Ecology and evolution in a host-

parasitoid system

Host search, immune responses and parasitoid virulence

Lisa Fors

©Lisa Fors, Stockholm University 2015

Cover photos: Robert Markus and Lisa Fors

Back cover photo: Anna Erlandsson

ISBN 978-91-7649-103-4

Printed in Sweden by Publit, Stockholm 2015

Distributor: Department of Ecology, Environment and Plant

Sciences, Stockholm University

Sometimes you’re the windshield,

sometimes you’re the bug

Mark Knopfler

List of Papers

This thesis is based on the following papers, which are referred to by their

roman numerals:

I. Fors L, Liblikas I, Andersson P, Borg-Karlson A-K, Cabezas N,

Mozuraitis R, Hambäck PA (2014) Chemical communication and

host search in Galerucella leaf beetles. Chemoecology doi:

10.1007/s00049-014-0174-1

II. Fors L, Verschut T, Hambäck PA. Host search and host

preference in Asecodes parviclava. Manuscript

III. Fors L, Markus R, Theopold U, Hambäck PA (2014) Differences

in cellular immune competence explain parasitoid resistance for

two coleopteran species. Plos One

doi: 10.1371/journal.pone.0108795

IV. Fors L, Markus R, Theopold U, Ericson L, Hambäck PA.

Geographic variation in parasitoid virulence and parasitoid host

race formation. Submitted manuscript

Contents

Introduction .................................................................................................. 9 Search behaviour and host preference................................................................ 10

Host search in herbivores ................................................................................. 10 Host search in parasitoids ................................................................................ 11

Insect Immunity ...................................................................................................... 12 Host defence strategies .................................................................................... 12 Parasitoid counter-defence strategies ............................................................ 15

Aim of the thesis ..................................................................................................... 16

Methods ....................................................................................................... 17 Study system and study area ............................................................................... 17 Chemical communication and host search (Paper I and Paper II) ................. 19

Paper I ................................................................................................................. 20 Paper II ................................................................................................................ 21

Host immune response and parasitoid virulence (Paper III and Paper IV) .. 22 Paper III .............................................................................................................. 22 Paper IV ............................................................................................................... 23

Results and discussion ............................................................................. 25

Concluding remarks .................................................................................. 32 Acknowledgements ................................................................................................. 32

References .................................................................................................. 33

Svensk sammanfattning .......................................................................... 40

Tack/Acknowledgements ......................................................................... 45

9

Introduction

In nature, much of evolution is coevolution between interacting species,

driven by natural selection [1]. Species can interact either through mutualistic

processes where all species involved benefit, as for example in specialized

plant-pollinator interactions [2, 3], or through antagonistic processes which

include either competitive or trophic interactions [4, 5]. A prerequisite for

coevolution is local adaptation, which can be defined as genetic change in a

population, due to a variation in selective pressures across the landscape.

Local adaptation can result in a higher fitness in local individuals at their home

site, compared to the fitness of nonlocal individuals, depending on their ability

to cope with local biotic and abiotic conditions [6]. As gene flow may prevent

populations of the same species to evolve independently, it can have great

influence on the process of local adaptation [7, 8].

In systems with antagonistic interactions, for example host-parasite systems,

there is a continuous coevolutionary arms race between the species, with each

species imposing strong selection pressure on the other. Parasites are

conventionally considered to be ahead of the hosts in the coevolutionary arms

race, due to short generation times and large population sizes [9].

Consequently, if the parasites have the ability to rapidly adapt to new defence

strategies in the host, it could result in local adaptation in the parasites, with a

certain parasite population showing higher virulence on local compared to

non-local host populations [10, 11]. However, there is a great variability in the

outcome of empirical studies concerning local adaptation in host-parasite

interactions [12]. In many studies, the parasites show local adaptation, but in

some cases there seems to be no local adaptation or even maladaptation of the

parasites [10].

A specific form of parasites are constituted by parasitoids; free-living adult

insects whose progeny feed on the body of another arthropod, which

unconditionally leads to the death of the host [13-15]. Parasitoids constitute a

diverse group of insects that can cause high mortality in many host

populations. All parasitoids are holometabolous with a four-stage life cycle of

egg, larva, pupa and adult. Most parasitoids attack a particular life stage of the

host insect. The juvenile stages (i.e. eggs, larvae, pupae) are most common to

attack, but a few species attack only the adult insects, for example conopid

wasps attacking adult bees of several genera [16]. Parasitoids are classified as

10

either idiobionts or koinobionts. Idiobiont parasitoids prevent further

development of the host once it has been infected, whereas hosts of

koinobionts continue their development after infection [17]. The parasitoids

can either develop inside the host (endoparasitoids) or on the exterior of the

host (ectoparasitoids). Most parasitoids fall into one or the other of these

categories, but there are examples of species showing a transition between the

strategies, most often from ectoparasitism to endoparasitism [18].

Endoparasitoids are usually considered to have a more complex relation to

their hosts, as they must overcome the immune defence of the host but at the

same time keep the host alive long enough for the parasitoid larvae to develop.

Parasitoids usually have a quite specific host range and the intimate

interactions between parasitoids and their hosts make host-parasitoid systems

particularly well suited for studying local adaptation and coevolution [19].

In order to better understand the evolution in host-parasitoid systems, different

aspects of the interactions must be taken into account, including search

behaviour and host selection in the parasitoid, development of defence

strategies in the host and counter-defence mechanisms in the parasitoid.

Recently, there has been a growing interest to gain a better understanding of

the complexity of trophic interactions by examining immunity-related traits in

relation to evolution and ecology, a field known as ecological immunology

[20]. However, there is still limited knowledge on the connections between

host immunity, parasitoid virulence, host race formation and speciation in

natural host-parasitoid systems. In this thesis, based on four papers, I have

investigated interactions and possible coevolution in a host-parasitoid system,

focusing on host search, parasitism success and host immune responses. The

first part of the thesis is a general background regarding search behaviour and

insect immunity, followed by a description of the four studies conducted.

Search behaviour and host preference

Host search in herbivores

Herbivore insects can use different methods to locate a host plant, including

visual, olfactory and gustatory search cues [21]. The most important stimuli

for many insects at a distance from the resource are olfactory and visual cues,

often used in combination. Shape, size and spectral quality are examples of

visual plant characteristics that may influence host selection [22]. Visual cues,

unlike odour cues, are not likely to be affected by abiotic factors such as wind

and temperature, and should thereby be quite stable [23]. However, in a dense

and complex vegetation, olfactory signals may be more reliable than visual

11

characteristics. Odour cues are airborne chemical compounds of different

origin, such as green leaf volatiles emitted by plants or pheromones emitted

by insects. Insect pheromones are primarily used as intraspecific signals for

social and sexual behaviour, but indirectly they are also used in the search for

host plants [24]. Both plant odours and insect pheromones are usually complex

blends of chemical components. Pheromones are often species specific,

whereas many green leaf volatiles can be produced by a variety of plant

species. However, many herbivorous insects have a highly evolved system for

olfactory reception, enabling them to distinguish specific plant volatiles in a

blend and translate them into a chemical message [25]. When herbivorous

insects feed on their host plant, the green leaf volatiles released differ

qualitatively and/or quantitatively from undamaged or artificially damaged

plants [26]. Consequently, insects feeding on a host plant may attract more

conspecifics and possibly also other herbivore species to the same plant,

leading to herbivore aggregation.

Host search in parasitoids

In host-parasitoid interactions, it is crucial for the parasitoid females to locate

suitable hosts and to select between host individuals of different qualities. The

process of host selection is conventionally divided into three steps: host

habitat location, host location and host acceptance [27-29]. Parasitoids most

frequently use odour cues when locating hosts [29], although some parasitoid

species use other types of signals for detection, such as sound [30, 31], visual

cues [32] and electromagnetic radiation [33]. The search strategies and the

cues used for host location may differ depending on whether the parasitoid is

a specialist or a generalist. The usability of a certain cue depends both on how

reliable the information is and how easily it can be detected by the parasitoid.

Odour cues can either be released directly from the host itself or derive from

the herbivore host plant, host products or the microhabitat [34]. Parasitoids in

tritrophic systems often use a combination of odour cues from both lower

trophic levels for host location. Many species are attracted to green leaf

volatiles released due to feeding activity of their host herbivore, but do not

show the same response to volatiles of mechanically damaged plants [35-40].

Some parasitoids respond to a mixture of odours from the host and the host

plant, even if the host is not actively damaging the plant. This is for example

seen in some species of egg parasitoids that respond to plant volatiles induced

by host egg deposition on the plant, whereas no response is observed when

volatiles are released by the same plant due to larval feeding [41, 42]. When

parasitoids exploit volatile chemicals directly emitted from the host as a search

cue, it is likely to be a selection pressure on the host species to avoid detection

by reducing the emission of the specific compound [36]. The parasitoids in

their turn are under selection to evolve more efficient ways to detect the

12

signals of their hosts, resulting in a coevolutionary arms race between host and

parasitoid [43]. However, if the host-emitted compound is also used by the

host to communicate with conspecifics (i.e. a pheromone) it is likely to be a

strong selection pressure for keeping the compound. Sex pheromones in

particular are expected to be under strong selection for stability, as even a

slightly modified version of the compound is less likely to attract potential

mates [44]. Usually, pheromones are produced in very small quantities, which

could be a mechanism to avoid eavesdropping by natural enemies [45, 46].

But even in small quantities pheromones are often detectable at quite long

distances and they are also often host specific, which would make them

reliable cues for parasitoid host location. However, the presence of the

preferred host stage is not necessarily indicated by adult pheromones [34].

Insect Immunity

Host defence strategies

In host insects there are a number of strategies developed in order to escape

parasitism, such as using enemy-free space, concealment or physical counter-

attack. In communities of social insects there are examples of external

strategies to avoid spreading of pathogens, something often referred to as

“social immunity”. One example of external defence is the uptake of

antifungal or antibacterial substances, such as the collection and use of plant

resins by honey bees [47] or conifer saw flies [48]. Other strategies can be

grooming to remove parasites from group members [49, 50], socially

generated fevers to limit the proliferation of natural pathogens [51], detection

and removal of infested and deceased individuals [52, 53] or relocation to

abandon infested areas [54].

Even when successfully parasitized, the host insect can still defend itself

through a potent immune defence. Insects only possess one level of immunity:

innate (or natural) immunity, which is present in both invertebrates and

vertebrates, but lack the adaptive (or acquired) immunity, which is present

only in vertebrates. Innate immunity refers to nonspecific defence

mechanisms that start immediately or within hours after an antigen has

appeared in the body. The innate immune system in insects consists of several

different defence mechanisms, with both cellular and humoral contributions

[55, 56].

13

Any organism trying to infest an insect first has to overcome the physical

barrier of the insect cuticle. The cuticle can be divided into two layers, the

outer epicuticle, which is a thin chitin-lacking layer, highly resistant to water

and other solvents, and the inner thicker procuticle. The procuticle can again

be divided into two layers, the outer exocuticle and the inner endocuticle,

consisting of a large number of protein and chitin fibre layers, which creates

a very tough and flexible substance [57]. Even if parasites or microorganisms

enter the insect body cavity through the mouth, they still have to overcome

physical barriers. The gut of most insects is lined by the peritrophic

membrane, which is a grid-like structure composed of chitin and proteins [58].

Furthermore, ingested microorganisms activate the production of reactive

oxygen species (ROS) in the insect midgut. ROS are multifunctional

molecules involved in host defence, hormone biosynthesis, apoptosis,

necrosis, and gene expression. The ROS response is strongly induced by

microbial infections [59, 60].

The two major immune organs in insects are the fat body and the hemocytes.

The fat body is the largest organ of the insect body cavity, playing an essential

role in nutrient storage and metabolism [61]. It is an endocrine organ unique

to insects that has been studied extensively in Drosophila [62, 63], whereas

there is still less knowledge of its specific functions in many other insects [64].

From the fat body, soluble effector molecules toxic to intruding parasites and

pathogens are secreted into the open circulatory system [57, 65]. The soluble

molecules recognize microbes and provide an early defence against pathogens

present outside host cells. They can act either directly on the invader or by

altering the insect’s immune response [66]. One type of soluble effectors are

antimicrobial peptides (AMPs), small molecules (12-50 amino acids) with

different target organisms (either bacteria or fungi). AMPs act by binding to

bacterial or fungal membranes, which leads to disruption of the membrane and

death of the cell [67]. The first induced AMP isolated from an insect was

Cecropin, fully characterized by Boman and co-workers, in bacteria-

challenged diapausing pupae of the moth Hyalophora cecropia [68]. Since

then, over 150 AMPs have been characterized in insects, but the number and

types vary between species [69]. Although most AMPs are secreted by the fat

body, some are also produced by hemocytes [65, 70]. Other examples of

processes mediated by soluble effectors are clotting and melanisation.

Clotting is the coagulation of hemolymph, leading to the rapid formation of a

plug which seals wounds and keep bacteria from entering the body cavity,

something that is especially important in the open circulatory system of insects

[71]. Melanisation plays a central role in insect defence against a wide range

of pathogens, participating in wound healing as well as in nodule and capsule

formation. The melanisation pathway leads to the formation of melanin, which

results in an immediate localized blackening of the tissue at the wound site or

around an encapsulated object [70] (Fig. 1).

14

The cellular immune defence mechanisms in insects are directly mediated by

the hemocytes, circulating freely in the insect hemolymph [72] or localized in

specific hematopoietic organs. Until recently, hemocytes have been studied in

most detail in Dipteran [73] and Lepidopteran [74] species, where several

hemocyte classes have been characterised. The cellular defences include

several mechanisms such as phagocytosis, encapsulation, clotting and

nodulation. Phagocytosis is the process where intruding objects are engulfed

and destroyed by individual, specialised hemocytes [75, 76]. Hemocytes can

phagocytise a variety of invaders, such as fungi, yeast, bacteria and apoptotic

bodies [72, 77]. Larger targets that cannot be engulfed by single cells, such as

parasitoids or nematodes, are instead encapsulated by aggregating hemocytes

(Fig. 1b-d). Following parasitoid attack, the capsule formation usually begins

within 4-6 hours and is completed after approximately 48 hours [78]. During

this process the capsule is often melanised [70] and due to crosslinking of its

protein compounds it also hardens, which leads to the death of the

encapsulated intruder [79, 80]. Another example of cellular defences, similar

to the encapsulation process, is nodulation. Insect nodules are multicellular

hemocytic aggregates, which are formed as a reaction against large numbers

of bacteria or fungi [81, 82].

Figure 1. Melanisation and encapsulation in Galerucella. A) Melanised wound site in the cuticle of G. calmariensis. B) G. pusilla larva with encapsulated parasitoid eggs visible through the cuticle (arrow). C) Encapsulated and melanised parasitoid eggs inside G. pusilla larva. D) Encapsulated parasitoid egg dissected from G. pusilla, with layers of hemocytes attached to the surface. Photos: Robert Markus and Lisa Fors.

15

Recognition of intruding pathogens is a crucial aspect of immunity. This task

is even more challenging when the pathogen is intracellular, as in viral

infections of a host [83]. One effective immune response against intruding

viruses is the destruction of infected cells by the tightly regulated process

called apoptosis or programmed cell death. Apoptosis is mediated by

caspases; proteolytic enzymes that are present in all cells as inactive

precursors. Another important immune response towards viruses and other

foreign genetic material is RNAi interference, first described in insects in the

model nematode Caenorhabditis elegans [84, 85]. RNAi interference is a

specific gene silencing process in which double-stranded RNA is used

to inhibit gene expression, mainly through the degradation of

specific mRNA molecules.

Parasitoid counter-defence strategies

Just like host insects have to develop defence mechanisms in order to avoid

parasitism, parasitoids are under strong selection to evolve counter-defence

strategies to protect their progeny from being rejected [86]. There is a variety

of mechanisms used by parasitoids in order to manipulate their host and create

a favourable environment for the developing parasitoid offspring [15].

Many parasitoid species have developed different methods to avoid the

encapsulation of eggs or larvae, which is otherwise the most common and

often successful defence against parasitoids. To avoid or reduce

encapsulation, the parasitoid female can hide her eggs in an organ that is

inaccessible to circulating hemocytes, such as the host brain, or produce eggs

that can adhere to the host fat body, which protects them from being

completely surrounded by hemocytes [86, 87]. Parasitoid eggs or larvae can

also be “camouflaged” by a protective layer in order to mimic the host tissue,

thereby preventing recognition by the host [36, 88]. Some parasitoid species

can even survive being encapsulated, by modifying capsule formation. Many

parasitic tachinid flies develop inside the host, but have spiracles armed with

hooks that penetrate the cuticle or trachea of the host. If encapsulated, the

parasitoid larva is not killed as long as it can use this respiratory funnel to

avoid asphyxiation [89].

The encapsulation process can also be disrupted by active destruction of host

immune cells that are required for capsule formation. Many parasitoids

manipulate the host and promote parasitism by injecting venoms or symbiotic

viruses into the host during oviposition [86, 90]. Injected substances can

suppress the immune response, cause tissue necrosis, or paralyze the host.

16

Venoms used by parasitoids typically consist of one or more proteins that

function by acting on nerve synapses or disrupting cell membranes. A

common strategy in many parasitoids is to quickly sting the host to inject

venom and then withdraw to let paralysis set in. Once the host is paralyzed the

parasitoid returns to oviposit undisturbed [91]. Some parasitoid species inject

venoms that keep the host alive but leave it immobile or incapable of moulting

for weeks after the attack [36]. Parasitoids belonging to the Braconidae and

Ichneumonidae families are known to form associations with viruses, which

have received the name polydnaviruses (PDVs). The name refers to the

arrangement of the viral genome, typically composed of 15-35 double-

stranded DNA circles. PDVs act as delivery vectors of genes that disrupt

humoral signalling pathways or hemocyte function, thereby altering the host’s

immune defence, growth or development to promote the development of

parasitoid offspring [15, 92]. Parasitoids from several families (in particular

Braconidae, but also Scelionidae and Trichogrammatidae) produce special

cells called teratocytes that are released into the host’s hemolymph by the

serosa membrane surrounding the parasitoid embryo [15]. Teratocytes do not

multiply once in the hemolymph, but they are often released in very large

numbers and they continue to grow, sometimes dramatically, within the host

body [93]. The main function of teratocytes is trophic; they absorb nutrients

from the host hemolymph and are eventually consumed by the developing

parasitoid larvae. However, the teratocytes can also secrete compounds that

may manipulate the host or interfere with the immune response in order to

favour parasitoid development [94].

Aim of the thesis

The overall aim of this thesis was to investigate interactions and possible

coevolution in host-parasitoid systems, by combining ecological studies with

chemical and cellular investigations. The studies have been carried out in a

system consisting of Galerucella leaf beetles (Coleoptera: Chrysomelidae)

and Asecodes parasitoids (Hymenoptera: Eulophidae). Specifically, I wanted

to investigate chemical communication in the host species (Paper I), parasitoid

search behaviour and host preference (Paper II) and parasitoid virulence in

relation to the immune response in the attacked host (Paper III and IV). In the

following part of the thesis, I will describe the study system and go through

the methods and results of each study, followed by a general discussion of the

combined results.

17

Methods

Study system and study area

There are five species of Galerucella included in the studies of this thesis: G.

calmariensis (L.), G. pusilla (Duftschmid), G. tenella (L.), G. sagittariae

(Gyllenhal) and G. lineola (Fabricius) (Fig. 2). The differentiation of the

species is fairly recent: G. pusilla and G. calmariensis are most closely related

with an estimated divergence date 77 000 years ago [95]. G. tenella is also

quite closely related to G. pusilla and G. calmariensis, whereas G. sagittariae

and G. lineola are further apart in the phylogeny. While G. pusilla and G.

calmariensis are monophagous and share L. salicaria as exclusive host plant,

the other species are polyphagous. G. tenella uses F. ulmaria as primary host,

but can also be found on other Rosaceae species. G. sagittariae shares some

host plant species with G. tenella, but also uses additional Rosaceae and

Primulaceae species, whereas G. lineola uses different species of Salicaceae

as host plants [95]. In many localities, several host plant species can be present

within close distance, which means that several Galerucella species can

sometimes co-occur in the same area. However, the geographic distribution of

the beetles in Sweden differs a bit between the species. For example, G. pusilla

is not present in the northern localities where G. calmariensis is found, even

though G. pusilla and G. calmariensis often co-occur in the same localities in

the south (often even on the same plant). For the studies of this thesis, beetles

and larvae from the five Galerucella species were collected from various

localities in Sweden each season of the experiments (see Fig. 3 for details).

Figure 2. Study system showing adults of the five Galerucella species and names of the attacking parasitoid species. A) G. calmariensis B) G. pusilla C) G. tenella D) G. lineola and E) G. sagittariae. Photos: Robert Markus and Lisa Fors.

18

The beetle species all have similar life cycles, over-wintering as adults and

emerging during spring. Mating takes place on the host plants and the eggs are

deposited directly on the leaves or stem in early summer, hatching after a few

weeks. Both larvae and adults feed on the plant, which can often lead to quite

severe damage. After 3-4 weeks the larvae pupate in the ground and the new

adults emerge from the pupae 2-3 weeks later [96].

Figure 3. Map of Sweden showing field localities used for collection of Galerucella adults and larvae. G. pusilla is not present north of the dashed line. The species were collected from the following localities, indicated by numbers: G. calmariensis (1, 3-11, 23-26), G. pusilla (2, 4-11, 21-31), G. tenella (1, 8-12, 14-22, 24, 26, 27, 29), G. lineola (8-12, 14, 15, 23, 24, 27, 29) and G. sagittariae (8-11, 13, 23, 24, 26-28).

19

The Galerucella spp. are attacked by three species of Asecodes (Hymenoptera:

Eulophidae): Asecodes parviclava (Thomson), A. lucens (Nees) and A.

lineophagum Hansson & Hambäck. Genetic studies of Asecodes suggest that

speciation of the parasitoid has followed the host speciation [95]. The

Asecodes parasitoids are small (<1mm) and morphologically similar but each

species has a unique host range. Asecodes parviclava parasitizes G.

calmariensis, G. pusilla and G. tenella, whereas A. lucens parasitizes only G.

sagittariae and A. lineophagum only G. lineola [97] (Fig. 2). Asecodes are

koinobiont endoparasitoids that can cause a high level of mortality in

respective host species. The parasitoids attack the beetles in the larval stage,

laying one or more eggs inside the larva. When the eggs hatch, the parasitoid

larvae start consuming the interior of the host. Parasitized larvae develop

normally until pupation, when they are unable to form pupae. Instead the

larvae turn into black mummies from which the adult parasitoids subsequently

hatch (Fig. 4), usually during the next summer [96, 98].

Figure 4. Development stages in Asecodes parasitoids. A) Parasitoid larvae dissected from an infected G. calmariensis larva. B) Live parasitoid larva. C) Parasitized, mummified G. calmariensis larva showing pupating parasitoids inside. D) Parasitoid pupae. E) Adult parasitoid (A. parviclava). Photos: Robert Markus and Lisa Fors.

Chemical communication and host search (Paper I and Paper II)

Earlier studies have shown that males of G. pusilla and G. calmariensis both

emit the same aggregation pheromone (dimethylfuran-lactone) when feeding

on their host plant [99, 100]. In other Galerucella species no pheromones have

previously been identified. Based on these previous observations, I wanted to

further investigate the pheromone production in Galerucella, and also study

the behavioural responses in the beetles to different search cues (Paper I).

20

Additionally, I wanted to find out more about the search behaviour in

Asecodes, and whether the parasitoids can exploit the pheromone of the

beetles as a search cue when locating a host (Paper II).

Paper I

The first part of this study was to investigate the production of and the

response to pheromones in Galerucella. In this part all five beetle species were

included: G. calmariensis, G. pusilla, G. tenella, G. sagittariae and G. lineola

(Fig. 2). As it had previously been reported that G. pusilla and G. calmariensis

produce the pheromone dimethylfuran-lactone only when feeding on their

host plant (Bartelt et al. 2006), the first step was to collect volatile compounds

from beetles of all five species feeding on their respective host plant. Thus,

volatiles were collected of G. calmariensis and G. pusilla feeding on L.

salicaria, G. tenella and G. sagittariae feeding on Fragaria x ananassa and

G. lineola feeding on Salix viminalis. To distinguish the compounds produced

by the beetles from those produced by the plants, half of the plants for each

species pair (beetle-host plant) were mechanically damaged and the other half

were damaged by feeding beetles (10 beetles/plant). There was also an

additional set-up with larvae instead of beetles. The plants (with or without

beetles or larvae) were then enclosed in polyester cooking bags and the

volatiles were collected during 24 hours using solid phase micro extraction

(SPME), which is a sampling technique where a polymer-coated fibre is used

to absorb analytes. After collection the chemical compounds were separated

and analysed by using a gas chromatograph-mass spectrometer (GCMS).

The next step was to test whether the beetles showed any attraction to the

pheromone. For this experiment, the pheromone was produced synthetically,

using a newly developed and improved method. The behavioural responses

were studied in two-armed olfactometers (see [101] for description), with a

cut out arena in the middle where the beetle was introduced. A small rubber

dispenser was placed in each arm of the olfactometer, one loaded with 100 µg

of synthetic dimethylfuran-lactone diluted in 50 µl hexane, and the other

loaded with 50 µl hexane (serving as control). The beetle’s position in the

arena was recorded every minute for a period of 30 min.

In the second part of the study the behavioural responses towards blends of

pheromone and respective host plant were investigated. This part included two

of the beetle species: G. pusilla (host plant: L. salicaria) and G. tenella (host

plant: F. ulmaria). The experimental procedure was the same as in the first

trial, but now the responses towards different odour combinations were tested.

In the first treatment there was a choice between a blend of pheromone and

host plant odour vs the pheromone alone and in the second treatment there

21

was a choice between the same blend vs host plant odour alone. The amount

of dimethylfuran-lactone was 100 µg diluted in 50 µl hexane for all

combinations. For treatments with plants, 10-15 cm branches of L. salicaria

and F. ulmaria were used. A few leaves on the branches were mechanically

damaged prior to the test, as there is usually a stronger response to damaged

compared to non-damaged plants.

Paper II

The aim of this study was to investigate the behavioural responses to different

host cues in A. parviclava, the parasitoid species attacking G. calmariensis,

G. pusilla and G. tenella (Figs. 4 and 5). First, the response to the pheromone

dimethylfuran-lactone, produced by males of G. calmariensis, G. pusilla and

G. tenella (Paper I) was investigated, in order to find out if A. parviclava can

exploit the pheromone as a host cue kairomone. In this part of the study

parasitoid females hatching from all three host species were included. The

behavioural responses of A. parviclava were studied in two-arm Y-tube

olfactometers, with an airflow of approximately 30 ml/s. Each arm of the Y-

tube was connected to a gas bottle, where a small rubber dispenser was placed.

In the first arm the dispenser was loaded with 100 g synthetic pheromone

(dimethylfuran-lactone) diluted in 50 l hexane and in the other arm the

dispenser was loaded with 50 l hexane (as a control). The parasitoids were

introduced to the Y-tube and given a couple of minutes to acclimatize without

airflow. Each parasitoid was observed for 5 min or until making a decisive

choice, which was recorded if the parasitoid passed two-thirds of an arm of

the Y-tube and stayed there for at least 5 seconds.

The second part of the study was to investigate the ability of the parasitoids to

distinguish between larvae of the different host species, based only on odour

cues. Due to insufficient numbers of parasitoids from G. pusilla, only

parasitoids from G. calmariensis and G. tenella were included in this part. The

general set-up was the same, using two-armed Y-tube olfactometers, but each

gas bottles was now loaded with a 10-15 cm host plant branch with 4 feeding

larvae (G. calmariensis and G. pusilla with L. salicaria and G. tenella with F.

ulmaria). Three different combinations were used: i) G. calmariensis vs G.

tenella larvae, ii) G. calmariensis vs G. pusilla larvae and iii) G. tenella vs G.

pusilla larvae.

22

Host immune response and parasitoid virulence (Paper

III and Paper IV)

Previous observations in the Galerucella-Asecodes system have shown

differences in parasitism rates between localities, with larvae in northern

localities generally showing a much higher parasitism rate (>70%) than larvae

in southern localities (<10%) [102], Hambäck, unpublished data. There have

also been observations of differences in parasitism rates between the species

[102, 103], indicating that G. pusilla (that does not occur in the north)

experiences a lower parasitism rate than the other two species. Based on these

observations, I wanted to investigate the structure of the immune system in

Galerucella (Paper III) and to detect whether the differences in the level of

parasitism were due to differences in the immune response in the three species

(Paper III and IV) or were caused by geographic variation in the ability of A.

parviclava to infect the hosts (Paper IV). I also wanted to investigate the

possibility that parasitoids hatching from one host species would have a higher

success when attacking larvae of the same species (Paper IV).

Paper III

In the first study (Paper III), only G. calmariensis and G. pusilla were

included. The A. parviclava used in the experiments all derived from northern

populations of G. calmariensis, as parasitoid abundance was much higher in

this area. The study was started by performing controlled parasitism

experiments, where laboratory-reared larvae of each species were put together

with a fixed number of A. parviclava females. After 24 h the parasitoids were

removed and the larvae were examined in a stereo microscope to detect

melanisation of wound sites (black dots) in the cuticle that would indicate

parasitoid attack. 96 h later the larvae were dissected in order to find out

whether they were successfully parasitized (containing live parasitoid larvae)

or showing a successful immune response (containing exclusively melanised

eggs).

The next step was to investigate the cell composition of the larvae, to find out

if there were any detectable differences between the species at the cellular

level. In connection to the dissections, hemolymph samples were prepared

from all larvae used in the parasitism experiments, as well as from non-

infested larvae reared in the laboratory and from larvae collected in the field.

To begin with, we had some troubles to establish a well-functioning method

for hemocyte preparation, as we found that the hemocytes of Galerucella

larvae rupture when in contact with air and the hemolymph coagulates very

quickly. To avoid hemolymph clotting the larvae were dissected submerged

in a well containing PBS mixed with a small amount of phenylthiourea (PTU).

23

All hemolymph samples were stained with blue-fluorescent nucleic acid stain

(DAPI) to reveal the nuclei in the cells. The samples were then studied in a

phase contrast, epifluorescent microscope connected to a Hamamatsu camera

with Axio Vision 4.6. Nine random images were taken from each individual.

As there were no previous cytological studies in these species, the first step

was to classify the different cell types in the hemolymph. Based on this

classification, differential hemocyte counts were performed for naïve and

parasitized larvae of both species. To identify phagocytic cells, fluorescently

labelled bacteria (E. coli) were injected into live larvae of both beetle species

30 min prior to dissection. To investigate which cell types were involved in

the encapsulation process, live and encapsulated eggs from infested

Galerucella were permeabilised with Triton-X and incubated with Phalloidin

and DAPI diluted in PBS.

Figure 5. Study species. A) The parasitoid A. parviclava attacking larvae of B) G. calmariensis C) G. pusilla and D) G. tenella. Photos: Robert Markus and Lisa Fors.

Paper IV

In the second immunological study (Paper IV), G. calmariensis, G. pusilla and

G. tenella were included, as well as A. parviclava originating from all three

host species (Fig. 5). This study had two aims: to investigate possible

geographic variation in parasitoid virulence and host immune response in the

Galerucella-Asecodes system, and to find out whether the former host species

of the parasitoid might have an effect on future parasitism success. As in Paper

III, the study was carried out by combining controlled parasitism experiments

with an investigation of the cell composition in the larval hemolymph. The

general set-up for the parasitism experiments was the same as in Paper III, but

in this study there was a distinction between parasitoids hatching from the

24

three host species, as well as between northern and southern populations of

both parasitoids and larvae. As far as possible, Galerucella larvae from all

three species and from both geographic areas (with an exception for G. pusilla

that is only present in the south) were parasitized with A. parviclava hatching

from all host species deriving from both geographic areas. However, there

were some gaps not possible to fill in the scheme of combinations, due to

insufficient numbers of parasitoids from southern populations of G.

calmariensis and G. tenella, as well as low numbers of G. tenella larvae. The

dissections and preparations of hemocyte samples were performed in the same

way as in Paper III, but only individuals that proved to be infected at dissection

were included in the cellular study.

25

Results and discussion

The first study, concerning chemical communication in the beetle host,

resulted in the finding that G. tenella both produces and responds to

dimethylfuran-lactone, the male aggregation pheromone previously observed

in G. pusilla and G. calmariensis [99], whereas G. lineola and G. sagittariae

were not found to produce or respond to the same pheromone (Paper I). Due

to these results, G. lineola and G. sagittariae were not included in the

behavioural study with pheromone and host plant odours. Unfortunately, G.

calmariensis also had to be excluded from this experiment, since the number

of beetles of this species available at the time of the experiment was too low

for meaningful analyses. The study showed that in the choice between the

blend and only the pheromone, males of G. pusilla preferred the blend. In G.

tenella, it was instead the females that preferred the blend over the pheromone

alone. In the choice between the blend and only the host plant, both sexes of

both species were significantly attracted to the blend.

It is likely that males of G. lineola and G. sagittariae produce other

pheromones even though they were not detected in this study. That the

pheromone was produced by G. tenella and not by G. lineola and G.

sagittariae seems fairly logical, since G. tenella is the species most closely

related to G. pusilla and G. calmariensis [95]. This suggests that pheromone

production and response may be connected to the phylogenetic relatedness

between the Galerucella species. The result was particularly interesting as the

three species producing the pheromone are all attacked by the same parasitoid

(A. parviclava), whereas G. lineola and G. sagittariae are attacked by two

separate Asecodes species. This led to the idea that A. parviclava might exploit

the adult host pheromone as a host cue kairomone. However, the results of the

following study suggested that the parasitoid females are unable to use the

pheromone to locate host larvae (Paper II). The lack of response to the

pheromone in A. parviclava could possibly be due to the fact that the

parasitoids attack the larval stage, which makes the male pheromone a less

reliable cue for locating a suitable host. Kairomones emitted by host adults are

commonly used by egg parasitoids, but more rarely by larval or pupal

parasitoids [34, 104, 105]. However, there are some studies showing that both

egg and larval parasitoids use pheromones for host habitat location and then

reside in the neighbourhood until the preferred host stage is available for egg-

laying [34, 106-108]. In Galerucella there is usually a period of 2-3 weeks

26

from pheromone emittance until the beetle larvae emerge, but there is

occasionally an overlap where adult beetles and larvae can be found

concurrently on the same plant, which could promote the idea of the adult

pheromone as a search cue.

The next part in Paper II suggested that A. parviclava hatching from both G.

calmariensis and G. tenella can detect hosts based on odour cues from the host

larvae and that they also have the ability to distinguish between host species

from a distance. These results were even more interesting in the light of the

results from the immunological studies, where larvae of G. pusilla were found

to have a much more potent immune defence towards A. parviclava than

larvae of G. tenella and G. calmariensis (Paper III and Paper IV).

Furthermore, parasitoids from both G. calmariensis and G. tenella were found

to have much higher success rates when attacking larvae of their former

respective host than larvae of G. pusilla (Paper IV). The results from Paper II

revealed that parasitoids from both G. calmariensis and G. tenella have a

preference for their former respective host species over the well defended host

G. pusilla. Parasitoids from G. calmariensis also had an ability to distinguish

larvae of G. tenella from larvae of G. pusilla, whereas parasitoids from G.

tenella did not distinguish between larvae of G. calmariensis and G. pusilla.

Notable is however that all A. parviclava individuals used in this study derived

from northern localities, where G. pusilla is not present. Thus, a positive

attraction for G. calmariensis and G. tenella larvae must have evolved in A.

parviclava in the north, since there cannot be any selection pressure on these

parasitoids to avoid larvae of G. pusilla.

At this point we have no information on which specific odour cues Asecodes

uses for host search and host selection, although our study indicates that the

most important cue is likely to be produced by the larvae. Some chemical

analysis have been performed on the odour from feeding larvae, but so far no

key differences have been found. Furthermore, the study does not show

whether A. parviclava can detect larval odours from a further distance. One

possibility is that the parasitoids use different cues to first locate the host

habitat and larval odours only when in closer range of the host. Even though

no response to the adult pheromone was observed in this study, it is still

possible that A. parviclava could respond to the pheromone in combination

with odours from the correct host plant. No such tests were performed (due to

low numbers of parasitoids) but it is something that could be worth

investigated further. A. parviclava has previously been shown to respond to

green leaf volatiles released from damaged L. salicaria and F. ulmaria, with

a strong preference for F. ulmaria [103]. However, in the study by Stenberg

et al., there was no distinction between parasitoids from the two species, which

makes it unclear whether the preference for F. ulmaria is true for A. parviclava

from both G. calmariensis and G. tenella. Further studies on search behaviour

27

in A. parviclava should preferably include also parasitoids from G. pusilla and

parasitoids from southern populations of G. calmariensis and G. tenella.

The immunological studies (Paper III and Paper IV) revealed large differences

in the level of successful immune response in the three host species G. pusilla,

G. calmariensis and G. tenella. Larvae of G. pusilla showed a much more

potent immune defence towards A. parviclava than the other two species, and

G. tenella showed a stronger defence than G. calmariensis (although still

much weaker than G. pusilla). This was in accordance with previous field

observations, suggesting that G. pusilla in general experiences a lower

parasitism rate than G. calmariensis and G. tenella. In the first study (Paper

III) where only G. pusilla and G. calmariensis were included, the difference

between the species was striking; no infected larvae of G. calmariensis

managed to suppress the parasitoid attack whereas all infected G. pusilla

showed a successful immune response. One thing that could potentially

influence the results of this study, was the fact that only parasitoids deriving

from G. calmariensis were used for the parasitism experiments. This

suggested an adaptation in the parasitoids to the former host species (G.

calmariensis), that would potentially explain the low parasitism success in G.

pusilla.

To a large extent, this idea proved to be true in the following study (Paper IV),

where G. tenella was included, as well as parasitoids deriving from all three

host species. In Paper IV it was clear that former host species of the parasitoid

had an effect on parasitism success. Accordingly, parasitoids with G. pusilla

as former host had much higher success rates when attacking G. pusilla larvae

than parasitoids from the other two species. Parasitoids from G. tenella also

had much higher success rates when attacking G. tenella larvae than

parasitoids from G. calmariensis. However, parasitoids from G. pusilla was

almost equally successful as parasitoids from G. tenella when attacking G.

tenella larvae. When combining data from all parasitism experiments

(regardless of parasitoid origin), the results showed an overall strong immune

defence in larvae of G. pusilla, a somewhat intermediate defence in G. tenella

and a very poor defence in G. calmariensis (Paper III and Paper IV). It is likely

that parasitoids from G. pusilla have developed an ability to overcome the

strong defence of the host and thereby become more effective also when

infecting larvae of the other two species. Accordingly, parasitoids from G.

calmariensis are generally less successful when attacking larvae of both G.

pusilla and G. tenella, as they developed in the host with the overall poorest

immune defence.

Although there was strong evidence that G. pusilla had a more potent immune

response than G. calmariensis and G. tenella, we did not know the underlying

cause for this difference, which led to further investigations on the cellular

28

level. Based on morphological characteristics, six types of hemocytes were

distinguished in non-infested and infested individuals: granulocytes,

phagocytes, prohemocytes, oenocytoids, lamellocytes and lamellocyte

precursors (Fig. 6). When studying the parasitoid eggs in the microscope, no

cell activity was found on the live eggs in G. calmariensis, whereas several

layers of cells were attached to the surface of the encapsulated eggs in G.

pusilla and G. tenella. The cytological studies revealed that the successful

encapsulation of parasitoid eggs in Galerucella involves at least three different

cell types: lamellocytes, phagocytes and granulocytes (Paper III).

Granulocytes could be detected by their autofluorescence in the green channel,

lamellocytes by phalloidin staining and phagocytes by engulfed fluorescent

bacteria. Large clusters of granulocytes were observed surrounding the

melanised capsules, as well as granulocyte content on the surface of the eggs,

suggesting cell rupture. When dissecting larvae in paraformaldehyde (PFA),

we found that the rupture of the granulocytes takes approximately 50 ms,

indicating that it is a rapid way to deliver the content of the cell to the wound.

Thus, we suggest that granulocytes in Galerucella, similar to crystal cells in

Drosophila [109], ruptures and delivers its cargo locally at the wound or

infection site. The results in Paper III indicated that lamellocytes are crucial

for the capsule formation to be completed, something that is supported by

previous findings in Drosophila melanogaster, where lamellocytes are

essential for encapsulation of parasitoid eggs [110]. Phagocytes also

contribute to the encapsulation in D. melanogaster, which further supports our

observations in Galerucella. We believe that also the oenocytoids may have

an active role in the immune defence, even if we have not yet been able to

reveal their specific function in Galerucella (Paper III and Paper IV).

Oenocytoids have been shown to participate in the melanisation process in

other species [111, 112].

Figure 6. Morphology of hemocytes in Galerucella. A) Granulocytes B) Phagocytes C) Prohemocyte D) Oenocytoid E) Lamellocyte F) Lamellocyte precursors. Cell nuclei are stained with DAPI (blue). Photos: Robert Markus and Lisa Fors.

The differential hemocyte counts showed that the hemocyte ratios differ

between the species and that there is strong connection between hemocyte

composition and the ability of the larva to mount an effective immune

response against A. parviclava (Paper III and Paper IV). The results in Paper

III revealed significant differences in hemocyte ratios between naïve and

parasitized individuals, indicating a cellular response. In particular, the levels

29

of the cell types shown to be active in the encapsulation process, phagocytes,

lamellocytes and granulocytes, changed upon infection. Lamellocytes and

phagocytes were found to increase in numbers following parasitoid infection,

whereas granulocytes decreased. There were also significant differences in

hemocyte composition between the two species (G. pusilla and G.

calmariensis), both in naïve and parasitized individuals, reflecting their ability

to defend against the parasitoid. The strong defence in G. pusilla was

connected to high levels of lamellocytes and phagocytes. In general, the same

pattern was found in the following study (Paper IV), but here only infested

individuals from G. pusilla and G. tenella were included, as the immune

response in G. calmariensis did not differ depending on former parasitoid host

species or geographic origin. Larvae of G. pusilla showed higher levels of

both lamellocytes and phagocytes when infected by parasitoids from G.

calmariensis or G. tenella, towards which the strongest immune response was

observed, than when infected by parasitoids from G. pusilla. Accordingly,

larvae of G. tenella showed a much higher level of lamellocytes when infected

by parasitoids from G. calmariensis, towards which the strongest immune

response was observed, whereas the level of phagocytes was equally high in

larvae infected by parasitoids from G. calmariensis or G. tenella.

Interestingly, the constitutive level of oenocytoids was found to be much

higher in G. tenella than in G. calmariensis and G. pusilla, indicating that the

cellular immune defence might be somewhat different in this species.

Moreover, the levels of oenocytoids were particularly high in G. tenella

individuals infected with parasitoids from G. calmariensis (towards which the

strongest immune response was seen), indicating a cellular response (Paper

IV). In some of the infected G. tenella larvae we also observed a type of large

cell structures which had not been seen previously in Galerucella. The

structures contained several nuclei and had the appearance of long ribbons of

connected cells (Paper IV). These cells we believe to be similar to the

multinucleated giant hemocytes that were recently described in Drosophila,

where they have a function in parasite elimination [113]. However, as no

functional tests were performed on the multinucleated cells observed in G.

tenella, we were unable to detect their potential role in the immune response.

Clearly, the very poor immune defence in G. calmariensis is reflected by the

cell composition in both naïve and parasitized larvae. However, there are some

things worth to keep in mind connected to the immune response. Even though

the cell ratios differ between the species, all cell types are shown to be present

in G. calmariensis (Paper III). This means that the poor immune defence

cannot simply be due to the lack of a crucial cell type, i.e. the lamellocytes.

When going through hemocyte samples from numerous individuals, we have

occasionally come across samples with fairly high ratios of lamellocytes also

in G. calmariensis, suggesting some variation within the species. Further, both

30

melanisation and encapsulation are possible processes in G. calmariensis.

Melanisation at the wound site is very common in G. calmariensis, in fact it

is often more easily detected than in G. tenella or G. pusilla. We have also

observed proper capsule formation and melanisation of the parasitoid eggs in

a few G. calmariensis individuals (both from the parasitism experiments and

from the field), even though the encapsulation is very rare compared to what

is observed in the other species.

There are also other aspects to take into account regarding the defence towards

the parasitoid. A strong immune defence is costly on two levels; one is the

cost of the actual defence after attack and one is the cost of having the ability

to mount a successful immune response [86]. Many studies have shown life

history trade-offs between growth, reproduction and immune competence in

insects [20, 114]. We know from previous studies that larvae of G.

calmariensis has a higher growth rate (15%) than G. pusilla in the field and

the adults are also larger in size [96]. The possible result of a high larval

growth rate is a shorter exposure of G. calmariensis larvae to parasitoids, and

consequently a lower risk for parasitism.

Even though the cellular immune response in G. calmariensis does not protect

it against the parasitoid, the larva is not completely defenceless. When

monitoring Galerucella larvae in parasitism experiments in the laboratory, we

have often observed a type of behaviour that resembles the active defence seen

in other parasitized species, for example the European pine sawfly,

Neodiprion sertifer [115]. If attacked by a parasitoid, N. sertifer larvae can be

seen vigorously flipping the upper part of the body in order to scare the

parasitoid away. In connection to this, the larvae sometimes regurgitate part

of the gut contents in sticky droplets. Galerucella larvae often show a similar

behaviour when encountered by Asecodes parasitoids; they flip or twist both

head and tail, sometimes in combination with blowing sticky bubbles from the

mouth (Fig. 7). Whether this behaviour differs between the Galerucella

species is not investigated, but so far we have mainly observed it in larvae of

G. calmariensis.

Figure 7. Active defence mechanism in G. calmariensis when attacked by A. parviclava. Photo: Robert Markus.

31

An additional aim with Paper IV was to investigate geographical variation in

parasitism success. Due to missing combinations in the parasitism

experiments the geographic effect on the immune response was a bit more

difficult to interpret. Previous field observations have shown that parasitism

rates on G. calmariensis are much higher in the north (>70%) than in the south

(<10%). Due to the results of Paper III, we initially suspected that this

difference was due to differences in the strength of the immune response.

However, as revealed in Paper IV, G. calmariensis has a very weak immune

defence regardless of geographic origin. In G. tenella, there was an indication

of a stronger defence in larvae from northern compared to southern

populations towards parasitoids from G. tenella and G. calmariensis, but no

significant differences could be detected. The immune defence in G. pusilla,

on the other hand, was found to be affected by the geographic origin of the

parasitoids, which suggests geographic differences in parasitoid virulence.

Larvae of G. pusilla (which only occurs in the south of Sweden) showed a

weaker defence towards parasitoids deriving from southern localities of G.

calmariensis than towards parasitoids deriving from northern localities. In

larvae of G. tenella, the effect of the parasitoids’ geographic origin on the

immune defence was hard to interpret due to missing test combinations. Thus,

it would be very interesting to further investigate the geographic variations of

immune defence and parasitoid virulence in this system, especially in

connection to further experiments on host preference in the parasitoid.

32

Concluding remarks

This thesis shows the importance of gathering information from different

fields, in order to better understand the interactions and evolution in natural

host-parasitoid systems. In particular, through combining studies of

behavioural ecology with chemical ecology and molecular biology, a lot of

knowledge can be gained, linking host selection to parasitoid virulence and

host immune response. In the studies performed, I have found that there are

large differences in immune competence between the Galerucella species,

which can be linked to differences in parasitism rates observed in the field.

The differences in immune defence closely correspond to the hemocyte

composition in the species. Further, the results suggest that parasitism success

in A. parviclava is strongly affected by former host species of the parasitoid.

In connection to this I have also observed a potential ability in A. parviclava

to select a host with weaker immune response from a distance. Taken together,

the results of this thesis suggest that there is an on-going evolution in both

parasitoid virulence and host immune responses in this system. Although

many questions remain to be answered, the Asecodes-Galerucella system has

proven to be a useful model system for investigating processes that may lead

to host race formation and speciation in host-parasitoid systems.

Acknowledgements

I want to thank Peter Hambäck and Ulrich Theopold for constructive

comments on earlier versions of this text, Robert Markus for help with editing

the photos and Mathilda Arnell for help with the map of field localities.

33

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Svensk sammanfattning

En viktig grundsten inom ekologisk forskning är att försöka förstå samspelet

mellan olika arter. Artinteraktioner beskrivs ofta som antingen mutualistiska

eller antagonistiska. En mutualistisk interaktion kännetecknas av att den är till

nytta för samtliga inblandade parter, vilket man till exempel kan se hos

specialiserade pollinatörer som är knutna till en viss växt. Antagonistiska

interaktioner förekommer istället mellan konkurrerande arter eller i system

med två eller flera trofiska nivåer. Trofiska samspel förekommer exempelvis

mellan herbivorer och växter, predatorer och bytesdjur eller mellan parasiter

och deras värddjur. I många trofiska interaktioner kan vissa arter vara

beroende av andra för att överleva. Förändras en art på något vis kan andra

arter kopplade till denna art behöva anpassa sig till förändringen. Det kan

medföra att interagerande arter följs åt i sin utveckling i en växelverkande

process, så kallad samevolution.

I parasit-värdsystem råder en ständigt pågående evolutionär kapprustning, så

kallad ”arms race” mellan arterna, där varje part utövar ett starkt

selektionstryck på den andra. Värden måste utveckla ett försvar mot den

attackerande parasiten och parasiten behöver i sin tur övervinna värdens

försvarsmekanismer. Parasiter anses ofta ha en viss fördel i den biologiska

kapprustningen, på grund av korta generationstider och stora populationer.

Om parasitarten har förmågan att snabbt anpassa sig till nya

försvarsmekanismer hos värdarten kan det leda till en lokal anpassning hos

parasiten, som då uppvisar en högre virulens (det vill säga en bättre förmåga

att infektera värdarten) i lokala värdpopulationer.

En särskild grupp av parasiter utgörs av parasitoider. De är frilevande insekter

vars avkomma uteslutande livnär sig på en annan insekt, vilket ovillkorligen

leder till värdinsektens död. Parasitoider kan delas upp i ektoparasitoider,

vilka utvecklas på ytan av sin värd och endoparasitoider, vilka utvecklas inuti

värden. Endoparasitoider anses vanligtvis ha en mer komplex relation till sin

värd, eftersom de måste övervinna värdens försvar men samtidigt hålla värden

vid liv tillräckligt länge för att själva kunna utvecklas. Parasitoider attackerar

vanligtvis ett relativt specifikt urval av värdarter och är ofta specialiserade på

ett visst levnadsstadium hos värden. Detta i samband med den nära relationen

till värdarten gör parasitoid-värdsystem särskilt väl lämpade för studier av

lokal anpassning och arters samevolution. För att få en bättre bild av hur

41

arterna i systemet påverkar varandra måste en rad olika aspekter tas i

beaktande, såsom sökbeteende och värdselektion hos parasitoiden, utveckling

av försvarsmekanismer hos värden och strategier hos parasitoiden för att

övervinna värdens försvar.

Precis som växtätande insekter behöver hitta rätt värdväxt måste också

parasitoider söka upp en lämplig värd för sin avkomma. Sökprocessen är i

stora drag densamma i båda fallen och kan delas upp i tre steg, där det första

steget är att lokalisera själva habitatet där värdväxten respektive värdinsekten

med stor sannolikhet kan påträffas. Nästa steg är att söka sig fram till rätt

värdart inom habitatet, för att slutligen avgöra om den aktuella värdindividen

är lämplig eller ej. Sökbeteendet hos växtätande insekter styrs av olika

signaler, såsom syn-, doft- och smakintryck. Vilken typ av signal som är

viktigast varierar mellan olika typer av insekter, men doftsignaler hör till de

vanligast förekommande. När en växt utsätts för angrepp av växtätande

insekter förändras de dofter som växten sänder ut. Detta innebär att insekter

som redan funnit sin värdväxt kan locka till sig fler insekter av samma art (och

eventuellt även andra insektsarter) genom att äta på växten. Doftsignaler kan

även komma från insekterna själva. Doftämnen som produceras och används

av insekter i avseende att kommunicera med individer av samma art kallas

feromoner. I likhet med växtätande insekter utnyttjar parasitoider ofta

doftsignaler för att hitta sin värd, även om en del arter har andra sökstrategier.

Många parasitoider utnyttjar samma söksignaler som värdarten, antingen

växtdofter eller feromoner från värdinsekten.

När parasitoiden väl lokaliserat en lämplig värd och lagt sina ägg gäller det

för värdinsekten att kunna försvara sig. Insekter har endast ett medfött

immunförsvar och saknar det adaptiva immunförsvaret som finns hos

ryggradsdjur. Trots detta finns det flera olika typer av försvarsmekanismer hos

insekter, vilka styrs antingen av hormoner eller av hemocyter (insektens

motsvarighet till blodceller). Det vanligaste försvaret mot parasitoider är att

hemocyter aggregerar runt parasitoidägget och kapslar in det. Under denna

process frigörs också ofta melanin, vilket gör att kapseln mörknar. I samband

med detta hårdnar kapseln och det inkapslade ägget dör. På samma sätt som

värdinsekten behöver undvika parasitering för att överleva är det också ett

starkt selektionstryck på parasitoiden att skydda sin avkomma från att bli

utslagen av värdens försvarssystem. Det finns en mängd olika strategier hos

parasitoider för att manipulera värdinsekten och skapa en gynnsam miljö för

utveckling av nästa generation. De kan till exempel gömma sina ägg genom

att lägga dem i ett organ eller i en vävnad som inte är tillgänglig för

cirkulerande hemocyter, vilket innebär att äggen inte kan bli inkapslade.

Jag har i denna avhandling undersökt interaktioner i ett parasitoid-värdsystem,

med fokus på värdselektion, parasiteringsframgång och immunförsvar.

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Systemet jag använt består av fem skalbaggsarter av släktet Galerucella

(Galerucella calmariensis, G. pusilla, G. tenella, G. lineola och G.

sagittariae) och tre parasitoidarter av släktet Asecodes (Asecodes parviclava,

A. lineophagum och A. lucens). G. pusilla och G. calmariensis delar

fackelblomster (Lythrum salicaria) som sin enda värdväxt, medan övriga arter

kan utnyttja flera olika växtarter. Parasitoiden attackerar skalbaggarnas

larvstadium och lägger sina ägg inuti larven. När äggen kläckts börjar

parasitoidlarverna att konsumera värdlarven inifrån. Larven utvecklas till

synes normalt, men när det är dags för den att förpuppa sig har den inte

förmågan att bilda en vanlig puppa, utan förvandlas till ett svart, mumifierat

skal. Från den mumifierade larven kläcks sedan nästa generation av

parasitoiden.

I min första studie undersökte jag produktionen av feromoner hos

skalbaggarna. Tidigare studier har visat att de två mest närbesläktade arterna,

G. calmariensis och G. pusilla, producerar samma feromon när de äter på sin

värdväxt. Jag började med att samla upp doftämnen från varje skalbaggsart

och respektive värdväxt med hjälp av SPME (Solid Phase Micro Extraction),

vilket är en teknik där en porös fiber används för att absorbera luftburna

ämnen, vilka sedan kan separeras och analyseras med hjälp av

gaskromatografi -masspektrometri. Analysen visade att även en tredje art, G.

tenella, producerar samma feromon. Nästa steg i studien var att undersöka

huruvida de olika arterna reagerade på feromonet. Försöket utfördes i en

tvåarmad olfaktometer, vilket är en konstruktion där skalbaggen (eller någon

annan insekt) får möjlighet att välja mellan två dofter som släpps in från olika

håll.

Det visade sig att de tre arter som producerar feromonet också var attraherade

av det, medan ingen reaktion kunde observeras hos de övriga två arterna, G.

lineola och G. sagittariae. Resultatet var särskilt intressant eftersom de tre

feromon-producerande arterna alla parasiteras av samma parasitoidart, A.

parviclava. Detta väckte frågan om feromonet var något som även

parasitoiden kunde utnyttja i sitt sökande efter en lämplig värd. I följande

studie undersökte jag därför hur A. parviclava reagerar på olika värdrelaterade

doftsignaler. Först testade jag preferensen för feromonet med hjälp av

tvåarmade olfaktometrar, men detta försök kunde inte påvisa någon attraktion

hos parasitoiden. En möjlig orsak till detta kan vara att parasitoiden endast

attackerar larvstadiet, vilket innebär att feromon från adulta skalbaggar inte

nödvändigtvis är en pålitlig söksignal. Nästa steg var att undersöka huruvida

A. parviclava har förmågan att skilja mellan larver från två värdarter enbart

baserat på doftsignaler. Detta försök visade att parasitoiden kan särskilja arter

och även till viss del tycks ha förmågan att välja en bättre lämpad värdindivid

framför en sämre.

43

I den första studien av immunförsvaret hos Galerucella var bara G.

calmariensis och G. pusilla inkluderade. Bakgrunden till denna studie var att

tidigare observationer indikerat ett lägre parasiteringstryck på G. pusilla i

Sverige, vilket ledde till frågan om det fanns skillnader i arternas försvar mot

parasitoiden. Första steget i studien var att utföra kontrollerade

parasiteringsförsök, där larver från respektive art sattes ihop med ett bestämt

antal A. parviclava under 24 timmar. Larverna dissekerades några dagar

senare för att fastställa om de var framgångsrikt parasiterade, dvs. innehöll

levande parasitoidlarver (och därmed kunde antas ha ett svagt immunförsvar).

De skalbaggslarver som uppvisade ett starkt immunförsvar innehöll istället

endast inkapslade, döda parasitoidägg. Studien visade en tydlig skillnad

mellan de två arterna, där G. pusilla visade sig ha ett mycket starkare försvar

mot parasitoiden. För att försöka finna orsaken till den stora skillnaden i

framgångsrikt försvar studerades också cellkompositionen hos

skalbaggslarverna. Resultaten visade att nivåerna av olika celltyper skilde sig

signifikant mellan arterna. I samband med detta undersöktes inkapslade

parasitoidägg från G. pusilla och levande parasitoidägg från G. calmariensis i

mikroskop. Jag kunde då finna att de inkapslade äggen hade flera lager av

hemocyter på ytan, medan ytan på de levande äggen var helt fria från celler.

Med hjälp av olika infärgningsmetoder kunde jag fastställa vilka celltyper hos

Galerucella som är inblandade i inkapslingen av parasitoidägg.

Nästa studie inkluderade G. calmariensis, G. pusilla och G. tenella, vilka alla

som tidigare nämnts parasiteras av A. parviclava. Denna studie hade två

syften: dels att undersöka om någon geografisk variation kunde påvisas i

parasitoidens virulens och i värdens immunförsvar och dels om

parasitoidhonans tidigare värdart kunde påverka framtida

parasiteringsframgång. Försöken utfördes i huvudsak på samma sätt som i den

föregående studien, men skillnaden var att parasitoiderna nu delades upp efter

vilken värdart de kläcktes från, samt att både parasitoider och skalbaggar

delades upp i nordliga och sydliga populationer. I den utsträckning det var

möjligt korsades sedan parasitoider från alla grupper med larver från alla

skalbaggsgrupper. Denna studie visade i likhet med föregående att G. pusilla

har det starkaste försvaret mot parasitoiden och G. calmariensis ett näst intill

obefintligt försvar, medan G. tenella visade sig ligga ungefär mitt emellan de

två andra arterna försvarsmässigt. Studien visade också att parasitoidens

tidigare värdart har en stor betydelse för parasiteringsframgången. Den

geografiska effekten var något svårare att tolka, men hos G. pusilla (som

endast förekommer i södra Sverige) kunde man se en tydlig skillnad i försvar

beroende på om parasitoiderna kom från sydliga eller nordliga populationer.

Även resultaten från denna studie kunde styrkas med cellstudier, där

skillnader uppvisades mellan de olika arterna, samt mellan individer av

samma art beroende på vilken parasitoid de attackerats av.

44

Sammanfattningsvis kan sägas att resultaten av studierna i denna avhandling

antyder att det råder en pågående utveckling av såväl parasitoidens virulens

som värdens immunförsvar i systemet Asecodes-Galerucella. Studierna visar

också nyttan av att samla information från olika vetenskapliga områden för att

bättre kunna förstå interaktioner och evolution i parasitoid-värdsystem.

Tack

Jag vill tacka Tommy Martinsson och Torbjörn Fors för konstruktiva

kommentarer på tidigare versioner av denna sammanfattning.

45

Tack/Acknowledgements

Jag vill börja med att tacka min handledare Peter som till stor del är ansvarig

för att jag halkade in i den spännande insektsvärlden. Tack för din eviga

entusiasm, för god handledning och för bra stöd när jag emellanåt blivit lite

uppgiven. Stort tack också till Uli som varit min närmaste biträdande

handledare. Tack för att du introducerat mig i den fascinerande

insektsimmunologin och för att jag fått husera i ditt lab. Tack till Anna-Karin

som varit min handledare på den kemiska sidan. Tack också till Jon. Tack

Lasse som varit till ovärderlig hjälp vid insamlandet av skalbaggar i norr. Tack

Johan S som lyssnat på många funderingar kring skalbaggar och parasitoider

och kommit med kloka råd.

Very special thanks to Robert, who has been my closest co-worker in this

project. Thanks for your patience in the beginning, for your excellent

photography, for your knowledge and your curiosity. Thanks for all the fun

(and exhausting!) times we have shared in the lab. Without you this would not

have worked. Best of luck in the future.

Thanks to all the nice and helpful people in Uli’s lab who always made me

feel welcome. Special thanks to Lucie for assisting me in the lab work towards

the end.

Det finns en hel massa människor från gamla Botan som jag vill tacka för att

de gjort min tid på institutionen så trevlig och givande. Till att börja med vill

jag tacka ”Oldies but Goldies”: Lenn, Ove, Johan E, Lena, Kristoffer och

Kjell. And welcome to Ayco, the newest addition to the adult crew! Särskilt

tack till Johan E för uppmuntrande tillrop när jag kört ”kvällspass”. Tack Ove

för kloka råd och intressanta litteraturseminarier. Tack Kristoffer för en

välorganiserad och intressant resa till Etiopien.

Tack till mina halvgamla doktorandkollegor: Johan D, Ellen, Elsa, Ulrika,

Alma, Tenna och Thomas. Och tack Tiina som varit med lite på distans!

Välkommen till den nya doktorandskaran: Matilda, Pil och Beate. Tack för

alla givande diskussioner, fikastunder, luncher och för att ni alla har bidragit

till att jag tyckt det känts så kul att gå till jobbet. Lycka till allihop! Good luck!

46

Särskilt tack till Elsa, för att du varit en fantastisk rumskamrat och för att du

stått ut med min alltmer stressade framtoning och mitt alltmer begränsade

ordförråd under de senaste månaderna. Tack också till mina gamla

rumskamrater Johan D och Alma. Tack Matilda för all uppmuntran och för

god hjälp med kartor och statistik. Stort tack till Tenna (och David!) för allt

roligt (jag menar morsomt) vi har gjort under de här åren. Resan till Grand

Canyon var en av höjdpunkterna under doktorandtiden!

Tack också till alla tidigare doktorander, tidigare och nuvarande post-docs,

assistenter och alla andra trevliga prickar som lyst upp tillvaron under min

doktorandtid: Malin, Karin L, Tove, Helena, Petter, Niklas, Debissa,

Veronica, Bryndis, Gundula, Jocke, Johan Dahlgren, Victor, Jörgen, Eric, Lisa

W, Maria J, Alicia, Jessica, Anna H, Anna L, Maria E, Karin K och alla ni

andra. Thank you all! Särskilt tack till Malin för all hjälp med statistikfrågor

och för kloka råd i det lätt panikslagna slutskedet av mitt

avhandlingsskrivande. Stort tack också till Petter för gott samarbete och

intressanta insektsdiskussioner!

Sen finns det ju några till…

Stort tack till Lina för ditt stöd, din sköna stil och för alla våra knasiga idéer

och fantastiska pubar. Det hade inte alls varit lika kul att doktorera utan dig!

Tack också till Johan K och Leila för tokiga upptåg, operabesök och

festligheter. Särskilt tack till Leila för all hjälp med att hitta rätt i den

administrativa djungeln. Du är fantastisk! Och Johan, tack för all hjälp med

datorerna… Och tack för musiken! Våra potpurrier förtjänar extra

uppmärksamhet. Tack också Johan D och Johan Eklöf som båda varit med på

ett musikaliskt hörn. Mer musik åt forskarna! Stort tack till Peter och Ingela i

växthuset för trevliga pratstunder, frön av olika slag och alla goda råd om

plantering och odling. Tack Erik för all hjälp med praktiska (och ibland rätt

opraktiska) saker.

Det finns också en hel massa människor utanför jobbet som jag vill tacka.

Tack till alla fina vänner för uppmuntran och för att ni får mig att tänka på

annat när det behövs! Tack Cajsa, Affe, Yonas, Pia och Anders för

folkmusiken. Tack Fredrik för alla roliga spelkvällar och för att du utan

protester agerat marsvinsvakt flera gånger. Tack Erik S för grammatiska råd

och intressanta diskussioner om det engelska språket. Tack Susanne för att du

är en så fin vän som är positiv till allt jag gör även när det är helt obegripligt.

Tack Jenny för ditt stöd, dina kloka råd och för alla trevliga pubkvällar. Tack

Katarina och Lasse för sushi och mysiga filmkvällar. Tack Anna Maria och

Mattias för middagar och brädspel och tack Anna Maria för att du emellanåt

har lika usel filmsmak som jag! Tack Helena och Erik för alla trevliga

pratstunder och goda luncher. Tack Kajsa och Pär för att ni är så roliga och

47

hjälpsamma. Snart har jag tid att hälsa på igen! Tack Malin för att du är så

positiv. Tack Maggan och Emil för trevliga middagar och tack Maggan för

alla stärkande promenader! Tack bästa Nina. Fast du är den av mina vänner

som befinner sig absolut längst bort känns du alltid nära. But mark my words,

if you are not coming home soon I will go under. Down under.

Many thanks to all my lovely friends in Brighton (my home away from home).

Very special thanks to Michal for all the fun times (not to forget our endless

“Brummy” tournament) and for putting up with me occupying your living-

room on a regular basis. Love you heaps.

Stort tack också till hela min härliga familj. Särskilt tack till Peter som lagat

mina bilar och därmed räddat mig från att bli stående långt ut i skogen under

någon fälttur. Stort tack till min syster Anna, David, Josef, Olle och Stina för

att ni är så fantastiska! Tack för all hjälp och uppmuntran och för alla festliga

middagar, fikastunder, skojiga aktiviteter och spelkvällar. Tack mamma och

pappa för att ni är de bästa och roligaste föräldrar man kan tänka sig, som alltid

ställer upp (även när det gäller att inhysa och utfodra hundratals skalbaggar).

Tack för ert eviga stöd och för att ni alltid tror att jag kommer lyckas.

Till sist, stort tack till världens bästa Tommy! Utan dig hade livet varit

ohyggligt trist. Du kanske är den enda som till fullo förstår vitsen med att lösa

gåtor på British Museum, laga hundratals hemliga små rätter, tillverka egna

brädspel och titta på samma filmer om och om igen. Tack för att du är min

bästa vän. You are the light in dark places, when all other lights go out.