also Wolinska et al. 2011). The water level of each

aquarium was refilled to 8 L. The number of infected and uninfected adult females was

counted under a stereomicroscope at a magnification of ×16.0 (Leica M 205 C) to assess

Daphnia density and parasite prevalence. In addition, from the samples collected at day 29

and 99, from each aquarium one subsample of 24 randomly selected adult females were

individually assessed for their body size (from top of the eye to the base of the spine),

fecundity (presence/absence of eggs in the brood pouch), infection status (presence/absence of

parasite spores in the body cavity) and taxon/clonal identity. Scoring presence/absence of

eggs in the brood pouch of Daphnia preserved in EtOH can lead to underestimation of

fecundity, but since all samples were treated the same, a possible underestimation is unlikely

to be treatment-biased.

Daphnia were genotyped at three diagnostic microsatellite loci (SWID 12, SWID 14 and

SWID 10, Brede et al. 2006). The DNA was extracted using HotSHOT DNA extraction

method (Montero-Pau et al. 2008). PCR was carried out using the following cycle profile: an

initial step of 15 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 1.5 min at 54 °C and

1 min at 72 °C; with a final extension step of 30 min at 60 °C. The total reaction volume was

12 μl, containing 6 μl Qiagen multiplex PCR mix, 3.4 μl water, 0.6 μl primer mix and 2 μl

DNA template. PCR products were analysed on a sequencer (Applied Biosystems 3730xl


48

DNA Analyzer). The microsatellite peaks were scored using Applied Biosystems

GeneMapper® software version 4.0.


Statistical analyses were performed using linear models in R 2.10.1 for Windows (R

Development Core Team, 2008). Our model simplification proceeded as follows: from each

initial model we removed higher order terms in order, starting with the least significant term.

We tested whether the exclusion of a term resulted in a significantly poorer model using

likelihood ratio tests as recommended by Crawley (2007). All relevant factors belonging to

the minimum adequate model for each analysis are summarized in Table 1. For the linear

mixed effects models, the highest posterior density (HPD) intervals and associated p-values

for fixed effect parameters were derived using Markov Chain Monte Carlo (MCMC)

sampling methods with the pvals.fnc command of the LANGUAGER library (Baayen et al.

2008). All data satisfied the assumptions of parametric tests (inspected with q-q plots and

plots of residual against fitted values). In the analyses of parasite prevalence and host

susceptibility, only data from aquaria exposed to parasite treatment were included. To correct

for repeated measures we included sampling date as random factor in all the performed

analysis (Pinheiro & Bates 2000; Crawley 2007).

Host density, parasite prevalence and host susceptibility. To examine treatment effects on

host density (measured as the number of adult females/L) or parasite prevalence (proportion

of infected adult females), two separate linear mixed effects models (with the lmer function)

were employed, using either host density or parasite prevalence as dependent variable,

exposure to parasite (yes/no; only in the host density model) and to pesticide (yes/no) as fixed

effects and sampling date as well as aquarium as random effects. To test for the influence of

pesticide addition on the susceptibility of certain taxa, a generalized linear mixed effects

model (glmer function) was used, with an individual being infected (yes/no) as binomial

dependent variable, exposure to pesticide (yes/no) and taxon identity as fixed effects and

sampling date as well as clone (nested within taxon) as random effects.

Host taxon and clonal composition. To test for treatment effects on taxon or clonal

composition (Agresti 2002), two separate log-linear models were used (with the glmer

function, family= poisson distribution). The counts of individuals were treated as dependent

variable, exposure to parasite (yes/no) and to pesticide (yes/no) and taxon/clonal identity as

fixed effects and sampling date and aquarium as random effects. For these and the following

tests, only data collected at day 29 and 99 were analysed.


49

Host fecundity and body size. A generalized linear effects model (with the glmer function)

was conducted with the presence of eggs (yes/no) as dependent variable to test for treatment

effects on host fecundity. Differences in the body size of adult Daphnia were examined using

a linear mixed effects model, with size of adult females as dependent variable. In both

models, exposure to parasite (yes/no) and to pesticide (yes/no) and taxon identity were treated

as fixed effects, whereas the sampling date and aquarium were random effects.

Results

Host density, parasite prevalence and host susceptibility. The density of adult females was

significantly reduced by exposure to parasites, but not to pesticides (Table 1; Fig. 1).

Although pesticide exposure had no effect on total parasite prevalence (Table 1; Fig. 2),

susceptibility of the different taxa was affected (see significant pesticide-by-taxon interaction;

Table 1). At day 29, D. galeata × longispina hybrids exposed to pesticides were more

infected than non-exposed hybrids, but this effect was not visible in D. galeata (Fig. 2).

Host taxon and clonal composition. Taxa and clonal composition changed when parasites or

pesticides were added to the aquaria (see the significant parasite-by-taxon/clone and pesticide-

by-taxon/clone interactions; Table 1). At day 29, hybrids were more abundant than D. galeata

across all treatments (Fig. 3). At day 99, however, the no hybrid taxon could be detected in all

the parasite and parasite + pesticide treatments (Fig. 3). At this same time point, six clones

Fig. 1: Changes in mean density (number of

adult females per litre ± SE) across the

different treatments: control (white circles),

pesticide (white squares), parasite (black

circles), and parasite + pesticide (black

squares).

Fig. 2: Changes in mean parasite prevalence

(proportion of infected adult females ± SE)

across the different treatments: parasite (black

circles), and parasite + pesticide (black squares).

Parasite prevalence per taxa at day 29 is shown

(empty symbols). Samples from day 99 were

also genotyped, but the community consisted of

D. galeata only (see Fig. 3).


50

were still present in the control treatment, five clones remained in the pesticide treatment, and

the same two D. galeata clones (AMM_47 & 66) remained in the parasite and

parasite + pesticide treatments (Fig. 4).

Fig. 3: Changes in mean taxon frequency (± SE) across the different treatments: (A) control, (B) pesticide, (C)

parasite, and (D) parasite + pesticide (grey: D. galeata; white: D. galeata × longispina hybrids).

Fig. 4: Changes in mean clonal frequency (± SE) across the different treatments: (A) control, (B) pesticide, (C)

parasite, and (D) parasite + pesticide. D. galeata clones are shown in grey (AMM_10, 47, 66), and D. galeata ×

longispina hybrid clones are shown in white (AMM_30, 39, 45).


51

Host fecundity and body size. In the parasite and parasite + pesticide treatments, females

were more fecund than in those treatments not exposed to parasites (Table 1; Fig. 5).

D. galeata were more fecund when additionally exposed to pesticide whereas hybrids were

more fecund when not exposed to pesticides (see significant pesticide-by-taxon interaction;

Table 1). Daphnia exposed to parasites were generally smaller than unexposed Daphnia

(Fig. 6). In addition, hybrids were smaller than D. galeata, but only in those treatments not

exposed to parasites (see significant parasite-by-taxon interaction; Table 1, Fig. 6).

Discussion

Parasite exposure had a high impact on the experimental Daphnia communities. As expected,

parasites reduced Daphnia host density, a finding consistent with other Daphnia-

microparasite experimental systems (Ebert et al. 2000; Decaestecker et al. 2005). It was

previously shown that Metschnikowia-infected Daphnia had fewer offspring and a higher

mortality rate than uninfected ones (e.g., Duffy & Hall 2008; Lohr et al. 2010). Conversely, in

our experiment, treatments involving parasite exposure resulted in a higher proportion of

fecund females. Given that fecundity of Daphnia is strongly dependent on resource

availability (Lampert 1978), this result could be due to parasites lowering Daphnia density

and consequently freeing up more resources per host individual.

Fig. 5: Changes in mean proportion of fecund

females (± SE) across the different treatments:

control (white circles), pesticide (white squares),

parasite (black circles) and parasite + pesticide

(black squares).

Fig. 6: Changes in mean body size (± SE) of

adult females across the different treatments:

control (white circles), pesticide (white

squares), parasite (black circles) and

parasite + pesticide (black squares).

21 99 21 99


52

Table 1: Statistical models and test statistics for the effect of parasites and pesticides on Daphnia host individual

traits and community composition. We used three kinds of linear models: linear mixed effect model (lmer),

generalized linear mixed effect model (glmer) and log-linear model. Details concerning the model construction

and simplification are provided in the text.

Dependent variable

(variable type) Model Relevant factors Estimate SE t

a/z

b MCMCp

a/p

bc,

Host density lmer parasite -66.670 14.430 -4.662 0.001

(continuous)

Parasite prevalence lmer pesticide 0.043 0.103 0.418 0.676

(continuous)

Host susceptibility glmer pesticide -0.640 0.262 -2.441 0.015

(binomial) taxon 1.708 0.446 3.834 < 0.001

pesticide × taxon 1.117 0.435 0.435 0.007

Taxon frequency log-linearc parasite × taxon -1.714 0.143 -11.979 < 0.001

(counts of individuals) pesticide × taxon -0.281 0.131 -2.143 0.032

Clonal frequency log-linearc parasite × clone

d χ

2:190.83 < 0.001

(counts of individuals) pesticide × cloned χ

2:25.617 < 0.001

Host fecundity glmer parasite 0.693 0.340 2.039 0.041

(binomial) pesticide 0.388 0.356 1.089 0.276

taxon -0.257 0.477 -0.539 0.590

pesticide × taxon -0.719 0.357 -2.017 0.044

Host size lmer parasite -0.223 0.033 -6.729 0.006

(continuous) pesticide -0.058 0.029 -1.995 0.001

taxon -0.092 0.035 -2.619 0.046

parasite × pesticide 0.106 0.040 2.674 0.590

parasite × taxon 0.126 0.030 4.246 0.004 a for lmer b for glmer and log-linear model (except for the clonal frequency model, where χ2 are presented) c single effects not included in the table because they are not meaningful in log-linear models d the overall effect of parasite/pesticide on clonal composition was tested by removing the corresponding interaction term and

comparing this new model with the model including all two-way interactions. ΔDf= 5

Parasites directly altered Daphnia species composition. Although two taxa coexisted in a

parasite-free environment, D. galeata × longispina hybrids went extinct under parasite

pressure. Differences in the susceptibility of the studied taxa may be the cause. At day 29,

D. galeata were less infected than hybrids, despite previous findings that Metschnikowia

infected different taxa and clones to the same extent (Stirnadel & Ebert 1997; Wolinska et al.

2009; Yin et al. 2011). Our result that hybrids had higher parasite prevalence may support the

„hybrid susceptibility‟ hypothesis (Fritz et al. 1994) or be explained by parasite-driven,

negative frequency-dependent selection (Hamilton 1980). At day 29, the more frequent taxon

(hybrids) were the most infected, and at the end of the experiment, hybrids had disappeared


53

and D. galeata were common. At this point, D. galeata had higher prevalence of infection.

Such a mechanism has been shown to be responsible for the promotion of genetic diversity in

Daphnia and other invertebrate host populations (Duffy 2008; Wolinska & Spaak 2009;

Berenos et al. 2011; King et al. 2011). As we did not look at the parasite genetic variation, we

cannot confirm, that the observed pattern is due to coevolution. Alternatively, the parasite

strain used in the experiment may have adapted to the hybrid taxon, as the parasite was

maintained for two years on a hybrid clone. This is unlikely, however. Yin et al. (2011) did

not find a different infection rate among D. galeata and hybrid clones, when exposed to a

parasite strain raised on a hybrid Daphnia clone. Also, Duffy & Sivars-Becker (2007) found

no evidence for Metschnikowia adaptation towards different host clones after five host

generations.

Contrary to our expectations that pesticide exposure would decrease Daphnia density due to

delay in reproduction, decrease in number of offspring or reduced survival (Sanchez et al.

1998), density did not differ from the control treatment. This could be the result of pesticide-

tolerant clones replacing sensitive clones, a mechanism described as pollution-induced

community-tolerance hypothesis (Blanck & Wangberg 1988). This mechanism should not

affect density, but only taxon and clonal composition. Indeed, in our experiment both taxon

and clonal composition were influenced by pesticide exposure. The observed increase in

D. galeata frequency in the aquaria containing pesticides, is plausible with respect to the

finding that D. galeata had higher fecundity in the pesticide treatments whereas the hybrid

fecundity was marginally lower when exposed to the pesticide. In contrast to our expectations,

the pesticide did not affect the number of the total infected adult females. However, we

observed that the hybrids (but not D. galeata) were more infected when exposed to the

pesticide compared to unexposed hybrids. Although there are examples that hybrids can be

more susceptible to parasites (i.e., „hybrid susceptibility‟ hypothesis of Fritz et al., 1994), the

sensitivity of hybrids and parental taxa towards pesticides has not previously been compared.

Our observations of reduced hybrid fecundity under pesticide exposure, as well as the higher

susceptibility of hybrids, indicate that hybrids are more affected by both environmental

stressors, parasites and pesticides. This combination of negative responses to both stressors

could lead to the observed higher infection rate under pesticide exposure.

Studies tracking genetic changes of a community exposed to a pesticide as well as studies

examining multiple stressors, especially those taking competition within and among taxa into

account are very rare. The here employed experimental design is a potentially promising tool

for a verification if effects on individual levels are also observed in communities as well as

help to interpret more complex observations of field studies (Marcogliese & Pietrock 2011).


54

Although previous studies did observe synergistic effect of the pesticide and parasite stressors

on individual level (e.g., Coors & De Meester 2008), this is a first study using a mixture of

taxa and several clones to investigate the relationship between the two stressors. Under the

tested experimental conditions (using pesticide concentration found in natural habitats)

parasite exposure was a stronger selection factor than pesticide exposure. However, since

there are often concentration effects of stressors (e.g., Little & Killick 2007; Ben-Ami et al.

2008; Oda et al. 2011), a larger experiment using several pesticide concentrations and spore

loads would better verify whether interaction effects were present. Nevertheless, our study

revealed that parasites, as well as pesticide exposure, alter the structure of Daphnia host

communities.

The observed changes in a Daphnia community are most likely due to varying sensitivity of

the taxa/clones to the biotic and abiotic stressors. Communities exposed to stress lose less-

tolerant taxa and clones, resulting in a decrease of diversity (Altizer et al. 2003; Relyea &

Hoverman 2008). Such a loss in diversity may make the community more vulnerable to

additional stress. Given that parasites are ubiquitous and anthropogenic stressors are

becoming increasingly pervasive, our study shows that the genetic diversity of Daphnia

populations might be at higher risk. Given Daphnia‟s key-role in aquatic food webs,

converting primary production to fish food, this may lead to a higher stress on aquatic

ecosystems.

Acknowledgment

We thank Esther Keller and Markus Möst for helping sampling the aquaria and Christine

Dambone for providing the Daphnia food. The Genetic Diversity Centre at ETH Zurich

allowed us to use their genotyping facility. Further we thank Christoph Tellenbach and Blake

Matthews for intense statistical discussions. The manuscript was substantially improved by

comments from Kayla C. King. This research was funded by the ETH Board (CCES-

GEDIHAP).

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58

Web appendix

S1: Sub-lethal effects of diazinon on Daphnia galeata

Material & Methods

We explored sub-lethal effects of diazinon on two Daphnia galeata clones following the

OECD guidelines for reproductive test (OECD/OCDE 1998). We tested five different

concentrations of diazinon (0.00256, 0.0064, 0.016, 0.04 and 0.1 µg/L). As controls, we used

1) lake water without diazinon and 2) lake water without diazinon but containing the same

amount of the solvent acetone as in the treatments. Nominal exposure concentrations of

diazinon were verified with LC-MS/MS (liquid chromatography-mass spectrometry/ mass

spectrometry) as described in Kretschmann et al. (2011). As measured diazinon

concentrations were within ± 20 % of the nominal concentrations, for data analysis nominal

concentrations were used. We distributed the offspring (<24 h old) of the third clutch singly in

50 mL jars with five different diazinon concentrations and two controls. Each treatment was

replicated 10 times, resulting in 140 experimental units (7 concentrations × 2 clones × 10

replicates). Under experimental conditions (20 °C, 16:8 light-dark photoperiod) the medium

was changed every third day and the Daphnia were fed with 1 mg C/L of the algae

Scenedesmus. The following parameters were recorded: survival after 21 days (experiment

was terminated then), time of first offspring released and number and body size of the

offspring of the first clutch.

To test for differences in the life history traits we conducted a univariate ANOVA using SPSS

19.0 with the life history trait (number of survived individuals, time of first offspring released

and number and body size of the released offspring as dependent factor, the different diazinon

concentrations as a fixed factor and clones as a random factor. All data were tested for the

assumptions of parametric tests (inspected with q-q plots and plots of residual against fitted

values) and, if necessary, transformed. The two sided Dunnett t-test treats one group as a

control and compares all other groups against it. From that we were able to figure out which

concentrations are different from the control and, consequently, determine the threshold

concentration.

Results

Different diazinon concentrations significantly affected survival rate (F= 13.35, df= 6,

p= 0.003), the number of offspring (F= 6.68, df= 6, p= 0.018), the size of offspring (F =4.66,

df =6, p =0.042) and the time of first offspring released (F= 5.79, df= 6, p= 0.025). We found


59

a clone effect on the average offspring size (F= 45.19, df= 1, p< 0.001) and the number of

offspring of the first clutch (F= 7.386, df= 1, p= 0.030).

Survival was only affected at the concentrations 0.016µg/L and 0.1µg/L. The time to release

the first juveniles was delayed and the number of offspring in the first clutch decreased when

the concentration was equal to or higher than 0.04µg/L (first juveniles released: 0.04 µg/L

(p= 0.024), 0.1 µg/L (p<0.001); number of offspring in first clutch: 0.04 µg/L (p= 0.003),

0.1 µg/L (p= 0.015)).

Fig. S1: The effect of different diazinon concentrations on survival rate (A), the number (B) and the time of

offspring of the first clutch released (C) and the size of offspring of the first clutch (D).

Conclusions

For further experiments the diazinon concentration of 0.1 µg/L was chosen. This

concentrations was above the threshold and showed significant differences compared to the

control for all measured traits.


60

S2: Degradation of diazinon under experimental conditions

Material & Methods

Measurements of diazinon degradation was carried out with 14C labelled diazinon

([Pyrimidinyl-6-14C]-Diazinon, 99.21 % radiochemical purity, supplied by American

Radiolabeled Chemicals, St. Louis, USA) under the same experimental conditions (20 °C,

16:8 light-dark photoperiod) and in the same room as the main experiment itself. Eight litre of

lake water (filtered through a membrane of 0.45µl) were filled into a 20 litre aquaria and

5 µg/L 14C labelled diazinon was added (concentration high enough to be measured with the

method described and below the limit of the permitted maximum dose 20 kBq). During

14 days, water samples (1 mL) were taken every 1 to 2 days and 10 mL of Ecosicint A

cintillation cocktail (National Diagnostics, Hessle, UK) immediately added to a water

subsample. Following the protocol of Ashauer et al. (2006), radioactivity was quantified with

liquid scintillation counting (Beckman LS6000 TA Liquid Scintillation Counter, Beckman

Instruments Inc., Fullerton, CA, USA) and blank controls were used to correct for background

activity.

Results

Measured diazinon concentrations over 14 days as well as the best fit function is shown in

Fig. S2. Diazinon is degrading 2.8 % per day.

Fig. S2: Degradation rate of 14C labelled diazinon during 14 days. Best fitted regression line and it‟s equation

are included in the graph.


61

Conclusions

To compensate for that loss, a corresponding amount of diazinon/acetone was added to the

aquaria every 2nd

week.

Acknowledgments

We thank Roman Ashauer and Andreas Kretschmann for helpful advices and measuring the

diazinon concentrations.

Reference supplementary material

Ashauer, R., A. Boxall, and C. Brown. 2006. Uptake and elimination of chlorpyrifos and

pentachlorophenol into the freshwater amphipod Gammarus pulex. Arch. Environ.

Contam. Toxicol. 51:542-548.

Kretschmann, A., R. Ashauer, T. G. Preuss, P. Spaak, B. I. Escher, and J. Hollender. 2011.



OECD/OCDE. 1998. OECD Guidlines for testing of chemicals, Daphnia magna, reproduction

test.21.

63

Chapter 5

Evolutionary and environmental effects of pesticide exposure on

parasite fitness and virulence

Claudia C. Buser, Justyna Wolinska & Piet Spaak

(Journal of Evolutionary Biology, in review)

Abstract

Parasite fitness and virulence can be altered by the environment, especially if conditions

persist for several parasite generations. In this study, we compared the evolutionary and

environmental effects of pesticide exposure to parasite fitness and virulence using the

planktonic crustacean Daphnia galeata and the yeast Metschnikowia sp. Environmental

effects of pesticide exposure resulted in host genotype specific interactions concerning

parasite spore load. Additionally, host fecundity was reduced. Under evolutionary pressure of

pesticide exposure, parasites evolved towards higher spore loads and lower virulence. Since

the concentration of pesticides reflected natural concentrations, our results indicate that

pesticide pollution might have an important impact on disease dynamics in aquatic systems.

Key words

Daphnia, experimental evolution, Metschnikowia sp., parasite, pesticide

Environmental-dependent parasite evolution Chapter 5

64

Introduction

The effect of the environment on host-parasite interactions depends on the sensitivity of hosts

to particular environmental conditions. Consequently, parasite fitness (measured, for example,

as infection rate and/or spore load: Ebert 1998; Jensen et al. 2006; Vale & Little 2009) and

virulence (measured as mortality of infected hosts and/or host sterilisation: Garnick 1992;

Poulin & Combes 1999; Alizon et al. 2009) depend on the environment in which the host is

living.

Parasite fitness can increase in stressful host environments due to reduced host immune

function (Sheldon & Verhulst 1996; Moret & Schmid-Hempel 2000). Empirical studies have

shown that stressed hosts are more frequently infected (Chu & Hale 1994; Oppliger et al.

1998; Rohr et al. 2008a) and parasites have higher spore production (Bedhomme et al. 2004;

Yin et al. 2011). Conversely, other studies have shown that parasites experience a fitness loss

when hosts are stressed, due to reduced resources available for parasite growth (Pulkkinen &

Ebert 2004; Tschirren et al. 2007; Hall et al. 2009). Parasite virulence can also be further

influenced by all variety of environmental conditions (reviewed in Thomas & Blanford 2003;

Lazzaro & Little 2009; Wolinska & King 2009). Generally, in environments which are

stressful for hosts, parasite virulence increases, as observed under higher host mortality rates

(Jokela et al. 1999; Coors & De Meester 2011).

The impact of environmental changes on parasite evolution could be especially important if

the altered conditions persist for several parasite generations. Aquatic habitats in particular are

extensively impacted by human activity, in ways that may affect the evolution of parasites.

For example, pesticide runoff from agriculture into water bodies is likely to select for fast-

growing, early-transmitted parasites (Mennerat et al. 2010) to overcome a short host life span

(Nidelet et al. 2009). Indeed, for obligate killers, parasite virulence can evolve to increase

when extrinsic host mortality is high (Ebert & Weisser 1997). Pesticides have been shown to

affect parasite fitness and virulence across many host-parasite systems; for example, in

parasites of amphibians (Gendron et al. 2003; Rohr et al. 2008b; King et al. 2010), fish

(Kreutz et al. 2010), oysters (Chu & Hale 1994) or planktonic crustaceans (Coors & De

Meester 2008; Coors et al. 2008; Coors et al. 2009; Coors & De Meester 2011). However, the

evolutionary consequences of long-lasting host exposure to pesticides on parasite fitness and

virulence have not been studied in great detail.

In the present study, we investigated environmental and evolutionary effects of host exposure

to pesticides on parasite fitness and virulence. We used the planktonic crustacean

Daphnia galeata (G. O. Sars, 1863) and the yeast Metschnikowia sp. (family:

Chapter 5 Environmental-dependent parasite evolution

65

Hemiascomycetes; Wolinska et al. 2009) as a host-parasite model system. Needle-like

ascospores accumulate in the body cavity and are released only after host death (Green 1974).

As a stressor, we applied diazinon, which is a commonly used pesticide (Konstantinou et al.

2006; Singer et al. 2010). Standard toxicological tests on D. magna showed that

environmental effects of diazinon exposure cause a delay in reproduction and earlier death, as

well as a decrease in offspring number and body size (Sanchez et al. 1998). D. magna is a

pond species whereas D. galeata inhabits large, permanent lakes (e.g., Keller et al. 2008;

Petrusek et al. 2008; Yin et al. 2010). Such habitats are frequently affected by pesticide

pollution (e.g., Konstantinou et al. 2006; Relyea & Hoverman 2006). Moreover, lake Daphnia

population are commonly infected by a variety of parasite species; with the parasites used in

the present study, Metschnikowia sp., being particularly common (Caceres et al. 2006; Duffy

et al. 2010; Wolinska et al. 2011). Metschnikowia sp. reduces D. galeata reproduction and life

span (Lohr et al. 2010). In addition, parasite fitness (i.e. infectivity and spore production) is

higher in D. galeata exposed to a natural stressor (a predator signal, Yin et al. 2011).

However, the effect of diazinon or any other pollutant on Metschnikowia sp. is not known.

Pesticide exposure has been already shown to increase parasite infectivity (Coors & De

Meester 2008, 2011), but to conversely decrease parasite spore load (Coors & De Meester

2011) in the D. magna-Pasteuria ramosa bacteria system. However, the above mentioned

studies investigated only the environmental effect of pesticide exposure over a maximum of

21 days. Here, we studied both the environmental and the evolutionary effects of pesticide

exposure on parasite fitness and virulence. We expected that parasites raised on hosts exposed

to the pesticide for several generations would evolve adaptations to that stressor, showing

higher infectivity and spore production than the parasites which has been raised in a control

treatment, when environmental effect of the pesticide was tested.


Origin and care of the host-parasite system and the pesticide

Two different Daphnia galeata clones were selected from a larger collection of clones

isolated in 2008 from the lake Ammersee in Germany (clone_10 and clone_66; taxon and

multilocus genotype assignment were based on 15 microsatellite loci, for methods see Yin et

al. 2010) and used as the experimental hosts. The clones were kept under standard laboratory

conditions (20 °C, 16:8 light-dark photoperiod, fed with 1.0 mg CL-1

of the green algae

Scenedesmus obliquus) for two years. The horizontally-transmitted yeast Metschnikowia sp.

was isolated from the same lake, also in 2008 (Yin et al. 2011) and used as model parasite.

Before the experiment, Metschnikowia sp. isolate was raised for 99 days in independent ten


66

experimental Daphnia communities (starting with a mixture of three D. galeata and three

D. galeata × longispina clones isolated from Ammersee, including clone_10 and clone_66).

The Daphnia communities were set up in ten 20L aquaria containing lake water filtered

through a membrane of 0.45µL. In five of those aquaria, Daphnia were exposed to the sub-

lethal dose of diazinon (CAS 333 41 5, 99.5 % purity, Ultra Scientific Analytical Solution N.,

Kingstown RI, USA), 0.1μg/L (similar diazinon concentrations have been measured in natural

systems; e.g. Konstantinou et al. 2006; Wittmer et al. 2010). In the other five aquaria, which

served as a control, Daphnia were exposed to the corresponding amount of the solvent

acetone. After day 99, only two D. galeata clones (clone_47 and clone_66) remained whereas

clone_10 disappeared in both treatments (Claudia C. Buser, Piet Spaak & Justyna Wolinska,

unpublished data). For the two experimental spore solutions, a mixture of heavily-infected

Daphnia collected from each aquaria of the corresponding treatment (i.e., with or without

diazinon) were ground up.


We conducted a life-history experiment with a fully crossed design, in which parasites

(Metschnikowia sp.) were raised for 99 days either on a host community exposed to diazinon

(hereafter referred to as PD: parasite-diazinon), or on a host community exposed to control

conditions (PC: parasite-control). In addition to using these two different parasite sources (PD

or PC), experimental Daphnia were either exposed to diazinon or to the solvent acetone

(control). This resulted in twelve experimental treatments: 2 (host clone) × 3 (parasite history:

PD, PC and no parasites: noP) × 2 (diazinon and no diazinon). The four treatments not

exposed to parasite spores were replicated 10 times, whilst the other eight treatments were

replicated 20 times, for a total of 200 experimental units.

Experimental Daphnia (younger than 24h, third brood of second generation) were kept

individually in 5 mL lake water with or without diazinon. On day 5, 80 individuals (40 from

each clone) were exposed to the parasites raised with diazinon (PD), and 80 to parasites raised

without diazinon (PC), while the remaining 40 animals were inoculated with the appropriate

amount of uninfected crushed Daphnia (noP), to avoid possible bacteria or alarm cue effects.

The concentration of spores in the parasite treatments was 450 spores/mL (similar as in Duffy

& Sivars-Becker 2007). Daphnia were fed daily with 1.0 mg CL-1

S. obliquus except on day 7

and 9, when no food was added to promote spore uptake (Hall et al. 2007). On day 8, 5 mL of

fresh diazinon or control medium was added, and on day 10, the Daphnia were transferred to

30 mL of the corresponding medium. From then on, the media was changed every third day

throughout the experiment. Juveniles were counted and removed daily. The body length (from

top of the eye to beginning of the spine) of three juveniles was measured from the first clutch


67

of each female. Dead Daphnia were ground up and the number of spores was determined

under a stereo microscope as described in Lohr et al. (2010), using a Bürker counting

chamber. The experiment lasted 44 days until all animals had died.


All statistical analyses were performed using SPSS 17.0. Twenty-eight individuals died

before day 10 (i.e., when infections first became visible) and were excluded from the

analyses. Differences in parasite infectivity were analysed using a generalized linear model,

and parasite spore load per lifetime was analysed using ANOVA; with diazinon (no/yes),

parasite history (PD/PC) and clonal identity as fixed factors. In the ANOVA analyses of host

life-history traits (survival, total number of juveniles, age at reproduction and 1st clutch

juvenile size), the same fixed factors were used with the exception that parasite treatment

included the third level (i.e. noP: no parasites, in addition to PD/PC), and only infected

animals were included in the parasite-exposure treatments. A Tukey HSD test was used to

determine if PD and PC treatments differed. All data were tested for the assumptions of

normality and, if necessary, transformed using the Rankit function (Conover & Iman 1982).

Results

Parasite fitness traits. Neither parasite history (PD, PC; i.e. evolutionary effects), nor

diazinon presence in the Daphnia medium (i.e. environmental effects), had a significant

influence on parasite infectivity (Fig. 1a). However, there was a main effect of clone, with

clone_66 being more frequently infected than clone_10 (Table 1; Fig. 1). Parasite spore load

was affected by evolutionary effects of diazinon exposure: spore load was higher when

Daphnia was exposed to parasites raised with diazinon (PD) than in Daphnia exposed to

parasites raised in control media (PC; Fig. 1b). Although there was no direct environmental

effect of diazinon exposure, parasite spore load was higher under diazinon exposure for

clone_66, whereas for clone_10 parasite spore load was higher without diazinon exposure

(see significant diazinon × clone interaction; Table 1).

Host traits to determine parasite virulence. Infection generally reduced Daphnia survival

(Fig. 2a, Table 1). However, Daphnia exposed to parasites evolved under diazinon exposure

(PD) died later than Daphnia exposed to parasites evolved under control conditions (PC);

Tukey test: p= 0.001. Environmental diazinon exposure did not affect Daphnia survival, but

the uninfected clone_66 died earlier in the diazinon treatment (parasite exposure × diazinon ×

clone interaction; Table 1).


68

Similarly, the production of juveniles was also reduced in the parasite-exposed Daphnia (Fig.

2b, Table 1). The evolutionary effect was important here as well: Daphnia exposed to

parasites evolved under diazinon exposure (PD) produced more offspring than those exposed

to parasites evolved under control conditions (PC); Tukey test: p= 0.02. Environmental

diazinon exposure resulted in lower production of offspring by the uninfected clone_66,

whereas uninfected clone_10 produced more offspring then (parasite exposure × diazinon ×

clone interaction; Table 1). This result reflects the clonal differences concerning survival (see

above).

Other host traits. Neither the reproductive age of Daphnia, nor the size of juveniles was

dependent on evolutionary or environmental effects of diazinon exposure (Table 1). However,

Fig. 1: Reaction norms (mean ±

SE) of parasite fitness traits (A:

proportion infected, B: spore

load) when exposed to parasite

raised with diazinon (PD) or

parasite raised in control media

(PC) for clone_10 (black circles)

and clone_66 (grey triangles).

Fig. 2: Reaction norms (mean

± SE) of host traits (A:

survival, B: total number of

juveniles, C: age at first

reproduction, D: juvenile size)

when exposed to parasite

raised with diazinon (PD),

parasite raised in control

media (PC) or no parasite

(noP) for clone_10 (black

circles) and clone_66 (grey

triangles).


69

there were clonal differences for both traits: clone_10 reproduced earlier (Fig. 2c) and

produced smaller juveniles (Fig. 2d), but the difference in size disappeared in juveniles from

infected mothers (parasite exposure × clone interaction; Table 1).

Table 1: Results of a generalized linear modela and several ANOVA‟s

b describing the influence of parasite,

medium and clone identity on the different dependent variables.

Dependent variable Source df X2a/F

b p

Parasite infectivity parasite history (PH) 1 0.358 0.550

diazinon (D) 1 0.124 0.725

clone (C) 1 3.981 0.046

PH × D 1 0.246 0.620

PH × C 1 0.033 0.856

D × C 1 0.056 0.813

PH × D × C 1 0.273 0.601

Parasite spore load parasite history (PH) 1 13.212 <0.001

diazinon (D) 1 1.136 0.289

clone (C) 1 0.105 0.747

PH × D 1 0.371 0.544

PH × C 1 2.470 0.119

D × C 1 7.592 0.007

PH × D × C 1 0.030 0.864

Survival parasite exposure (PE) 2 56.093 <0.001c

diazinon (D) 1 0.490 0.485

clone (C) 1 2.728 0.101

PE × D 2 0.704 0.496

PE × C 2 2.600 0.077

D × C 1 0.010 0.920

PE × D × C 2 3.328 0.038

Total no. of juveniles parasite exposure (PE) 2 51.282 <0.001c

diazinon (D) 1 5.325 0.022

clone (C) 1 41.014 <0.001

PE × D 2 0.199 0.820

PE × C 2 0.589 0.556

D × C 1 0.011 0.915

PE × D × C 2 4.424 0.014

Age at reproduction parasite exposure (PE) 2 0.432 0.650

diazinon (D) 1 0.873 0.352

clone (C) 1 19.164 <0.001

PE × D 2 0.003 0.997

PE × C 2 0.339 0.713

D × C 1 0.131 0.718

PE × D × C 2 2.324 0.102

1st clutch juvenile size parasite exposure (PE) 2 0.088 0.916

diazinon (D) 1 2.782 0.098

clone (C) 1 4.942 0.028

PE × D 2 0.216 0.806

PE × C 2 3.883 0.023

D × C 1 2.376 0.126

PE × D × C 2 0.461 0.632 c PC and PD differ (Tukey test: for exact p-values see the main text)


70

Discussion

The parasite strains that were confronted with long-lasting diazinon exposure (PD) produced

significantly more spores than those raised on a host community exposed to control

conditions (PC). In contrast, such an effect was not observed under environmental (short-

lasting) exposure to pesticides. Apparently, the parasites had evolved during the long-lasting,

pre-culturing conditions. Although in our study the parasites were evolving on host

communities originally consisting of six different clones, by the end of the 99 days only two

clones (clone 66 and 47) were left. However, as the clonal composition of experimental host

communities did not differ between the communities exposed and unexposed to pesticides

(Claudia C. Buser, Piet Spaak & Justyna Wolinska, unpublished data), we are confident that

the observed evolutionary effects are due to the pesticide treatment and not due to clonal

differences. Therefore, diazinon might have selected for parasite genotypes with high spore

production in the pre-culturing conditions. Indeed, the parasite selection experiment for either

pesticide or control conditions was likely to be started with a mixture of parasite strains. This

is because a parasite was originally isolated from a field-infected Daphnia. Co-infections with

multiple strains and species are a rule and not an exception in nature (e.g., Read & Taylor

2001), and Daphnia are often co-infected by several strains of a given parasite taxon (Ben-

Ami 2008). Similarly to our results, an increase in parasite reproductive success was

commonly observed in the field of parasite control. For example, it was suggested that drug

therapy should eventually lead to more fertile nematodes (i.e., higher parasite fitness), and

therefore can act as a selection pressure on parasite evolution (Leignel & Cabaret 2001). In

contrast, malaria protozoan parasites adopted reproductive restraint when stressed by drugs

(Reece et al. 2010).

Although there was no main effect of environmental (i.e. short-lasting) diazinon exposure on

parasite spore production, there was a difference between the two tested clones: one clone

produced more spores without pesticide stress, whereas the other clone produced more spores

under diazinon exposure. A similar effect of host genotype-by-environment interaction on

parasite fitness has been detected across other host-parasite systems (reviewed in Wolinska &

King 2009). The other studied parasite fitness trait, infectivity, also differed among the clones,

but was neither affected by evolutionary nor by environmental pesticide exposure. This

suggests that genetics are more important than host physiology, as potentially influenced by

environment (Vale & Little 2009).

Overall, host fitness was reduced due to parasite exposure; infected Daphnia had lower

reproductive output and shorter lifespan (as in Lohr et al. 2010; Yin et al. 2011). However,

parasite virulence differed according to the parasite evolutionary history: parasites that


71

evolved with diazinon (PD) were less virulent than parasites raised on a host community not

exposed to diazinon (PC). Hosts infected with PD produced more offspring and had higher

survival. Such long-lasting pesticide exposure leading to evolution of lower virulence

contrasts with most studies testing for environmental effects of pesticide exposure, which

have observed an increased parasite virulence, particularly in Daphnia-microparasite systems

(e.g., Coors et al. 2008; Coors & De Meester 2011). Moreover, an increase in parasite

virulence under conditions stressful for hosts is commonly observed in other systems, also as

a consequence of evolutionary treatment. For example, Mennerat et al. (2010) suggested in

their review that intensive fish farming is likely to select for more virulent parasites.

However, repeatedly used imperfect vaccines do not affect parasite virulence at all (Gandon et

al. 2001; Alizon & van Baalen 2005). Thus, most studies testing for environmental or

evolutionary effects on parasite virulence are contrary to our findings. It could be that the

environmental stressor applied here, diazinon, suppresses the transmission of parasites which

consequently leads to lower virulence (e.g., Frank 1996). Indeed, transmission-blocking

vaccines can select for lower virulence if there is competition among the parasite strains

(Gandon et al. 2001).

Parasite fitness and virulence seem to be interconnected; the later the host dies the longer the

duration of spore production. Also in our experiment, parasites showing an elevated spore

load (the PD parasites) caused less host mortality. Diazinon somehow causes a decoupling of

parasite growth and virulence. The conventional wisdom (avirulence hypothesis) suggests that

lower parasite virulence, and therefore increasing parasite fitness, should be favoured by

natural selection as there is more time to exploit hosts (discussed for example in Anderson &

May 1979; Alizon et al. 2009). Alternatively, the “virulence-transmission trade-off”

hypothesis, in which an increased transmission rate increases virulence is now commonly

accepted (Anderson & May 1982; Ewald 1983). Although our pattern of virulence evolution

is more in line with the avirulence hypothesis, it is still possible that virulence decreases and

transmission rate increases assuming a trade-off between those two traits. Alizon & van

Baalen (2005) modelled that an additional activation of the hosts immune response leads to a

decrease in parasite virulence and increase of transmission rate. It is possible that diazinon

could have activated hosts immune response. Organophosphosphate pesticides have been

shown to affect the immune function of invertebrates, fish, and higher vertebrate wildlife

(reviewed in Galloway & Handy 2003).

Previous work has focused on acute effects of pollution on host and parasite fitness, as well as

on parasite virulence (e.g., Coors & De Meester 2008; Coors et al. 2008; Kreutz et al. 2010).

Here, we additionally considered the evolutionary implications of parasite exposure to


72

diazinon (host community exposed for 99 days) by using a mixture of parasite spores from

parasites evolved in five independent experimental lines. We therefore tested for a mean

evolutionary response in the presence/absence of the pesticide. The evolutionary effects of

pesticide exposure (resembling natural concentrations) increased parasite fitness, but

decreased parasite virulence, and might therefore have an important impact on disease

dynamics in aquatic systems. More empirical studies testing the effect of a persistent

environmental stress on parasites would improve our understanding of the evolution of

virulence in nature.

Acknowledgment

We thank Esther Keller and Christine Dambone for assistance in the lab and providing food

for the Daphnia as well as Christoph Tellenbach and Otto Seppälä for statistical advice. The

manuscript was substantially improved by comments from Hanna Hartikainen, Kayla C.

King, Tom Little and Samuel Alizon. This research was funded by the ETH Board (CCES-

GEDIHAP).

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77

Chapter 6

Environment-dependent virulence affects disease spread in host

populations

Claudia C. Buser & Kayla C. King

(In preparation)

Abstract

Parasite virulence can be mediated by variation in the host environment. Empirical work has

documented the environmental conditions under which virulence is expected to increase or

decrease. However, less attention is given to how condition-dependent virulence affects the

epidemic spread of disease in a host population. Here, we examine epidemiological models in

which parasite virulence (i.e., infected host mortality rate) is environment-dependent. We

explicitly explore how eight qualitatively different relationships between parasite virulence

and environmental quality alter the ability of a parasite to invade in “poor” and “good” host

environments. Our results suggest that these relationships can drastically affect whether

highly virulent parasites can spread in the population. The study highlights the consequences

of environmental quality for epidemics. We further discuss how variation in the shape of the

virulence-environment relationships may also affect the strength of selection on natural host

populations across time and space.

Keywords

Condition-dependent virulence, environment, host-parasite interactions

Environment and disease spread Chapter 6

78

Introduction

The spread of disease epidemics is highly dependent on certain host and parasite traits, such

as reproduction, transmission and virulence (e.g., Anderson & May 1981; Anderson & May

1986). Parasite virulence, which can refer to parasite-induced mortality rate or reduction in

host fitness, may play an especially important role (Frank 1996). Disease dynamics can be

affected by changes in virulence caused by relatedness among infecting strains (Frank 1992,

1996), host population structure (Boots et al. 2004) and parasite transmission mode (Day

2001), for example.

Parasite virulence has also been repeatedly shown to be influenced by environmental variation

in a variety of host-parasite systems (Lazzaro & Little 2009; Wolinska & King 2009).

However, the effects on virulence differ depending on the environmental factor of interest.

Most empirical data to date show that infected hosts die more in environments where hosts are

stressed in poor conditions: mortality of infected hosts increase because of exposure to near-

starvation conditions (Jokela et al. 1999; Ferguson & Read 2002; Brown et al. 2003; Tseng

2004; Jokela et al. 2005), pesticides (Coors et al. 2008; Coors & De Meester 2011) and high

temperatures (Mitchell & Read 2005). Virulent effects may also be reduced if hosts are better

able to tolerate infection in improved environmental conditions (Vale et al. 2011).

Conversely, when conditions are good, parasites might also induce a higher host mortality

(see "high food" example in Vale et al. 2011). Overall, the relationship between virulence and

environmental quality can take many directions (see additional examples in Thomas &

Blanford 2003) and likely varies among host-parasite interactions. The exact shape of the

relationship, however, over a range of conditions remains largely speculative and untested for

most systems.

Epidemiological models are increasingly integrating the environment to determine how local

conditions affect epidemiology (e.g., Lafferty & Holt 2003; Hall et al. 2007; Hall et al. 2009),

as well as host-parasite coevolution (Mostowy & Engelstaedter 2011). Indeed, theoretical

studies have found that abiotic conditions can accurately describe some host-parasite

population dynamics in nature (e.g., Hall et al. 2006; Hall et al. 2009). However, little is

known of the influence of condition-dependent virulence on the spread of disease in a host

population. Here, we explored how eight different relationships between virulence and

environmental quality alter the ability of a parasite to invade across a gradient of host

conditions.

Chapter 6 Environment and disease spread

79

Model and Results

We use a „classic‟ differential SI model (Anderson & May 1981), with disease-induced

mortality and mass-action transmission, to follow how different functional relationships

between virulence and environmental quality affect the spread of disease across an

environmental gradient (ranging from bad to good). We are interested in virulence from an

ecological perspective, as the immediate effect of parasite infection on host mortality, not as a

parasite trait.

The change in susceptible host (S) and infected host densities (I) was tracked as follows:

(1A)

(1B)

The rate of change in the densities of susceptible hosts (equation 1A) is dependent on the

number of susceptible and infected hosts, the strength of density dependence on birth rate (c),

the birth rate of uninfected and infected hosts (our parasite does not affect birth rate of the

infected animals) (b), the transmission rate (β) and the natural background mortality (d). The

rate of change in densities of infected hosts (equation 1B) depends on transmission rate (β) as

well as host death, as a result of natural background mortality (d) or mortality due to

infection, otherwise known as virulence (α).

We calculate all possible equilibria: dS/dt=dI/dt=0, the equilibrial density of susceptible hosts

with no infected hosts (disease-free or boundary equilibrium, S*bnd) and persisting with

parasites (the endemic or interior equilibrium, S*int):

(2)

To examine the conditions under which disease can spread in a host population, we calculate

R0, the parasite reproductive ratio. This is the expected number of secondary infections

generated from a single infected host individual. Calculating R0 is a standard approach to

analysing similar epidemiological models (Anderson & May 1986).

(3)

dSSIIScISbdt

dS ))(1)((

IdISIdt

dI

bc

dbSbnd

*

dS

*

int

*

int

*

0S

SR bnd


80

Parasites can spread in a population when R0 > 1. As R0 increases, so does disease spread. By

substituting S*bnd and S

*int from equation 2 into equation 3, we see that R0 increases when β

and b are high and c, α and d are low. It is never the case that d > b, which excludes the

possibility of R0 < 0.

(4)

We assume our environment (E) varies between 0 (“bad”) and 1 (“good”) and affects birth

rate (equation 5) in a positive linear fashion. As the quality of environment improves (as E

approaches 1), more resources are available for reproduction. Therefore, br is the innate birth

rate in an ideal, good environment.

(5)

We set environmental conditions to alter virulence/the mortality rates of infected hosts (α) in

eight mutually exclusive functional relationships (see also Fig. 1):

(6)

We set the following parameters: β= 0.001, c= 0.01 and d= 0.01. The reference birth rate of

hosts (br) is 0.9. Five levels of peak reference parasite virulence are examined to explore

parameter space between 0.01 and 0.2 (1-20 % of infected animals die at peak virulence).

Values of α vary from 0 to αr. As a reminder, the influence of the environment on R0 depends

dbc

dbR

)0

Ebb r

Er 1

32 Er

)816(31

E

r

e

Er 14

35 Err

81661 E

rr

e

)-(14 r7 EE

rr8 +1)-(4 EE


81

on how the environment affects the ability of the parasite to harm the host or on the ability of

the host to resist the parasite. Virulence may increase as the availability of more host

resources can boost parasite exploitation rates (α1: positive linear, α2: positive cube root,

α3: positive sigmoidal). In contrast, virulence may decrease in a good environment from

higher host resistance/tolerance and less conflict over shared resources (α4: negative linear, α5:

negative cube root, α6: negative sigmoidal). Some parasites may have a fitness optimum in an

intermediate environment (α7: negative quadratic). However, others may be successful in a

bad environment because of weakened host resistance (Coors et al. 2008; Rohr et al. 2008)

and also in a good environment as hosts may have higher resource levels to exploit (Seppälä

et al. 2008; α8: positive quadratic).

Fig. 1: Relationships between virulence and host environmental quality, from “bad” to “good”. Environmental

conditions can have a (a) linear, (b) cube root, (c) sigmoidal, or (d) quadratic relationship with virulence.


82

We delineate changes in the reproductive ratio (R0) with environment (E) by calculating the

partial derivative. Changes in R0 with environmental quality depend on two components:

(7)

Component A encompassed the dependence of birth rate on environmental quality and

component B, the dependence of virulence. With increasing birth rate and virulence,

component A and B become smaller, respectively. As component B increases relative to A

disease spread decreases. To examine the specific changes in R0 with improved environmental

quality, we consider the different functional relationships between virulence and

environmental quality: positive relationship (α1, α2, α3), negative relationship (α4, α5, α6),

intermediate optimum (α7) and intermediate minimum (α8). For α1, α2 and α3, parasite spread

reduces as a consequence of improved environmental conditions (Fig. 2a, b, c). The reduction

in R0 is less pronounced in the positive cube root relationship (α2) (Fig. 2b), but more so in the

positive sigmoidal relationship (α3; Fig. 2c). For the negative virulence-environment

functional relationships (α4, α5 and α6), R0 increases with environmental quality (Fig. 2d, e, f).

Virulence with an intermediate optimum (α7), allows for those parasites to spread in either

poor or pristine environments only (Fig. 2g). Conversely, parasites with minimal virulence in

intermediate environments (α8) are inhibited from spreading in environments of either bad or

good quality (Fig. 2h).

Less virulent parasites were found to spread in all conditions (Fig. 2). However, the ability of

highly virulent parasites (i.e., αr= 0.2) to spread strongly depended on the environmental

conditions and the virulence-environment relationship. Under the positive cube root (α2)

relationship, virulent parasites are barely able to reproduce and transmit in poor environments

and not at all as conditions improved. In poor environments, virulent parasites with a positive

linear (α1), a positive sigmoidal (α3), and a negative quadratic relationship (α7) could spread,

and the positive sigmoidal relationship (α3) resulted in the best conditions for virulent

epidemics. In good environments, however, virulent parasites were successful given negative

relationships (α4, α5, α6) and the negative quadratic relationship (α7). Virulent parasites were

able to spread across the largest range of environments under the positive quadratic

relationship α8, but not under extremely poor or good conditions.

B

Ed

A

E

b

bdb

dR

E

R

110

0


83

Fig 2.: Spread of parasites (R0) of low to high virulence (five lines from lightest grey to black represent αr =

0.01, 0.05, 0.10, 0.15, 0.20) in “bad” (0) to “good” (1) environmental conditions. Eight relationships between

virulence and environmental quality are shown. Virulence increases with quality – (a) linear, (b) cube root, (c)

sigmoidal – or decreases with quality (d) linear, (e) cube root, (f) sigmoidal. Parasite virulence may also be

highest in intermediate environments (g) negative quadratic, or lowest in intermediate environments (h) positive

quadratic.

Discussion

There is mounting empirical evidence that environmental conditions can play an important

role in mediating host-parasite interactions (Lazzaro & Little 2009; Wolinska & King 2009).

Our results suggest that while less virulent parasites easily spread over a range of

environments, as virulence increases, parasites depend more on environmental quality and the

conditions under which they can optimally exploit hosts (i.e., virulence-environment

functional relationship). In addition, it is when their virulence is reduced by environmental

conditions that otherwise highly virulent parasites are able to spread. For example, a larger

intensity of disease spread requires low virulence caused by condition-dependence: among

functional relationships α1-α3 (positive relationship), R0 is highest in α3 (positive sigmoidal,

Fig. 2c) because poor environments maintain the lowest levels of virulence. Virulence limits


84

disease spread in the present model, a finding in agreement with other epidemiological

models without a trade-off between virulence and transmission (see Keeling & Rohani 2007),

albeit in a constant environment. However, we show how virulence interacts with the

environment and plays out across a range of environmental conditions.

The best conditions for disease spread may not always be optimal for parasite fitness over the

long term. If parasite virulence has a positive functional relationship with environmental

quality (Fig. 2a-c), epidemics are more likely to occur in a poor environment. The

combination of poor conditions and parasite spread could be devastating for a host population

and suboptimal for parasite fitness, given that bad environmental conditions may additionally

impair other aspects of host health (e.g., growth, maintenance; Strong & James 1993;

Boersma & Vijverberg 1994). Therefore, negative relationships with environment quality

(Fig. 2d-f) may be the most optimal for parasite epidemics in the long term. Indeed, the

majority of studies examining condition-dependent virulence show parasite virulence at its

peak under stressed host conditions (e.g., Jokela et al. 1999; Ferguson & Read 2002; Tseng

2004). As a result, compared to conditions in a laboratory setting which are ideal, parasites

may do more damage to hosts, and thus have greater impact on host populations, in the wild

where hosts experience stressful, natural environmental conditions (Jaenike 1992; Jaenike et

al. 1995).

By mediating the impact of infection on hosts, environmental factors can alter the strength of

parasite-mediated selection (see examples in Lazzaro & Little 2009; Wolinska & King 2009).

Our results suggest a mechanism by which the environment may do this: favour the spread of

virulent parasites in some environments and not others. Environments conferring an

advantage to epidemics of highly virulent parasites may impose stronger selection than those

in which they cannot spread. Under relationship α3 (increasing sigmoidal), for example,

epidemics of virulent parasites can occur in a poor host environment, but are inhibited in a

good host environment (Fig. 2c). In addition, while some relationships yield little change in

R0 across the range of environmental conditions (Fig. 2b, 2g), parasite reproduction can

greatly vary in others (Fig. 2c, 2f, 2h). Rapid changes in the strength of selection may result

from environmental fluctuations or geographic clines depending upon the environment-

virulence relationship. Thus, variation in environmental conditions across populations or

within populations over time may contribute to host-parasite coevolutionary dynamics

(Mostowy & Engelstaedter 2011) or the geographic mosaic of coevolution (Thompson 2005).

Our model makes important predictions of how the interaction between parasite virulence and

environmental condition affects disease spread. It emphasizes the need for studies that


85

examine the relationship between environmental factors and virulence in natural systems

vulnerable to environmental variation over space or time. Ideal candidates would be

organisms and parasites which interact across seasons or a range of habitats, perhaps even

those that are anthropogenically-degraded.

Acknowledgments

We thank J. Engelstädter, J. Jokela, C. Lively, P. Spaak and especially S. Hall and M.

Kwiatkowski for their advice and support. We acknowledge funding from the ETH Board

(CCES-GEDIHAP to C. Buser), ThinkSwiss (to K. King) and NSERC Canada (to K. King).

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Boersma, M., and J. Vijverberg. 1994. Resource depression in Daphnia galeata, Daphnia

cucullata and their interspecific hybrid: Life history consequences. J. Plankton Res.

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Boots, M., P. J. Hudson, and A. Sasaki. 2004. Large shifts in pathogen virulence relate to host

population structure. Science 303:842-844.

Brown, M. J. F., R. Schmid-Hempel, and P. Schmid-Hempel. 2003. Strong context-dependent

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Day, T. 2001. Parasite transmission modes and the evolution of virulence. Evolution 55:2389-

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Ferguson, H. M., and A. F. Read. 2002. Genetic and environmental determinants of malaria

parasite virulence in mosquitoes. Proc. R. Soc. B. Biol. Sci. 269:1217-1224.

Frank, S. A. 1992. A kin selection model for the evolution of virulence. Proceedings of the

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Hall, S. R., C. J. Knight, C. R. Becker, M. A. Duffy, A. J. Tessier, and C. E. Caceres. 2009.

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Hall, S. R., A. J. Tessier, M. A. Duffy, M. Huebner, and C. E. Cáceres. 2006. Warmer does

not have to mean sicker: Temperature and predators can jointly drive timing of

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resistance in an invertebrate. Proc. R. Soc. B. Biol. Sci. 272:2601-2607.

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87

Chapter 7

Concluding remarks and recommendations for future research

Claudia C. Buser

Discussion & Conclusion Chapter 7

88

With this thesis, I aimed to detect pesticide-mediated effects on host-parasite interactions.

Specifically, I examined the influence of pesticide pollution on host and parasite fitness, as

well as virulence, and tested the effect of multiple stressors on host population dynamics and

disease spread.

Firstly, I would like to discuss the effect of pesticide on host fitness and host community

structure without taking parasites into account. I showed that both acute and chronic exposure

to the pesticide diazinon reduced host fitness. By exposing Daphnia to acute effect diazinon

concentrations (Chapter 2), test species and clones varied in survival, and therefore in

sensitivity, towards the toxicant. Also, the exposure to chronic and sub-lethal diazinon

concentrations reduced the number of offspring (Chapter 5), but for only one of the two

clones, pesticides caused higher mortality and lower fecundity. In Chapter 4, I found that the

fecundity of the competing taxa depended on the pesticide: D. galeata was more fecund when

additionally exposed to pesticide whereas the hybrids were more fecund when not exposed.

Pesticides have been previously shown to affect host life-history traits (see for example

Hanazato 2001), but pesticide concentrations used in most experimental tests are often higher

than concentrations found in aquatic systems (Relyea & Hoverman 2006). Thus, I aimed to

use ecologically-relevant diazinon concentrations (e.g., Konstantinou et al. 2006; Wittmer et

al. 2010), and I found that such sub-lethal concentrations drastically reduce host fitness. In

general, variation in response to pesticide exposure can be due to variation in traits, such as

size, mode of respiration, feeding type, habit, and life-cycle duration (Baird & Van den Brink

2007; Rubach et al. 2010), as well as variation in biotransformation processes and in

sensitivity of the target site (e.g., a certain enzyme) inside an organism (Barata et al. 2001).

Additionally, it can be because of adaptation towards the chemical compound (Klerks & Weis

1987). Consistently observing this genetic variation in sensitivity towards the pesticide

diazinon suggests that more than one genotype should be used in toxicology studies. This is

necessary to take into account the variation within and between species and to minimize the

risk of underestimating the effects of a chemical in the wild (Cairns 1984; Chapman 2000).

Differences in the sensitivity of single genotypes to anthropogenic effects can be manifested

at the community level. Pesticides did not reduce host community density, but changed the

genetic structure (Chapter 4), indicating that pesticide-tolerant clones replace sensitive

clones. In the Chapter 4 experiment, pesticide concentrations measured in natural habitats

were able to change the genetic structure of a mesocosm experimental community. Similarly,

Hanazato & Yasuno (1987) observed changes in zooplankton species composition after

Chapter 7 Discussion & Conclusion

89

ecologically-relevant exposure to insecticides, and Relyea & Hovernan (2008) demonstrated

that pesticide exposure lowered zooplankton diversity and abundance in mesocosms. A loss in

diversity, through exposure to a single stressor can highly impact communities making them

more prone to future stressors (Spielman et al. 2004; Reusch et al. 2005). For example,

reductions in genetic diversity may influence host or parasite evolutionary rates (Altizer et al.

2003). A reduction in host population diversity can also increase the risk of getting infected

(Elton 1958; Sherman et al. 1988; Schmid-Hempel 1998; Altermatt & Ebert 2008) or lead to

increased severity in the effects of epidemics on host populations (Duffy & Sivars-Becker

2007; Duffy & Hall 2008; Lively 2010). Furthermore, in a population exposed to pesticides,

resistance towards this chemical compound may evolve. Evolving resistance can be

associated with evolutionary costs and can lead to further synergistic interactions between a

pesticide, and for example, parasite exposure (Jansen et al. 2011).

In this thesis, I further examined whether pesticide exposure affected parasite fitness and

virulence upon single (environmental) or several (evolutionary) host generation exposures to

the pesticide diazinon. In Chapter 3, exposure to the pesticide for one generation increased

virulence. Pesticides has been shown to increase parasite fitness and virulence in several host-

parasite systems; parasites of amphibians (Gendron et al. 2003; Rohr et al. 2008; King et al.

2010), fish (Kreutz et al. 2010), and oysters (Chu & Hale 1994). Also virulence of a bacterial

parasite, Pasteuria ramosa, was demonstrated to increase when it‟s host, Daphnia magna,

was exposed to the pesticide carbaryl (Coors & De Meester 2008; Coors et al. 2008; Coors et

al. 2009; Coors & De Meester 2011). Interestingly, in Chapter 5, whether diazinon exposure

caused lower or higher parasite virulence (i.e., production of host offspring) and/or parasite

fitness (i.e., spore load) was highly dependent on the Daphnia clone infected by the parasite.

In Chapter 5, the evolutionary consequences of long-lasting pesticide exposure on parasite

fitness and virulence were more obvious than the environmental effects due to parasite

evolution. Persistent pesticide exposure over several host generations resulted in higher

parasite fitness (i.e., spore load) and lower virulence (i.e., host mortality rate and host

reproduction). It would be very interesting to test, if the observed decreases in virulence is

stable over time.

In general, parasite virulence is increased when hosts are exposed to pesticides in the short-

term. From our virulence-environment relationships built into the epidemiological model in

Chapter 6, this would correspond with a negative (decreasing) relationship (see Fig. 1d, e or f

in Chapter 6). Following the assumptions of the model, I would expect that disease would be


90

less likely to spread in a host population inhabiting an environment of bad quality than in a

population in good condition. Higher background mortality, as well as reduced transmission,

select for parasites to adapt and reduce their virulence (Garnick 1992; Bull 1994; Ebert &

Herre 1996; Frank 1996; Ebert & Weisser 1997; Day 2001). This was observed in Chapter 5,

in which parasites under pesticide stress evolved towards lower virulence, and consequently

higher fitness. Changes in parasite fitness and virulence can have consequences on disease

dynamics, as well as host population dynamics. In Chapter 4, we were not able to detect such

effects likely because the chosen timescale of 3 months was too short. Nevertheless, the

results of Chapter 5, in addition to the simulations of Chapter 6, improve our understanding

of external factors that may affect the evolution of virulence and the spread of disease.

A major goal of thesis was to determine the effect of multiple stressors on host fitness and

community structure and the consequences of pesticide-meditated effects on host-parasite

interactions, as well as the spread of disease. In our studies, a first clear effect of simultaneous

exposure to parasite spores and pesticide was found in Chapter 3: Whereas each stressor

alone did not affected host survival and parasite spores itself were not infectious, I found a

reduced survival of Daphnia by exposure to parasite spores which did not cause infection in

combination with pesticides. It has been shown that a combination of abiotic and biotic

stressors is often more harmful than either stressor alone (e.g., Hanazato & Dodson 1995; Folt

et al. 1999; Relyea 2003; Sih et al. 2004; Marcogliese & Pietrock 2011). The effect of

multiple stressors can be synergistic, antagonistic or additive, varying by response level

(Crain et al. 2008). Mostly synergistic or no effect of simultaneous exposure has been

observed (Holmstrup et al. 2010). In the Daphnia-endoparasite system clear interactions

between pesticide and parasites were found, namely increased host mortality and host

castration (Coors & De Meester 2008; Coors et al. 2008; Coors & De Meester 2011).

Pesticides, including diazinon have been shown to have immunomodulatory effects (Oostingh

et al. 2009; Holmstrup et al. 2010). The immune system seems to be condition- and genotype-

dependent (Carius et al. 2001; Mucklow & Ebert 2003; Rolff & Siva-Jothy 2003) which can

lead to genetic variation in resistance (e.g., Dybdahl & Lively 1998; Baer & Schmid-Hempel

1999) and infection (e.g., Lively 1989; Ebert 1994). I repeatedly found that if the host is

susceptible, and if pesticides enhance parasite virulence depends on the host genotype. For

example, in Chapter 5, I observed interactions between host genotype, parasite and pesticide:

in one clone, parasite spore load was higher when the clone was exposed to diazinon, whereas

for the other clone, parasite spore load was higher when the clone was not exposed to


91

diazinon. G×E×E interactions were also observed when looking at taxon susceptibility in

Chapter 4. I observed that the Daphnia hybrids (but not D. galeata) were more infected

when exposed to the pesticide compared to unexposed hybrids. In this experiment, reduced

hybrid fecundity under pesticide exposure, as well as the higher susceptibility of hybrids,

indicates that hybrid Daphnia species are more adversely affected by the environmental

stressors, parasites and pesticides. Interactions between host-parasites and environment are

increasingly being considered, particularly at the genotype level, to affect host-parasite

interactions and coevolution (Lazzaro et al. 2008; Wolinska & King 2009).

In the studies conducted in this thesis I found effects of multiple stressors on an individual

level, but host density and community composition was not affected by simultaneous

exposure (Chapter 4). At that level, parasite stress was the stronger selection factor. This

could be due to the low pesticide concentrations used in that experiment. In general, a dose

effect is very important. For example, depending on the amount of parasite dose (e.g., Ebert et

al. 1998) or chemical concentrations (see Chapter 2) used in the experiment, the harm of a

single stressor on host fitness can vary, and this could also affect the interplay between those

stressors.

In summary, the effects of anthropogenic activities can be manifested at the genotype,

population, and community levels of host-parasite interactions. Both parasite and pesticide

stress can mediate host fitness and community structure. Pesticides can affect key parasite

traits: fitness and virulence, and parasites can act together with ecologically-relevant levels of

pesticide pollution to enhance harm to host health. However, the strength of the combined

exposure to pesticides and parasites are highly variable, and must not be generalized. Effects

depend on host genotype, as well as the pesticide type, parasite dose, and the target host level

(individual, community). Nevertheless, given that parasites are ubiquitous, and anthropogenic

stress is a major cause of disease emergence in wildlife populations (Daszak et al. 2001),

predicting joint effects of stressors are important. In fact, combined effects of multiple

stressors must be considered for risk assessment of environmental pollution or conservation

biology, to make predictions more ecologically realistic and useful. Moreover, the genetic

diversity of exposed populations might be at higher risk, resulting in changes in the genetic

structure and opportunity for selection in whole ecosystems.


92

Recommendations for future research direction

Resurrection ecology

Using hatchlings from dormant eggs is a strong tool to study environmental effects on a

decade-long evolutionary scale. In Chapter 3 I failed to show evolutionary responses to

multiple stressors over time, as I did not have a visible sign of infection and therefor I could

not quantify infection success (spore density) over time. Nevertheless, from this study I

learned that for investigating multiple stressors over several decades it would be an advantage

to use several stressors which are all historically well documented or measureable in the

sediment. For example, using an approach as in Chapter 3, the anthropogenic stressor could

be a heavy metal (e.g., lead) which is accumulating in the sediment. In general, resurrection

ecology creates great opportunities to answer questions related to disturbance ecology and can

be applied to questions concerning disturbance effects on natural populations on a multi-

decade scale. Having knowledge of the distribution of environmental stressors in the past

provides a mechanism to follow changes in community structure, for example, by linking

genetic variation through time to environmental changes along extended time axes.

Combining field studies with mesocosm and laboratory studies

Another approach is to do field studies to examine the influence of abiotic stressors on host-

parasite interactions (e.g., Minguez et al. 2009; King et al. 2010; Marcogliese et al. 2010;

Wenger et al. 2010). Such field studies reflect realistic conditions, but are sometimes difficult

to interpret; it is difficult to control for confounding factors. The combination of field studies

and controlled mesocosms or laboratory studies helps to better interpret effects of multiple

stressors. Furthermore, potential confounding factors (e.g., trait-mediated effects or density-

mediated effects) can be disentangled and accounted for. Moreover, genes involved in host-

parasite interactions can be identified from laboratory experiments and reconciled with studies

in natural populations in stressed versus non-stressed habitats. For example, one can examine

the difference in expression of the candidate genes in more or less polluted habitats.

Interaction of disease and anthropogenic stress factor

When testing the effect of multiple stressors on host fitness in Chapter 3 we observed that

combined exposure of parasite spores and pesticide-reduced host survival significantly

compared to when exposed to a single stressor. This suggests that under certain conditions the

immune system can cope with one stress, but is compromised with multiple stressors.

Moreover, I demonstrated that host clones vary in susceptibility to parasites based on their


93

ecological habitat. The next step is to test on a range of clones varying in susceptibility to one

parasite strain (genetic diversity of susceptibility to parasites) if an anthropogenic stress factor

can influence the development of the disease. Are less susceptible clones fitter when exposed

to combined stress factors than highly susceptible clones? The expectation would be that less

susceptible individuals may have a stronger immune system, one that is less affected by a

second stressor, like diazinon, than those with high parasite susceptibility. Such a study could

be further expanded by using several parasite strains instead of one strain and exposing them

in a matrix (e.g., Carius et al. 2001), but under varying concentrations of pesticide. Revealing

G×G×E effects, the results would show if anthropogenic stress affects host-parasite

specificity.

Epidemiological models including environment

Epidemiological models are increasingly integrating the environment to determine how local

conditions affect disease spread (e.g., Lafferty & Holt 2003; Hall et al. 2007; Hall et al. 2009),

as well as host-parasite coevolution (Mostowy & Engelstaedter 2011). Our model from

Chapter 6 could be expanded to ask: (1) What is the optimal level of virulence in a

fluctuating environment (when virulence is environment-dependent), and (2) How does the

optimal level change with different functional relationships between virulence and

environmental quality? In my opinion, models have to be tested with empirical data.

Therefore, the relationships between environmental factors and virulence should be quantified

in natural systems vulnerable to environmental variation over space and time. Ideal candidates

would be organisms and parasites which interact across seasons or a range of habitats, perhaps

those that are anthropogenically-degraded.

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Curriculum vitae

Hubring 42 • 8303 Bassersdorf • Switzerland

Phone : +41 (0)78 840 02 01 • E-mail: claudia.buser@eawag; [email protected]

CLAUDIA CAROLINA BUSER

EDUCATION

2008 – present Ph.D. position, Department of Aquatic Ecology, Eawag, Dübendorf,

and Institute of Integrative Biology, ETH Zürich, Switzerland

2007 – 2008 Master of Science in Biology, Systematics and Evolution, Zoological

Museum, University of Zürich, Switzerland

2003 – 2007 Bachelor of Science in Biology, University of Zürich, Switzerland

LANGUAGES

German First language

Spanish Fluent in spoken and written

English Fluent in spoken and written

French Fluent in spoken and written

PUBLICATIONS

Demont, M., Buser, C.C., Oliver, M.Y., Bussière, L.F. Polyandry in the wild: Differential

sperm storage and temporal changes in sperm competition intensity in yellow dung flies.

Functional Ecology, doi: 10.1111/j.1365-2435.2011.01861.x, in press.

Jansen, M., De Meester, L., Cielen, A., Buser, C.C., Stocks, R. The interplay of past and

current stress exposure on the water flea Daphnia. Functional Ecology, doi: 10.1111/j.1365-

2435.2011.01869.x, in press.

CV Claudia C. Buser

98

MANUSCRIPTS IN REVISION

Buser, C.C., King, K.C. Environment-dependent virulence and transmission affect disease

spread in host populations.

Buser, C.C., Ward, P.I., Bussière, L.F. How maternal perception of larval environments

influences clutch size, larval performance, and paternity in yellow dung flies.

SUBMITTED MANUSCRIPTS

Buser, C.C., Spaak, P., Wolinska, J. Disease and pollution alter Daphnia community

structure. Submitted to Freshwater Biology.

Buser, C.C., Wolinska, J, Spaak, P. Evolutionary and environmental effects of pesticide

exposure on parasite fitness and virulence. Journal of Evolutionary Biology, in review.

Kashian, D., Buser C.C. The role of diapausing eggs in disturbance ecology. Book chapter in

Resurrection ecology, C. W. Kerfoot, ed.

MANUSCRIPTS IN PREPARATION

Buser, C.C., Fox, J.A., Kretschmann, A., Brede, N., Spaak, P. Variation within and between

taxa cannot be ignored in toxicity test.

Buser, C.C., Jansen, M. Pauwels, K., De Meester, L. Spaak, P. Combined exposure to

parasites and pesticides causes increased mortality in the water flea Daphnia.

Wüst, C., Demont, M., Buser, C.C., Bussière, L.F. How female reproductive tract

morphology affects sperm movement in yellow dung flies.

Dupont, L., Viard, F., Pérez Portela, R., Buser, C.C., El Nagar, A., and Bishop, J.D.D.

Paradox revisited: severe cyto-nuclear founder effects in rapidly expanding Corella eumyota

(Urochordata: Ascidiacea) populations in NW Europe.

INTERNSHIP

02/2009 - 06/2009 Laboratory of Aquatic Ecology and Evolutionary Biology, Katholieke

Universiteit Leuven, 3000 Leuven, Belgium

Claudia C. Buser CV

99

WORK EXPERIENCES

10/2006 – 04/2007 Research assistant, Zoological Museum, University of Zürich,

Switzerland

07/2006 – 10/2006 Research assistant, Marine Biological Association of the United

Kingdom, Plymouth, United Kingdom

07/2003 – 09/2003 Voluntary service, Foundation for information and research on marine

mammals (firmm), Pedro Cortés, 11380 Tarifa, Spain

TEACHING EXPERIENCES

2008 Supervising undergraduate student, Department of Aquatic Ecology,

Eawag, Switzerland

2007 Tutor in the reproduction course, Zoological Museum, University of

Zürich, Switzerland

2007 Tutor in the systematic zoology course, ETH Zürich, Switzerland

2007 and 2005 Tutor in systematic zoology course, University of Zürich, Switzerland

2004 Tutor in chemistry course, University of Zürich, Switzerland

INVITED PRESENTATION

10/2010 Bloomington, USA: Buser C.C., Wolinska J., Spaak P. “The

influence of a pesticide on host-parasite interactions”

CONFERENCE PRESENTATIONS

08/2011 ESEB meeting, Tübingen, Germany: Buser C.C., Ward P.I., Bussière

L.I. “Maternal perception of the larval environment affects offspring

performance but not paternity” (poster)

03/2011 GEDIHAP- annual meeting, Zürich, Switzerland: Buser C.C.,

Wolinska J., Spaak P. “Disease and pollution alter Daphnia

community structure” (talk)

CV Claudia C. Buser

100

02/2011 Eco-Evolutionary Dynamics, Leuven, Belgium: Spaak P, Buser C.C,

Schöbel C, Wolinska J “The coexistence of hybrid and parental

Daphnia – the role of parasites” (talk)

11/2010 SETAC, Portland, USA; Buser C.C., Wolinska J., Spaak P. “The

influence of an insecticide on Daphnia individuals and populations

exposed to parasites” (poster)

06/2010 ASLO Summer Meeting, Santa Fe, USA: Buser C.C., Wolinska J.,

Jansen M., Spaak P. “The influence of a pesticide (Diazinon) on

Daphnia-parasite interactions” (talk)

09/2009 Resurrection ecology, Hertzberg, Switzerland: Buser C.C., Jansen M.,

Pauwels K., De Meester L., Spaak P. “Effect of two stress factor on

host fitness” (talk)

08/2009 ESEB meeting, Turin, Italy: Buser C.C., Jansen M., Pauwels K., De

Meester L., Spaak P. “Effect of two stress factor on host fitness”

(poster)

10/2008 Symposium on Sexual Selection, Sperm Competition & Cryptic

Female Choice in Honour of Paul Ward, Zürich, Switzerland: Buser

C.C., Bussière L.I. “Do female yellow dung flies adjust oviposition

and paternity according to perceived levels of larval competition?”

(talk)

04/2008 BEES, Zürich, Switzerland: Buser, C.C. “Do female dung flies adjust

paternity according to the level of larval competition?” (talk)

09/2007 Fortgeschrittenenseminar in Evolutionsbiologie, Kilchberg,

Switzerland: Buser C.C. “Oviposition behaviour of the yellow dung

fly“ (talk)

WORKSHOP

09/2009 Host-Parasite Coevolution Workshop, Frauenchiemsee, Germany

Claudia C. Buser CV

101

GRANTS

2010 ETH travel fund (CHF 725)

SETAC-Portland travel award (USD 265)

2009 Eawag Mobility support for the realization of experiments in a foreign

institute (CHF 5600)

SKILLS

Molecular techniques DNA and RNA extraction (different methods), PCR (touch down

PCR, semi-quantitative PCR), genotyping (with different sequencers

and gels), gel electrophoresis (polyacrylamide, agarose), microarrays

Software experience Statistical analysis software (R, SPSS, Statistica), graphing software

(SigmaPlot), genotyping software package (GeneMapper®),

population genetics software packages (Genepop, FSTAT) and

modelling software (Mathematica)

Relevant techniques Microdissection, microscopy and image analysis, laboratory culture of

sessile invertebrates, insects and crustacean

Field work Insect and crustacean sampling, marine invertebrate sampling on

rocky shores, marina and an offshore breakwater, sediment core

sampling

Licences Driving licences (car, motor bike, motor boat, sailing boat),

cardiopulmonary resuscitation (CPR), rescue swimming (Brevet 1,

ABC 1), diving (Paddy advanced, nitrox diving)

103

Acknowledgments

For sure, this PhD would not have been possible nor would have been so much fun without all

the people around me! I would like to thank the following people for the great support during

my doctorate:

Piet Spaak for giving me the opportunity to do my PhD at Eawag, for supervising me

during the whole period, for always having his door open to discuss new

ideas/projects/problems, for being open to all my nice collaborations and for the support

to go for some months to the University of Leuven. I really appreciated that he always

trusted me and gave me the freedom to work independently.

Justyna Wolinska for being my co-supervisor, for discussing and improving ideas, for

giving hints for practical work and for her patience in reading, commenting and polishing

my manuscripts.

Christoph Vorburger, Jukka Jokela , Justyna Wolinska and Piet Spaak for being part of

my PhD committee, for many fruitful scientific discussions, for excellent advises and for

always having an open door for questions.

Christoph Haag and Paul Schmid-Hempel for being my examiners.

Andreas Kretschmann, Christoph Tellenbach, Donna Kashian, Jennifer Fox, Joost

Vanoverbecke, Kayla King, Kewin Pauwels, Luc De Meester, Luisa Orsini, Mieke Jansen,

Nora Brede, Piet Spaak and Justyna Wolinska for all the lovely collaborations in the past,

present and near future. It has been very inspiring to work with you. I am looking forward

to continue working with you.

Blake Matthews, Christoph Vorburger, Jukka Jokela and especially Christoph Tellenbach

and Otto Seppälä, for taking the time to discuss and check my statistical analyses.

Andi Bruder, Chris Robinson, Christian Moser, Christoph Tellenbach, Hanna

Hartikainen, Jennifer Fox, Justyna Wolinska, Kayla King, Katja Räsänen, Marek

Kwiatkowski, Markus Möst, Mat Seymour, Piet Spaak, Silke Van den Wyngaert, Spencer

Hall and the co-authors for reading earlier versions of my manuscripts and other texts to

polish the style and content.

Esther Keller for always answering my nasty questions, for introducing me to the practical

world of Daphnia, for helping me to set up experiments, for measuring animals, for

helping with molecular work and for nice non-scientific chats.

Christine Dambone and Nadja Tschopp for preparing the algae and taking care of my

clones.

Nadja Tschopp and Kirsten Klappert for organizing the laboratory and ordering materials

and chemicals.

Esther Keller, Markus Möst and Silke Van den Wyngaert for helping setting up big

experiments.

Andreas Kretschmann and Roman Ashauer for being my experts of the pesticide diazinon.

Acknowledgments Thank you!

104

The Genetic Diversity Center of ETH, the Werkstatt and the Aua-Labor of Eawag.

The inhabitants of G17 for being great office mates, for the good mood and for having

great discussions.

Chris Robinson, Markus Möst, Remo Freimann and Silke Van den Wyngaert for keeping

me healthy with all the fruit breaks.

Andi, Anja, Aline, Christoph, Diego, Dorota, Katja, Katri, Maria, Markus, Mat, Nora,

Olivier, Otto, Remo, Sandra, Silke, Vicenç and the rest of the Eco department for funny

and unforgettable moments in and outside Eawag.

Eawag for providing the money of the Mobility Support to go to Luc De Meester‟s Lab

for 4 month.

Luc De Meester and his group, especially Evelyne, Luisa, Kevin, Mieke, Nelly, Joachim,

the two Joosts, Sarah, Silvia, Steven and Wendy, for discussing and helping with

experiments and for contributing to a great time in Leuven.

Luc Bussière for introducing me to the nice world of science and for great discussions,

inputs and motivation.

My mother Olivia Buser for her support, for being concerned about my welfare, for

providing me with excellent food, for occasionally giving me a lift to Eawag and for

listening to and supporting my moods.

My father Ruedi Buser for supporting me during the whole studies, for interesting

discussions and for the beautiful weekends in Saanen.

Christian Moser for the extraordinary support during the whole PhD, for listening to my

concerns, impressions and experiences, for being patient when I had difficulties to turn off

thinking about science, for all the helpful advises and for facilitating decisions. I am

looking forward to our shared future!

Thank you all!

dirty water and disease: pesticide mediated interactions ...4612/eth... · pesticide mediated...

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