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Unraveling the ecology of the dune aphid Schizaphis rufula

(Hemiptera: Aphidoidea):

Ecological preferences and Parasitoids (Hymenoptera)

Charlotte Van Moorleghem

Promotor: Eduardo de la Peña

Thesis submitted to obtain the degree of Master of Science in Biology

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Image on front cover: Schizaphis rufula on its host plant Ammophila arenaria. © Eduardo de la Peña

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Table of contents

Abstract ……………………………………………………………………………………………………………………………........ 2

Introduction

1. Sand dune landscape …………………………………………………………………………………………………… 2 2. Sand dune community …………………………………………………………………………………………………. 4 3. Schizaphis rufula ………………………………………………………………………………………………………….. 6 4. Parasitoids …………………………………………………………………………………………………………………… 7

Aims …………………………………………………………………………………………………………………………………………. 8

Material and methods

1. Study design

a. Ecological preferences

Study sites …………………………………………………………………………………………… 9

Study design ……………………………………………………………………………………….. 9

b. Plant suitability experiment

Preparations ………………………………………………………………………………………… 11

Experiment ………………………………………………………………………………………….. 12

c. Identification of parasitoids

Morphological identification ……………………………………………………………….. 12

Molecular identification ………………………………………………………………………. 13

2. Statistical analysis

a. Ecological preferences …………………………………………………………………………………….. 14

b. Plant suitability experiment …………………………………………………………………………….. 14

Results

1. Ecological preferences

a. Probability of aphid encounter ………………………………………………………………………… 15

b. Aphid population dynamics …………………………………………………………………………….. 17

2. Plant suitability experiment …………………………………………………………………………………………. 17

3. Identification of parasitoids

Morphological identification ……………………………………………………………………………. 18

Molecular identification …………………………………………………………………………………… 21

Discussion

1. General findings ………………………………………………………………………………………………………….. 22

2. Ecological preferences: underlying mechanisms …………………………………………………………. 22

3. Ecological preferences and their meaning for the functioning of a dune ecosystem…… 25

4. Parasitoids: the importance of finding new associations …………………………………………….. 27

5. Overall conclusions ………………………………………………………………………………………………………. 29

Summary …………………………………………………………………………………………………………………………………. 29

Acknowledgements …………………………………………………………………………………………………………………. 31

References ………………………………………………………………………………………………………………………………. 31

Appendix

1. Tables …………………………………………………………………………………………………………………………. 37

2. Glossary ……………………………………………………………………………………………………………………… 41

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Abstract Schizaphis rufula is an aphid species which is highly abundant in coastal sand dune landscapes across

Europe. In these habitats it is usually found on sand stabilizing dune grasses like Ammophila arenaria

and Leymus arenarius. These species have a large impact on their surroundings through their ability

of fixating large sand masses. Despite S. rufula being a potential candidate for influencing local

populations of such sand stabilizing grasses, the ecology of this herbivore is relatively unknown. Our

aims were to determine 1) the influence of the surrounding plant community on population densities

of S. rufula associated with A. arenaria, 2) the specificity of the aphid’s host choice within and

between species and 3) the parasitoid community associated with S. rufula. Through a field study and

a plant suitability experiment it was found that the presence of S. rufula on its host plant A. arenaria

is mainly determined by biotic environmental factors that also indicate dune fixation, namely species

richness and percentage cover by Festuca rubra within a one meter radius from the monitored plant

individual. S. rufula is more often present on A. arenaria in a species poor environment with a low

presence of the dune grass F. rubra. These conditions are characteristic for mobile dunes where only

specialized plants occur that can endure the extreme circumstances linked to these kind of habitats.

In addition to these findings, a preference of the aphid for dune stabilizing grasses, like A. arenaria

and L. arenarius, was found. These mobile dune species also showed a decline in biomass when

occupied by aphid populations in the plant suitability experiment. Both the result of the field survey

as those from the plant suitability experiment indicate the potential of S. rufula to impact population

dynamics of primary dune fixating grasses and consequently also dune dynamics. The parasitoid

wasps Aphidius rhopalosiphi and A. avenae (Hymenoptera: Aphidiinae) where for the first time

observed to parasitize on S. rufula. Also hyperparasitoids from the genus Apoanagyrus sp.,

Dendrocerus sp. and Pachyneuron sp. (Hymenoptera) were found.

Introduction Studying species ecology does not only give us greater knowledge of the species itself, but also of the

functioning of the environment and habitat quality (Memmott, 2009). When studying species

ecology, however, there must also be attention for the abiotic components of the environment next

to the biotic component (e.g. interactions between species). This is particularly the case when the

environment has very specific characteristics, like in a coastal sand dune landscape (Bertels et al.,

2005). The specific abiotic factors which shape coastal dune systems contribute to its uniqueness

through their effect on species ecology and evolution. However, this uniqueness makes coastal dune

systems also very vulnerable (Provoost et al., 2011a & 2011b). Research within coastal dunes and on

coastal dune species with special focus on dune landscape structure and dynamics is thus important

to better understand and protect this environment. Further on in this introduction, more information

will be given about a sand dune landscape, its functioning and the community it harbors to

eventually end up with elucidating the species of focus within this study.

1. Sand dune landscape

A coastal sand dune landscape is a dynamic system with specific abiotic characteristics which shape

the biological community it harbors (Bigot et al., 1982). The main factor that distinguishes it from

most other habitats is the great importance of wind dynamic for the appearance and functioning of

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the habitat. It determines whether or not there is soil development. This soil development is largely

responsible for the way in which plant succession in a dune landscape occurs. When the soil is not

well developed or fixated dry sand is continuously repositioned and can bury whole plant

communities. This creates a self regenerating landscape which contains a mosaic of several plant

successional stages, each successional stage representing a so called dune habitat (Bonte & Provoost,

2005; Crevits, s.d.). Between these dune habitats there can be occurrences of transitional areas.

Scientific research within a dune system must be performed taking into account the specific dune

landscape structure (Bertels et al., 2005). Figure 1.a shows a scheme of coastal dune structure and in

what follows the different habitats will be discussed. Mainly Belgian dune habitats will be considered,

but the general lines are applicable to similar dune habitats in Atlantic Europe.

When sand is repositioned from the beach more land inwards either by wind dynamics or the

working of tides, it is first captured by Elymus farctus. This species, through limited vertical spread of

rhizomes, can endure moderate sand accumulation and it aids in the formation of embryonic shifting

dunes (Crevits, s.d.; Bertels et al., 2005; European Commission, 2013). These embryonic shifting

dunes are the first step in dune formation and are mostly situated on strandlines. Other vegetation

that can occur here are salt-tolerant pioneer species like Cakile maritima. Embryonic shifting dunes

form a harsh and unpredictable habitat that can easily be blown apart again by the wind or be

washed away during high tides. When these embryonic dunes do manage to become higher and

when freshwater is present in the underground E. farctus gets replaced by Ammophila arenaria. This

Yellow dune

Embryonic dunes

Yellow dunes

Beach

Dune scrub

a.

b.

Fig. 1: a. Scheme of successional relations between coastal dune vegetation types within a landscape

ecological framework. (altered from Bertels et al., 2005); b. pictures that illustrate the main dune habitats that

were considered during this study with the frames being color codes that will be maintained in further graphs.

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grass species has a more developed system of vertically growing rhizomes which allows it to prevail

on large sand masses (Huiskes, 1979). At this stage, the habitat is called a yellow dune habitat or

even a mobile dune habitat (also called dynamic or shifting dune habitat), when sand is not fixated

entirely by A. arenaria so that large masses of sand can still be relocated by wind dynamics

(European Commission, 2013). In Belgium these kind of mobile dunes are only present in the area

between the French-Belgian border and Nieuwpoort, being particularly remarkable in extension and

natural value in the Westhoek (Doody, 2008). Leymus arenarius can be present in both embryonic

shifting dunes and yellow dunes at the Belgian coast, but becomes a more important dune grass in

regions north of latitude 63°N, where A. arenaria becomes less dominant (van der Meijden, 2005;

Huiskes, 1979). The more sand gets fixated by A. arenaria, the more plant species can settle that are

not adapted to dynamic yellow dune conditions. The environment becomes more species rich and

Carex arenaria and Festuca rubra are more often encountered while A. arenaria populations

deteriorate (European commission, 2013; Bertels et al., 2005; Crevits, s.d.). It is in these more species

rich conditions that build up of organic matter and nutrients is promoted. This leads to the

establishment of strong competitors such as Calamagrostis epigejos (Maun, 2009b). Eventually the

habitat can become a dune scrub with the main species being Hippophae rhamnoides. External

factors such as trampling or natural blow outs can again lead to soil destruction and initiate

secondary vegetation patterns (Bertels et al., 2005).

The dune habitats mentioned above are not the only possible habitats occurring at the Belgian coast.

Under specific conditions, other succesional stages can develop. Where sand is blown out up until

groundwater level, moist or even flooded dune slacks are formed (Crevits, s.d.). Or when extended

rains follow extreme dry conditions the soil is acidified, giving rise to a vegetation cover of acid-loving

species like Viola canina, Potentilla palustris and Carex arenaria (Maun, 2009b). Moss dunes are

formed at the lee slope of mobile dunes were the environment is less dynamic. This dune habitat is

however very sensitive to trampling and must be managed closely at the Belgian coast to prevent

further succession towards dune scrub (Crevits, s.d.; Maun, 2009b). These are some examples of

other dune habitats. However, this study’s main focus is on embryonic dunes, yellow dunes and

transitional zones between them and towards dune scrub.

2. Sand dune community

Insect biodiversity in a dune landscape is surprisingly high and stands in contrast with the harshness

and relatively limited diversity in vegetation. This insect diversity is however highly vulnerable

because of the restriction of these organisms to a dune habitat. Many dune species exhibit

evolutionary adaptations to dune conditions. Some species for example switch to a nocturnal

lifestyle to avoid the high summer temperatures, while A. arenaria rolls up its leaves during warm

periods to retain water (Maun, 2009b; Bonte & Provoost, 2005; Huiskes, 1979). The evolutionary

adaptations that make living within a dune environment possible, however, at the same time

constrain their distribution to other habitats (Bonte & Provoost, 2005). This is partly because of the

large investment they have to put in these adaptations. An investment that makes it impossible for

these organisms to put energy in certain other traits. These other traits may not be important in a

dune environment, but they can be essential for living in other habitats. Besides this, the large

amount of energy that flows to adaptations to coastal sand dunes also impacts insect development.

It takes a much longer time span for dune species to become an adult compared to closely related

species from other habitats. The dune beetle Polyphylla fullo has for example a larval development of

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two to three years (Bonte & Provoost, 2005). This makes them more vulnerable to the high rate of

tourism at the Belgian coast nowadays.

In the light of the recent deteriorations of European sand dune landscapes, the vulnerability of dune

organisms and the restriction to their environment makes the urge of protecting and managing

coastal sand dune systems particularly clear (Provoost et al., 2011b). In Belgium it is even more

compelling because its coastline is almost 50% urbanized and the remaining dune habitats are very

fragmented and damaged (Doody, 2008). Because of extensive landscape changes, mobile dunes

become fixated and the regenerating character of the system is lost (Provoost et al., 2011a).

Eventually this makes that the whole mobile sand dune ecosystem disappears and becomes replaced

by dune scrub or forest.

Various environmental factors impact sand dune communities. The previously mentioned factor of

wind dynamics can influence sand dune fauna directly by causing stress through the lashing of

overblown sand (Bonte & Provoost, 2005). Also an indirect impact is present from its influence on

the vegetational succession. Covering with sand of older successional stages takes the vegetation

back to a pioneer stage. Other plant species will occur on these places with other specialist

herbivores and their predators. Another indirect effect is through the prevention of soil

development. This causes nutrient scarcity which also influences the sand dune community.

Although these factors are of major importance within the system, there are still other factors that

shape a dune community. The sea influences the ecosystem by buffering seasonal temperatures,

causing salty conditions through sea spray and flooding and supplying the calcareous remains of

marine organisms which increase soil pH. Also soil surface temperatures can reach extremes of up to

70°C on warm days during the summer months, while at night there is a very rapid cooling due to the

low heat retention capacity of sand (Bonte & Provoost, 2005). Living in these harsh and dynamic

habitats is thus challenging. Still, many species seem to cope with this challenge often through their

specific evolutionary adaptations to dune environmental conditions. A dune community is thus a very

specialized community, but above all a variously changing community, especially in and near the

most dynamic dune habitats such as mobile sand dunes (Bigot et al., 1982).

Major taxa of dune herbivores are aphids (Hemiptera: Aphidoidea), leafhoppers (Homoptera:

Ciccadellidae), certain beetles (Coleoptera), grasshoppers (Orthoptera: Caelifera) and snails

(Gastropoda) (Maun, 2009a; Bonte & Provoost, 2004). In a yellow dune environment, with A.

arenaria being the main plant species, a diverse herbivore community is rather remarkable. Different

species can however live next to one another by the aid of different life strategies. Niche segregation

is an example of this. An example of this can be found when considering the aphids Schizaphis rufula,

Laingia psammae and Metopolophium sabihae which are all associated with A. arenaria. Schizaphis

rufula is mostly present on the leaves, while L. psammae can be found living between the flowers or

fruits. These aphids are relatively closely related species. It is not surprising that species that are

more distantly related show an even larger array of feeding strategies. The larvae of the dune beetle

Otiorrhynchus atroapterus (Coleoptera: Curculionidae) feed on the roots and larvae of the moth

Apatetris kinkerella are leaf miners of A. arenaria (Weeda et al., 2003c). Herbivores can also cope

with competition for a certain plant species by extending their host range to other plant species.

Schizaphis rufula is besides on A. arenaria also found on Leymus arenarius and L. psammae is also

known feeding on C. epigejos. Metopolophium sabihae was first only known from the species F. rubra

and Vulpia membranacea (Vandegehuchte et al., 2010). Aphids are predated on by Syrphid larvae

and Coccinellidae but also by other predators present in the vegetation like certain dune spiders

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(Araneae). Another kind of predators within dune habitats are ground dwelling arthropods like wolf

spiders (Araneae: Lycosidae) and carabids (Coleoptera: Carabidae). The dune predator community is

like the herbivore community surprisingly diverse.

3. Schizaphis rufula

Aphids (Aphidoidea) are an economically important group of herbivores because they are a major

pest on a wide range of crops. Some examples are the cherry tree aphid Myzus cerasi, the cereal

aphids Sitobion avenae and Rhopalosiphum padi which are major insect pests in northern Europe and

Nasonovia ribisnigri (Mosley), specific to lettuce (Stutz & Entling, 2011; Hansen, 1995; Höller et al.,

1993; Barrière et al., 2014). In natural systems aphids do not develop into a pest species. The

maintenance in diversity of the predator community in these more complex systems is usually put

forward as the main reason for this observation but it remains a subject of discussion (Chaplin-

Kramer & Kremen, 2012; Lithourgidis et al., 2011, Müller & Godfray, 1999; Altieri & Letourneau,

1982). Although aphids and insect herbivores in general do not often show outbreaks in natural

systems, this does not take away their importance in ecosystem functioning. Chronic herbivory can

for example significantly influence forest ecosystems (Weisser & Siemann, 2004; Hunter, 2001).

Indeed, leaf area loss to insects in general can reduce tree growth (Wagner et al., 2008; Marquis &

Whelan, 1994) and redirects primary production into the herbivore food chain (Cebrián, 1999;

McNaughton et al., 1989), altering material flows from the canopy to the forest soil (Metcalfe et al.,

2014; Hartley & Jones, 2004; Hunter, 2001)(Mazía et al., 2012). Other examples are the influence of

herbivory on interspecific competition of grasses (Ibanez et al., 2013), pollination success (Strauss et

al., 2001; Lehtila & Strauss, 1999) and seed production and seedling recruitment (Maron et al., 2002),

which can all influence vegetative composition within the habitat. The study by Maron et al. (2002)

was conducted in coastal and continental dunes but mainly focused on moths (Lepidoptera).

Aphids in natural systems are often overlooked within ecological research when not being a pest

species or they are regarded within a broader community of herbivores, e.g. in general herbivore

exclusion experiments. Neglecting aphids in plant herbivore research can however be unjustified.

Allan and Crawly (2011) put forward the aphid Diuraphis holci as a potential promoter of vegetative

diversity in an acid, mesotrophic grassland and even more aphid species are expected to be

important actors within the herbivore community. Within a dune habitat an important extra factor is

added to the aphid’s potential range of influence. Namely, influence on dune vegetative structure

manifests itself further into dune dynamics, causing more drastic changes of landscape appearance

and functioning.

Within the European dune landscape, several aphid species have been observed feeding and

reproducing on the important dune grasses that form a dune habitat, Schizaphis rufula (Walker 1849)

being most abundantly present (Vandegehuchte et al., 2010). The genus Schizaphis contains mainly

grass inhabiting aphids which in some cases can be deleterious for food crops like the much more

investigated S. graminum, which is a severe pest species on small grains all over the world (Evidente

et al., 2009; Pettersson, 1971a & b; Börner & Heinse, 1957). The distribution of S. rufula is restricted

to Europe and ranges from northern countries like Finland, Sweden and Denmark to more southern

regions such as Corsica and Sicily. In central and western Europe it is known to occur in Germany,

Poland, the Netherlands, Britain and Ireland (Nieto Nafría, 2007). It was only until recent, in the

summer of 2007, that S. rufula was for the first time reported in Belgium indicating the relatively low

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amount of taxonomical research conducted in Belgian dune systems (Vandegehuchte et al., 2010). It

can be found from June or even earlier up until October feeding and reproducing on dune grasses

including A. arenaria, E. farctus and L. arenarius. In contrast with the species’ large range and

potential importance for the ecosystem, little is known about its ecology. In articles published around

1970, Pettersson made first attempts to clarify the species biology based on research conducted in

Sweden. He found S. rufula feeding on L. arenarius which is more abundantly present at Swedish

than at Belgian coasts (Pettersson, 1971a; van der Meijden, 2005). He stated that superficially there

was no pronounced damage observed on L. arenarius plants infested by the aphid, which is in

contrast with observations on other plants from the genus Hordeum were S. rufula seems to reduce

the survival of plants considerably, implying that the aphid’s effect on plants is species specific. To

understand the way in which S. rufula influences dune grasses or the dune ecosystem, its host plants

and environmental preferences need to be clarified. Pettersson (1971c) already did a host choice

experiment with four Schizaphis species, among which S. rufula, investigating sixty different host

species coming from a very diverse range of habitats. In this study he did not focus on dune habitats

when choosing plant species to be tested. In our study, dune species and the successional stage or

dune habitat they represent get a more pronounced position.

4. Parasitoids

Parasitoids can play a major role in controlling aphid populations (Thies et al., 2005; Schmidt et al.,

2004). That is why they are often used as biocontrol agents in agricultural crops. Parasitoids regarded

in this study are insect parasitoids. These are insects that live a part of their lives at the expense of

other organisms and nearly always kill their host. This is opposed to true parasites that keep their

host alive (Raper, 2001). Insect parasitoids are spread over various orders, but are very well

represented within the Hymenoptera. All together they attack a very large range of hosts spread over

numerous taxa worldwide (Sanz & Leverton, 2010; Salvo & Valladares, 2007; Guerrieri, 2006;

Kopelke, 2003; Huang & Polaszek, 1998; Hedqvist, 1998; Godfray, 1994; Hàgvar & Hofsvang, 1991). A

special case of parasitoids are hyperparasitoids, which parasitize other parasitoids within their host.

Parasitoids can be subdivided into two groups according to their lifestyles, namely koinobionts and

idiobionts, the biggest difference being that in koinobionts the host is (at most) only partially

paralyzed by the wasp’s venom and soon recovers whereupon it continues to develop and is only

killed when the parasitoid reaches maturity. Idiobionts on the other hand totally paralyze their host

with their venom, terminating its development (Raper, 2001). For the dune aphid S. rufula there are

indications of parasitism by koinobionts. In a field survey conducted in 2007 by Vandegehuchte et al.

(2010) parasitized S. rufula individuals called mummies were found. These parasitized aphids have a

swollen, brown appearance which is caused by parasitoid wasps (Hymenoptera) that lay their eggs

inside a healthy aphid by penetrating through its surface with an ovipositor. The egg develops into a

larva, which stays in the aphid until it is fully grown. When developed into an adult, it emerges

(Hàgvar & Hofsvang, 1991). There are only two families of Hymenoptera that contain parasitoids of

aphids, namely the Aphelinidae (only two genera) and the Braconidae (27 genera from the subfamily

Aphidiinae) (Turpeau et al., 2010a). The family of Braconidae is the second largest family in the

Hymenoptera containing approximately 40 000 species, the largest being its sister family

Ichneumonidae with approximately 60 000 species (Goulet & Huber, 1993). The identity of the

parasitoids found on S. rufula as well as the way in which they influence aphid population dynamics is

not yet clear.

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Aims It is known that the dune aphid S. rufula uses host plants that are of high importance for the

functioning of a dune ecosystem. The main aims of this master thesis are to determine which biotic

factors impact population dynamics of S. rufula present on these host plants and under which

conditions and on which plant species this aphid’s populations do best. By investigating this, it can be

determined whether S. rufula has the potential to impact ecosystem dynamics through its interaction

with its host plant and under which circumstances this impact is the most pronounced. Research

questions were tackled while giving coastal sand dune structure a pronounced place within the

study. More specifically we determined 1) the influence of the surrounding plant community on

population dynamics of S. rufula associated with A. arenaria in the field, 2) the specificity of the

aphid’s host choice within and between plant species and 3) the parasitoid community associated

with S. rufula. Two different research strategies were used. First of all a more observational strategy

was conducted during the course of a field survey. Secondly, this field data was complemented with

the more experimental setup of a host suitability experiment.

Field survey: The main aim of the field survey was to determine how S. rufula populations behave on

A. arenaria host plants with varying intrinsic plant characteristics and located in different dune

habitats. It was expected that S. rufula populations would be more abundantly present on young

plants of A. arenaria, due to the higher nutritional value of younger compared to older plants

(Pettersson, 1971c; Kennedy, 1958). The aphids should also do better in dune habitats where stress

from predation and overblowing sand is minimal. These conditions are probably more prominently

present in areas with a more dense vegetational cover. This vegetation can be mainly composed by

A. arenaria itself like in yellow dunes, but can also include other dune plants in transitional zones

towards more fixated dune habitats. Hypotheses were based on field observations and studies from

de la Peña (2011), Vandegehuchte et al. (2010) and Pettersson (1971c). During the field survey, the

species composition of the parasitoid community associated with S. rufula was also determined to

have a better view on the community of natural enemies surrounding this aphid species. Similar

studies on parasitoids of S. rufula have to our knowledge not yet been performed.

Plant suitability experiment: In previous field observations S. rufula is besides being abundantly

present on A. arenaria also seen on other dune grasses like E. farctus, F. rubra, L. arenarius and even

on the sedge species Carex arenaria. As stated in the introduction under subheading “Sand dune

landscape” these plants are important players in the vegetative succession within a dune landscape.

In the plant suitability experiment, it was tested if there were observable differences in aphid

population dynamics considering mainly the variable “plant species”. In doing so, there is a

decoupling of the aphid-plant interaction from any other habitat specific influence. This gives a

better view of the aphid’s host preference. The broadness of the aphid’s host choice was also tested

by putting plant species from other habitats under experiment. It is expect that S. rufula will not be a

true specialist species regarding the fact that it is already observed feeding on various host plants in

the field. Still pronounced differences between plant species are expected because of their various

identities and observed variation in aphid population dynamics from previous field observations.

This experiment complements the field survey in a way that the aphid’s potential impact can be

shown within other dune habitats besides the ones containing host species A. arenaria. Embryonic

shifting dunes are an example of a dune habitat which could not be investigated in the field survey

because A. arenaria is not present in this kind of environment. By following aphid population

9

dynamics on the embryonic dune species E. farctus it can be shown whether or not there is the

potential for an impact from the aphid on the functioning of this dune habitat.

Material and methods 1. Study design

a. Ecological preferences

Study sites: When selecting the study sites for conducting the field survey, it was assured that

certain criteria were met. First of all, study sites had to comprise the dune habitats that are typical to

a European dune landscape. This means that natural dune dynamics had to be influenced as little as

possible by human influences. Due to the urbanization of the Belgian coast this already reduced our

options significantly (Doody, 2008). Most dunes that are still more or less intact have to be closely

managed in order to maintain dune dynamics (Provoost et al., 2011a; Herrier & Killemaes, 1998;

Crevits, s.d.). Eventually three nature reserves were selected where sand is still sufficiently mobile to

maintain the specific character of the dune landscape. These nature reserves were situated along the

Belgian coast with the third one being just across the border with the Netherlands. These three sites

are included in the Natura 2000 network and have been designated as Sites of Community

Importance (figure 2; Herrier & Van Nieuwenhuyse, 2005). One of the sites chosen was located in

nature reserve Westhoek in De Panne, Belgium, near the French border. It is the oldest nature

reserve in Flanders. Mobile sand masses are still present in this area to a large extent, although they

are significantly declining over the years (Provoost et al., 2011a). The nature reserve is accessible to

the broader public, be it only on fenced hiking trails. Another nature reserve that still contains mobile

sand dunes is Ter Yde in Oostduinkerke, Belgium (Bonte & Provoost, 2005). Here, public access

should also be restricted to hiking trails, but because of the fences that are occasionally overblown

by sand, people sometimes do not know that they stray from the paths (own observation, 2012). The

last location was situated just across the border with the Netherlands in Retranchement and is part

of nature reserve Zwin of which the biggest part is present at the Belgian side of the border. Our

study location in this area was fully accessible for the broader public. All three nature reserves are

managed by the Agentschap voor Natuur en Bos (ANB) or the Forest and Nature Agency which is an

agency of the Flemish government.

Study design: In July 2012, a field study was conducted on the ecological preferences of S. rufula in

nature reserves Westhoek and Ter Yde in Belgium and the part of nature reserve Zwin in the

Netherlands (figure 3). In each of the three reserves, twenty individuals of the dune grass species A.

arenaria were marked with little numbered flags and GPS-coordinates were noted in order to easily

find back each individual plant in the weeks that followed (figure 3e). The relative occurrence of bare

soil and plant species present within a radius of one meter around the marked A. arenaria individual

and plant characteristics of this individual were determined once at the beginning of the study. These

plant characteristics include the diameter at bottom and top of the plant, the length of the longest

leave and the amount of leaves. Population densities of S. rufula were determined on a weekly base

for a period of four weeks.

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Fig. 2: Situation map of sites of community importance SCI’s on land (1=De Panne, 2=Koksijde, 3=Nieuwpoort,

4=Middelkerke, 5=Oostende, 6=Bredene, 7=De Haan, 8=Zuienkerke, 9=Blankenberge, 10=Zeebrugge, 11= Knokke-

Heist) (from Herrier and Van Nieuwenhuyse, 2005). Study sites from the field survey are indicated with arrows.

Fig. 3: a. Nature reserve Westhoek in De Panne, Belgium; b. Nature reserve Ter Yde in Oostduinkerke, Belgium; c. Study

location at nature reserve Zwin in Retranchement, the Netherlands; d. Schizaphis rufula Walker 1949 (Hemiptera:

Aphidoidea); e. One of the numbered flags is seen on the right side of the picture, standing between A. arenaria plants; f.

several S. rufula aphids on a leave of A. arenaria.

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b. Plant suitability experiment

Preparations: To complement the results from the field survey, where focus was only on host plant

A. arenaria, a plant suitability experiment was performed. This kind of experiment has as a main aim

to decouple environmental influences from host plant – aphid interactions and purely focus on the

suitability of the plant species to maintain populations of S. rufula. Both dune species and grass

species from other habitats were tested. Seeds of the embryonic shifting dune species E. farctus as

well as the yellow dune species A. arenaria and representatives of more fixated dunes like C.

arenaria and F. rubra and a species of coastal tall grasslands and dune scrubs C. epigejos were

collected in nature reserve Westhoek in De Panne, Belgium. Seeds of the mobile dune species L.

arenarius originated from nature reserve Zwin in Retranchement, the Netherlands. Seeds from non-

dune species were obtained from the Dutch company Cruydt-hoeck, which is a distributer of wild

plant seeds. The species Deschampsia cespitosa was used as a representative of moist, nutrient rich

grassland, Lolium perenne mostly situated on moist, fertilized or highly treaded soils and Poa

pratensis on nutrient rich, mostly treaded soils, on walls and between pavers. Also Deschampsia

flexuosa was used as a representative of heathland habitat which more closely resembles a dune

system.

All seeds were first subjected to a surface sterilization treatment. This involves a procedure of

washing the seeds in 30 % EtOH for 2 minutes, than in a 10 % bleach solution for 5 minutes and

finally with a large amount of sterile dH2O. After this treatment, seeds were kept overnight in a

sufficient amount of distilled water to soak of any remaining bleach residues before they were plated

onto petri-dishes containing a 1% agar growth medium. These petri-dishes were then put before a

window, subjecting them to the prevailing night/day rhythm during the month of October. When

seeds germinated, seedlings were planted in polypropylene cups containing sand collected from

nature reserve Westhoek, De Panne. Because all plants were raised in similar Westhoek soils a

potential variation in soil biota was eliminated. For five weeks these were maintained under

fluorescent lamps with a summer night:day regime of 8:16 hours with day temperatures reaching

22°C . By our own observations in the field and also supported by results published by Pettersson

(1971c) based on a survey conducted by Kennedy (1958), five to six weeks is a plant age that allows

optimal feeding by the aphids. This is linked to a higher intensity of amino acid transport at this stage

in the plant’s life. Schizaphis rufula individuals taken from nature reserve Westhoek in De Panne,

Belgium, were further reared in the lab on A. arenaria plants under the same conditions as the re-

potted plants.

Fig. 4: study design of the plantsuitability experiment. On eachexperimental plant individual, a single

adult S. rufula individual from our ownaphid culture was placed. To prevent

aphids from escaping, plastic longcocktail glasses that were punctured atthe bottom were placed upside down

over each plant individual.

12

Experiment: Of each species (dune species E. farctus, A. arenaria, C. arenaria, F. rubra and C.

epigejos as well as non-dune species D. flexuosa, D. cespitosa, L. perenne and P. pratensis) twenty

individuals were randomly chosen from all successfully lab grown plants. Ten were assigned to being

a control plant and ten would undergo the experiment. Because of the low amount of successfully

grown L. arenarius individuals, only 7 replicas of this species could be put under experiment and no

plants were available to set up a control. All plants were put under fluorescent lamps which were

connected to a timer that kept a summer night:day rhythm of 8:16 hours. On each experimental

plant individual, a single adult S. rufula individual from our own aphid culture was placed. To prevent

aphids from escaping, plastic long cocktail glasses that were punctured at the bottom were placed

upside down over each plant individual (figure 4). No aphids were put on the control plants. First

counts of the amount of aphids per plant replica were performed two days after manually adding

aphids to the experimental plants. From then on every two days a counting took place for a period of

four weeks. On a regular basis, plants with and without aphids were randomly repositioned under

the fluorescent lamps. After the four week period, the experiment was terminated and each plant

was weighed. Also dry weight was determined after putting all plants for 24 hours in an oven set to

60°C.

c. Identification of parasitoids

Morphological identification: Within the field survey and during extra field trips in August of the

same year and from July up until August in 2013 parasitized aphids were collected from A. arenaria

as well as F. rubra and C. arenaria. Most parasitoid wasps (Hymenoptera) were found in nature

reserve Westhoek in De Panne, Belgium, with only sporadic observations in nature reserves Ter Yde

in Oostduinkerke, Belgium, and Zwin in Retranchement, the Netherlands. Parasitized aphids, called

mummies (see figure 5), were taken to the lab in eppendorf tubes together with part of the attached

plant. These eppendorf tubes were put in a quiet environment in front of a window in order to

minimize disturbance and to maintain a natural night/day rhythm. Between two to five days after

collection, the parasitoid wasps emerged. They were preserved in eppendorf tubes containing

ethanol with a purity of 99,9%. A first identification was performed using morphological and

morphometric characteristics of the wasps. To examine the organisms an Olympus stereomicroscope

system SZx7 with the magnification being 57x, was used at the Terrestrial Ecology Unit of Ghent

University. Measurements of the specimens were taken with an ocular micrometer and with ImageJ

version 1.47 (Wayne Rasband, National Institutes of Health, USA) when appropriate photo’s were

available. These pictures were taken using a digital camera attached to the stereomicroscope with a

trinocular tube. More detailed images were made using a Hitachi Tabletop Scanning Electron

Microscope TM-1000 located at the VIB Department of Plant Systems Biology (UGent) in Zwijnaarde,

Belgium. For the identification, keys were used from Goulet and Huber (1993), Tomanović et al.

(2012), Kavallieratos et al. (2013), Gibson et al. (1997) and also the sites of Encyclop’APHID

(http://www4.inra.fr/encyclopedie-pucerons), BugGuide.Net and Phylogenetics of Ceraphronoidea

(http://ceb.csit.fsu.edu/ronquistlab/PCCP/ceraphronoidea/index .html ) were consulted.

13

Molecular identification: DNA barcoding was conducted to support the morphological

identifications. First the DNA was extracted from each individual wasp using the NucleoSpin®

Tissue

(Macherey-Nagel) kit and following the support protocol for purification of genomic DNA from

insects (January 2010/Rev. 11). DNA amplification and further analysis was performed based on the

16S ribosomal RNA mitochondrial gene and the long wavelength rhodopsin (LWRh) nuclear gene.

There are two ways of amplifying these DNA regions differing only in the identity of the reverse

primer. The first method is focused on amplifying DNA from adult wasps that have already emerged

out of their aphid host. Primers used are 16S-F (forward) 5’-CGCCGTTTT ATCAAAAACATG T-3’,

developed by Simon et al. (1994), and 16S-R (reverse) 5’-TTACGCTGTTATCCCTAA-3’ by Kambhampati

& Smith (1995) for the amplification of the 16S gene. The primers LWRh-F (forward) 5’-

AATTGCTATTAYGARCANTGGGT-3’ and LWRh-R (reverse) 5’-ATATGGAGTCCANGCCATRAACCA-3’, both

developed by Mardulyn & Cameron (1999) were used for the amplification of the LWRh region. The

second method aims on amplifying the DNA of immature parasitoids still inside their aphid host

(Derocles et al., 2012). Forward primers used here were identical to the ones from the first method.

The reverse primers, however, were developed by Derocles et al. (2012) and are specific to the

Aphidiinae subfamily. For the amplification of the 16S region 16S-Rspe (reverse) 5’-

TCTAWAGGGTCTTCTCGTCT-3’ was used and for LWRh LWRh-Rspe (reverse) 5’-

GATGCAACATTCATTTTTTTAGCTTG-3’. PCR amplifications were carried out as described in Derocles et

al. (2012) using a 25 µL reaction volume which contains 18.5 µL of H2O, 2.5 µL of buffer (final

concentration, 1x), 1 µL of MgCl2 (final concentration, 1 mM), 0.5 µL of dNTPs (final concentration,

0.2 mM), 0.175 µL of each primer (final concentration, 0.07 µM), 0.125 µL of Taq (final concentration,

0.63 U/µL) and 2 µL of extracted DNA. PCR amplification conditions for both genes were also as

described by Derocles et al. (2012): 94 °C for 180 s; 40 cycles of 94 °C for 30 s, 56 °C for 60 s, 72 °C for

90 s, with a final elongation of 72 °C for 10 min. A volume of 3 µL of PCR product was run through a

3.6% agarose gel through gel electrophoresis (100V, 35min) and afterwards stained using the

fluorescent nucleic acid stain GelRedTM. The eventual product was visualized using the Gel DocTM

2000 from BIO-RAD Laboratories, Inc. (Hercules, CA, U.S.) and the Quantity One® software package.

Before performing any sequencing, PCR products were purified with Exonucleas I + fastAPTM

(Fermentas Molecular Biology Products, Thermo Fisher Scientific Inc., U.S.) and send to Macrogen

Inc., Europe. Service performed was Macrogen’s EZ-seq service. Sequences were edited using BioEdit

Fig. 5: an aphid mummy with the parasitoid larvae still inside (darker part).

14

version 7.2.5 (Hall, 1999). Specimen identification was done by performing a standard nucleotide

BLAST of the sequences on the website of the National Center for Biotechnological Information

(NCBI) (National Library of Medicine, U.S.; http://blast.ncbi.nlm.nih.gov/Blast.cgi?). Felsenstein-

Tajima-Nei distances were calculated between our specimens and specimens from GENBANK to

resolve specimen identities that stayed unclear after running the BLAST procedure.

2. Statistical analysis

All Statistical analyses were conducted using the statistical package SAS® version 9.4 (SAS Institute

Inc., Cary, NC, U.S.). Models were built based on sequential removal of variables with non-significant

Wald statistics (SAS Institute Inc., 2014). To avoid putting covariant independent variables in the

models a correlation matrix was made which showed the Pearson correlation coefficients between

all variables in our dataset (see table 1 in Appendix).

a. Ecological preferences

From the plant community data the biodiversity index “richness” (R) was calculated for the

vegetation in a one meter radius from each studied plant individual to be integrated with all other

variables in statistical analyses. Intrinsic plant characteristics and surrounding plant community

composition (incl. richness R) were analyzed separately. Generalized linear mixed models, GLMM’s,

were used to identify independent variables that influence the probability of aphid encounter and

population dynamics of S. rufula on its A. arenaria host plant. Location (Westhoek, Ter Yde and

Zwin), plant individual and date were included in the models as random effects.

Probability of aphid encounter: An extra variable was added to our dataset indicating the

presence or absence of S. rufula with the values one and zero, respectively. This was implemented in

further analyses as being binomially distributed.

Aphid population dynamics: Also population dynamics on plants where S. rufula was present was

considered. In doing so, plant individuals that had no aphids present on their leaves were left out.

The eventual data was Poisson distributed and was as such implemented in the analysis. The relative

amount of aphids was calculated as being the amount of aphids divided by the amount of leaves the

host plant individual had. This was also implemented in models as being Poisson distributed.

b. Plant suitability experiment

A plant individual was regarded as accepted by S. rufula when minimally one aphid prevailed up until

the second counting date, that is for four days. Persistence was defined as the amount of days that

minimally one aphid individual could still be seen alive. This was not necessarily on the plant

individual itself, but could also be on the soil surface or the side of the cup. To determine if the plant

species has a significant impact on the rate of acceptance by S. rufula, the longitude of persistence or

the amount of aphids present on the plant a GLMM was again used with the dates on which aphids

were counted put in as a random effect. A Tukey test was performed in order to pairwise compare

plant species means of the amount of aphids present on each plant on a given date. To see whether

or not S. rufula had an impact on plant fitness, biomass and dry weight of plants with and without

aphids, measured at the end of the experiment, was analyzed using a General Linear Model or GLM.

Here, the maximum amount of S. rufula on the host plant was put in the model as a random effect.

15

Results 1. Ecological preferences

a. Probability of aphid encounter

It was found that the chance of encountering S. rufula on its host plant A. arenaria is influenced by

certain biotic environmental factors. The presence of aphids on A. arenaria is negatively correlated

with the percentage of F. rubra situated within a one meter radius from the A. arenaria host plant

(F1,180 = 11.89, P = 0.0007) and also with plant species richness R within that same area (F1,180 = 21.08,

P < 0.0001; figure 6). These two independent variables are mutually covariant and were therefore

analyzed separately in SAS®

version 9.4.

Regarding intrinsic plant characteristics of the A. arenaria host plant it is found that the more leaves

the host plant had the more chance there was to encounter S. rufula feeding on the plant (F1,179 =

5.16, P = 0.0243). However, the estimate for the fixed effect “amount of leaves” was relatively close

to zero (0.01096) and as seen in figure 7, that shows a box-whisker plot of the variation in plant sizes

for plants that carried (1) as well as lacked (0) the aphid on their leaves, variability in both groups is

rather high. These results should therefore be regarded with care. Further elucidation on this subject

can be found in the section “Discussion”.

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7

Pres

ence

of S

. ruf

ula

(%)

Plant species richness

TrendlineDatapoint

Trendline formula:Y = 83.77 - 15.85X

p < 0.05R² = 0.85

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7

Pres

ence

S. r

uful

a(%

)

Festuca rubra (%)

Trendline formulay = 201.18e-1.638x

p < 0.05R² = 0.83

Dune fixationless more

Fig. 6: Both graphs show the trend in chance of finding S. rufula on A. arenaria along gradients in variables that indicatedune fixation, namely plant species richness (above) and percentage plant cover of F. rubra within a one meter radius(below). The trendline formula, the goodness of fit to this trendline (R²) and the p-value are indicated.

16

No. of

leaves/A

. are

naria

ind.

Presence of S. rufula

p < 0.05 Fig. 7: Box plot that shows the variation inplant sizes (measured in amount of leaves/A.arenaria indivdual) within the group of plantson which S. rufula was absent (0) as well asplants on which the aphid was present (1). Thelength of the box represents the interquartilerange (the distance between the 25th and 75th

percentiles). The diamond shape in the boxinterior represents the group mean. Thehorizontal line in the box interior representsthe group median. The whiskers issuing fromthe box extend to the group minimum andmaximum values. Group means differsignificantly, but variation is equally widespread.

0

10

20

30

40

50

60

70

80

90

Popula

tion

density

S.

rufu

la(i

nd.) E. farctus

A. arenaria

L. arenarius

C. arenaria

F. rubra

C. epigejos

D. cespitosa

D. flexuosa

L. perenne

P. pratensis

Embryonic dunes Yellow dunes Dune scrub Other environments

Fig. 8: The variation in population densities of S. rufula is shown during the four week duration of the plant suitability experimentfor each plant species that was tested. Color codes are as indicated in figure 1 and summarized in the upper right corner. Errorbars represent standard error (SE) around each plant mean on a given date.

0

5

10

15

20

25

30

35

Per

sist

ence

(day

s)

a

b b

c

d d,ee

a

ef

Embryonic dunes Yellow dunes Dune scrub Other environments

Fig. 9: The mean persistence is shown (amount of days that minimally one aphid persisted on the plant) for every plant species. Whichhabitat each species represents is indicated through the color of the bars. Letters situated above each bar illustrate the outcomes of atukey-test for comparing mean persistence between plant species. When bars are indicated with the same letter, they showed nosignificant difference in aphid persistence time. Error bars represent standard error (SE) around each mean.

17

b. Aphid population dynamics

Aphid population densities were not influenced by both surrounding plant community as intrinsic

plant characteristics when plants with no aphids on their leaves were left out of the analysis.

However, when going from plants with a low amount to plants with a higher amount of leaves a

slightly deteriorating trend was found in the relative amount of aphids (intercept = 0.1855; estimate

= - 0.02297; F1,93 = 9.39; P = 0.0029).

Out of the sixty plant individuals that were studied there was one individual situated in nature

reserve Zwin were there was an association of an ant species (Formicidae) with S. rufula in week four

of the survey. The aphid population counted 68 individuals which was the biggest observed

population at Zwin during the four week span with the second biggest comprising only 26 individuals.

There thus seems to be a positive influence from this aphid-ant interaction. Although this is an

interesting observation, in this study, we could not go further on the matter.

2. Plant suitability experiment

The amount of aphids present on a host plant at a given moment was dependent on the host plant

species (F9, 1 = 951.41, P = 0.0252) with the highest amounts of aphids found on L. arenarius and A.

arenaria (see figure 8). When taking these two species out of the analyses, significance is lost (F7,1 =

36.67, P = 0.1265). Also the persistence of the aphids on the plant individual is highly dependent on

the plant species (F9,77 = 8.75, P < 0.0001). A longer persistence was seen on species A. arenaria and

L. arenarius, with no significant differences between the two (t77 = -1.03, P = 0.3065, see figure 9).

When however comparing plant least squares means of these two with all other plant species,

significant differences were seen (all P-values < 0.0002). These results illustrate a clear separation of

A. arenaria and L. arenarius from all other plant species within our analysis. Considering the non-

dune grasses, aphids on D. flexuosa persist significantly longer compared to all others and even

compared to C. arenaria, F. rubra and C. epigejos. No significant differences were found between D.

flexuosa and E. farctus (t77 = 1.27, P = 0.2066). When comparing the dry weight of the control plants

with the dry weight of the grasses that underwent the experiment, only a significant decrease in

biomass was seen for A. arenaria (F1,8 = 18.21; P = 0.0027, see figure 10). For all other plants, a

negative impact from S. rufula could not be proven.

0

0.01

0.02

0.03

0.04

0.05

Elymus farctus Ammophila arenaria Festuca rubra Calamagrostis epigejos

Dry

weig

ht

(g)

control

experiment

Embryonic dunes Yellow dunes Dune scrub

*

Fig. 10: The graph shows the comparison between the means in dry weight of the control plants versus the plants underexperiment per dune species (without L. arenarius, for which we had no control plants). The asterisk indicates a significantdifference (p<0.05) within mean dry weight of the control versus the experimental plants. Significance could only be provenfor yellow dune species A. arenaria. Color codes are according to figure 1 with blue shades representing an embryonic dunespecies, yellow shades a yellow dune species and green shades more fixated dune or dune scrub species.

18

3. Identification of parasitoids

Morphological identification: Parasitoids found on S. rufula belonged to the genus Aphidius, which

is a genus within the family of Braconidae. Figure 11 shows the most important morphological

characters that support our identification to genus level. A glossary can be found in the Appendix.

Two Aphidius species were found, namely A. rhopalosiphi and A. avenae, which are shown in figure

12 along with their morphological and morphometrical characteristics. For the species of A. avenae

two individuals showed a remarkable difference in the appearance of the anterolateral part of the

petiole which was distinct from all other specimens belonging to this species and from what was

described in literature.

Besides the parasitoids, also hyperparasitoids of Aphidius sp. were found. Through morphological

study, specimens could be identified to generic level. Three genera were found: Apoanagyrus sp.

(Hymenoptera: Encyrtidae), Dendrocerus sp. (Hymenoptera: Megaspilidae) and Pachyneuron sp.

(Hymenoptera: Pteromalidae). These genera could however not be verified through molecular

analysis because the protocols used here for amplification of DNA did not support these taxa. The

morphological characteristics that support the identifications are shown in figures 13, 14 and 15. The

family of Pteromalidae, to which the genus Pachyneuron belongs, is not defined by any unique

attribute or combination of attributes. Membership is largely determined by elimination (Goulet &

Huber, 1993).

Braconidae

C+Sc+R

2m-cu absent

RS+M absent

Aphidiinaetergum 2 & 3

sec. flexible

- brown mummy- pupation inside aphid

cp

lb

Aphidius sp.

See figure 11b.

a. b.

c.

d.

e.

Fig. 11: (bottom of previous page) Characters that assign specimens extracted from S. rufula to the family of

Braconidae are the concave apical margin of the clypeus (cp) with the anterior surface of the labrum (lb) concave and

exposed (a.), the absence of the C cell caused by fusion of the C and R vein and the absence of the 2m-cu vein in the

forewing (b.) and a greatly reduced hind wing venation (c.). Metasomal tergum 2 and 3 are secondarily flexible (d.),

which is only the case in subfamily Aphidiinae (Goulet & Huber, 1993), a subfamily of parasitoids parasitizingspecifically on aphids. Aphidiinae specimens gathered within this study all had normally developed wings, pupated

within the aphids skin and caused the aphid to develop into a brown mummy (e.). Together with the absence of the RS

+ M vein (b.) this indicates that the specimens belong to the genus Aphidius (Tomanović et al., 2012).

19

50 µm

100 µm

500 µm

100 µm

Aphidius rhopalosiphiDe-Stefani Perez 1902

Aphidius avenae Haliday 1834

stigma narrow

- Costulate (fine ribs)- Petiole 3.5-4 x as long as wide

Antenna 16-17segments

Dark body

Costate (broad ribs)

R1 shorter than stigma

Variation in petiole type

a.

b.

c.

e.

Fig. 12: Pictures of the three different morphologies of parasitoid wasps retrieved from S. rufula mummies, the second and thirdcolumn being the same species, namely A. avenae, according to molecular research (see also table 1 and glossary in appendix).Most important characteristics are indicated in the figures. a. female parasitoids. b. head in lateral view. c. petiole or metasomaltergum 1 in lateral view. e. wing venation.

20

Apoanagyrus sp.

3

2

1

4

c.

b.

a.

Dendrocerus sp.

12

3

45

a.

b.

c. d.

Fig. 13: Microscopic and scanning electron photographs of specimensidentified as the hyperparasitoid Apoanagyrus sp. a. Female in dorsalview. b. Head and part of the thorax in ventrolateral view. c. Head inanterior view. Features that supported our identification are indicatedwith numbers and are as follows: (1) head and body are steel-blackwith greenish lustre, (2) all segments of the funicle are longer thanbroad, (3) mesoscutum and scutellum are dark, (4) forewings arehyaline. There is also a reticulate structure on the frontovertex butthis is not indicated in the figure (see Appendix for a glossary).

Fig. 14: Microscopic and scanning electron photographs of specimens identified asDendrocerus sp. a. Female in lateral view. b. Distal part of the mesotibia with basal part ofthe mesotarsus. c. Head in dorsal view. d. Thorax in dorsolateral view. Features thatsupported our identification are indicated with numbers and are as follows: (1) forewingwith large stigma, (2) mesotibia with two spurs, (3) antenna with nine flagellomeres, (4)mesoscutum with three longitudinal grooves, (5) anterior margin of metasoma in dorsalview with neck-like constriction (see Appendix for a glossary).

21

Molecular identification: DNA sequences could be retrieved from 21 specimens of which

sequences of 15 specimens were appropriate to run in BLAST. From seven specimens, both the 16S

and the LWRh region could be used. Table 2 (see Appendix) gives an overview of the specimens from

which DNA could be successfully retrieved and amplified and shows the percent of similarity and

Felsenstein-Tajima-Nei divergence distances with sequences of other specimens retrieved from

Genbank. With these sequences the presence of A. rhopalosiphi and A. avenae within the studied

ecosystem could be supported.

Pachyneuron sp.

1

2

3

no1tga. b.

c.

Fig. 15: Microscopic and scanning electron photographs ofspecimens identified as Pachyneuron sp. a. tibia and tarsi. b. headand thorax. c. female in lateral view. The family of Pteromalidae isnot defined by any unique attribute or combination of attributes.Membership is largely determined by elimination (Goulet & Huber,1993). Some important characteristics are indicated with numbersand are as follows: (1) tarsi with five tarsomeres, (2) stigmareduced, (3) ring-like flagellomeres and the pronotum (no1) isseparated from the tegula (tg) (see Appendix for a glossary).

22

Discussion Knowing how dune biota function and interact with one another will aid in our understanding of the

unique and dynamic dune system. Greater knowledge can contribute to better management. Within

the light of Belgium’s very fragmented and damaged coastline and also the deteriorating trends on

European scale, the importance of dune ecosystem research is stressed (Provoost et al., 2011a &

2011b). From the results stated above it can be concluded that we have succeeded in our aim to

assign factors that impact population dynamics of a dune grass herbivore which is prominently

present within European dune landscapes. These influential factors are tightly linked to dune

landscape structure, elucidating the importance of taking this landscape structure into account when

performing research or management in a coastal dune environment. Also parasitoids were found

that were to our knowledge not yet known to be associated with S. rufula. This knowledge reveals a

previously unknown trophic level within the network surrounding S. rufula and in the dune

ecosystem in general. There is still much to be learned about parasitoid impact on S. rufula and in

general, but here a first step has been taken. In what follows, results will be discussed in more detail.

1. General findings

It can be deduced from the outcomes of the field survey that S. rufula on A. arenaria host plants has

a preference for a yellow dune habitat. When a dune becomes more species rich and thereby fixated,

the presence of the aphid declines. This is the case for transitional dune habitats towards dune scrub

and also tall grassland with main species C. epigejos (see figure 1.a). However, because in the field

survey’s main focus lay on the host plant A. arenaria, certain dune habitats could not be investigated.

This is because A. arenaria does not occur in such environments. Aphid population dynamics could

therefore not be followed for habitats like moss dunes, embryonic dunes and their transitional zones.

From the experiment it is however known that S. rufula prefers primary dune fixating grasses. It is

thus expected that patterns seen for aphids on host plant A. arenaria, which is such a primary dune

fixater, can be generalized and occurrence of the aphid in (transitional zones towards) moss dunes

should be rather low. This is firstly because moss dunes are highly fixated dunes and secondly

because primary dune fixating grasses do not usually occur in this kind of environment. A proper host

plant for S. rufula is thus absent in such a moss dune habitat. For embryonic dunes, however, it

would have been more interesting to investigate aphid population dynamics. This is especially so

because of the occurrence of important host plant L. arenarius in both embryonic as yellow dunes.

To compare aphid population dynamics between embryonic and yellow dune habitats another field

survey could be done, but this time with main focus on host plant L. arenarius. Results from such a

survey could be especially interesting for regions north of latitude 63°N where L. arenarius replaces

A. arenaria as most important dune fixating grass. It is expected that in embryonic dunes the even

more pronounced presence of the sea should have an impact on aphid population dynamics.

2. Ecological preferences: underlying mechanisms

Why S. rufula has a preference for yellow dune habitats is not yet clear. As stated in the introduction,

such a dune habitat forms a very harsh and unpredictable habitat. Why should herbivores settle here

instead of taking cover within nearby more fixated dune habitats? When considering the outcomes

of the field study, a decrease can indeed be seen in the percentage of plants harboring S. rufula

individuals when going from a mobile sand dune habitat, with the presence of A. arenaria, to fixated

23

dunes where more plant species can persist and F. rubra is more abundantly growing (see figure 6).

Because population densities themselves do not differ significantly along gradients of plant species

richness or amount of F. rubra in the proximity of the host plant, it seems that plants in more fixated

parts of the dunes are not necessarily of a lesser food quality. When aphids manage to colonize a

plant, they are equally able to develop relatively high population densities in different dune habitats

and thus appear to be equally fit when comparing these different habitats. The observation that

fewer plants are infested in more fixated regions could be due to the lower abundance of A. arenaria

and a potentially higher isolation of individual plants. Schizahis rufula could have problems with

locating A. arenaria host plants within fixated dunes where A. arenaria individuals are less

aggregated. The effect that specialist insects are more likely to find, remain and reproduce on their

hosts when these plants grow in dense patches in pure stands is called a resource concentration

effect (Otway et al., 2005). This effect could prevent aphids from seeking cover on host plants in

more fixated dunes. It can also be that other more divers and complex vegetation types harbor a

greater community of natural enemies, putting aphids on host plants nearby more at risk (Straub et

al., 2013; Altieri & Letourneau, 1982).

This does not take away that the host plant’s intrinsic food quality could still have an impact on S.

rufula. Indeed, no direct measurements for host fitness or host plant quality were at hand. Also, the

decreasing trend in chance to find S. rufula on A. arenaria when going to more fixated dunes occurs

parallel with the observation that A. arenaria exhibits strongly suppressed growth as sand accretion

ceases, generally called “the Ammophila problem” (Marshall, 1965). Knowing the underlying

mechanisms supporting the Ammophila problem could thus give us an indication of which factors

prevent S. rufula of being abundantly present in more fixated dune habitats and thus influence dune

ecosystem dynamics. Various possible solutions for the Ammophila problem have been proposed.

These are based on both abiotic and biotic factors. Vandegehuchte et al. (2010) even stated that the

underlying cause could be region specific, giving the Ammophila problem a more complex twist. On

the one hand you have the biotic component of soil biota like nematodes, bacteria and fungi which

can have either a mutualistic or antaganostic impact on plant growth. On the other hand there are

still abiotic factors that seem to have an influence which can even suppress the effect of soil biota.

This was for example the case in nature reserve Westhoek (Vandegehuchte et al., 2010). Which

abiotic factor dominated plant growth in Westhoek soils could, however, not be stated.

For nature reserve Ter Yde, the soil biota were the most important factor. Suppose these soil biota

have an antagonistic relationship towards A. arenaria. Antagonist soil biota can be biomass reducing

soil biota or biota that influence aboveground plant nutritional quality. When these features are not

significantly altered, the soil organism can still trigger plant defense mechanisms. Herbivores feeding

on plants that interact with antagonists mostly show a reduced performance (Vandegehuchte et al.,

2010; Bezemer et al., 2005; Hanounik & Osborne, 1977; Bardgett et al. 1999; Wardle, 2002; Van Loon

et al., 1998). The “escape hypothesis” (Van der Putten et al., 1988) states that in dynamic sand dunes

the continuing supply of fresh sand triggers vertical growth of A. arenaria and allows it to escape

harmful soil biota. Aphids could benefit greatly from this escape through better food quality. They

don’t have the indirect negative impact of the antagonist soil biota anymore. This could explain the

finding why S. rufula leaves the protection of a more dense vegetation cover in fixated dunes. Under

fixated circumstances, the aphid’s food source is poor. It is thus better to move to mobile dunes to

get a sufficient amount of nutrients to survive and reproduce. It can be stated that studying factors

causing the Ammophila problem can give us a better view of how the aphid-plant interaction works.

24

When looking at the results of the plant suitability experiment it appears that mobile dune grasses A.

arenaria and L. arenarius are the most suitable hosts for supporting S. rufula populations. The

importance of this result should be stressed, because this is found independent of any other factor

that could have had an influence on population dynamics of S. rufula in the field. Schizaphis rufula

thus also shows greatest proliferation on plant species that are characteristic for this environment

when external environmental influences like soil biota are controlled for. This suggests that these

plants have evolved a similar characteristic that not only link them to a yellow dune or similar

habitats, but also make them more attractive to S. rufula. The observation that of the non-dune

species D. flexuosa does best considering the persistence of the aphids could be an extra indication

for this. Results for persistence on D. flexuosa were comparable with the embryonic dune grass E.

farctus. As stated above, D. flexuosa is found in heathland habitats that more closely resemble a

dune environment with its dry, sandy but also acidic soils. This apparent impact of the environment

on plant traits differs from the short term impact causing the Ammophila problem.

It is not yet clear which plant characteristics cause the preference of S. rufula for representatives of

sandy and dry environments. Either way, a remarkable feature of the aphid is that it has a relatively

long rostrum which is rather unique within the genus Schizaphis (Heie, 1986). This long rostrum could

be an adaptation to the dense covering of the leaves of host plant A. arenaria with trichomes. These

hairlike structures make it easier for the plant to prevent dehydration under dry circumstances by

retaining a thin layer of moist air on the leave’s surface. That is the reason why the Mediterranean

subspecies A. arenaria ssp. arundinaceae has a higher density of trichomes on its leave surface then

the Atlantic subspecies A. arenaria ssp. arenaria (de la Peña, 2011). Trichomes can take many

different forms depending on their function for the plant. In the case of Galium aparine they are for

example hooked to prevent stems from slipping from their support or in the carnivorous plant genus

Drosera they secrete a sticky substance to catch insects (Weeda et al., 2003a; Weeda et al., 2003b).

Studying differences in morphology of these trichomes for the plant species investigated in this

master thesis could give more insight in what way S. rufula is linked to psammophytic plant species,

i.e. species that grow on sandy soils.

It seems that both the field survey and the plant suitability experiment indicate independent from

one another that the aphid has the greatest potential of influencing dune dynamics in yellow dune

habitats. The result that D. flexuosa does better than other non-dune species could, however, also be

a consequence of our study design. For all plants sandy soils were used from nature reserve

Westhoek to rear them on. D. flexuosa could thus have been of a better quality as a feeding source in

comparison with all other non-dune plants that are not linked to sandy soils, because it was the most

adapted to these soils. Even in this case, however, the overall conclusion that S. rufula has the most

potential of influencing dune dynamics in yellow dune habitats will still hold because the soil is

evenly optimal for all dune grasses among which the same tendency could be seen.

25

3. Ecological preferences and their meaning for the functioning of

a dune ecosystem

The observation that S. rufula was mostly found on A. arenaria when it stood in dynamic yellow

dunes implies that the aphid has the greatest potential of impacting its surroundings in these kind of

environments. From the results found in the plant suitability experiment it could also be concluded

that primary fixating grasses supported the largest aphid populations. Interestingly, these grasses are

also the ones that suffered the most when S. rufula populations were present on their leaves.

Ammophila arenaria showed a significant decrease in dry weight when comparing the control with

the experimental plants in the plant suitability experiment. There was no control for L. arenarius but

the same results as for A. arenaria are expected because at the end of the experiment these plants

had a clearly withered appearance similar to that observed with the A. arenaria individuals under

study (own observation, 2013). This observation indicates that S. rufula has the potential of having a

significant impact on important dune grasses which could result in an impact on the dune landscape

as a whole.

However, although these dune grasses seem to suffer the most from the presence of the aphid

within the plant suitability experiment, these observations were not yet seen in the field. This must

not come as a surprise because interactions in the field are much more complex compared to those

artificially put up under lab conditions. Examples of additional factors that have to be taken into

account are predation rate, physical stress by windblown sand and as previously stated the impact of

below ground fauna on above ground herbivory. Considering for example the predation rate. Syrphid

larvae and Coccinellidae have been seen predating on S. rufula, but it is expected that also other

generalistic predators, like e.g. certain dune spiders (Araneae), occasionally feed on S. rufula. Similar

with the parasitoid wasps, it is not known for certain what the impact is of these organisms on

population dynamics of S. rufula. It was hypothesized that aphids on shoots of dune plants are more

easily detected by these predators and thus more easily subjected to predation than aphids in more

concealing tussocks of grass. This implies that the chance of finding aphids in the latter is higher.

Although a greater chance was indeed found of encountering aphids on plants with more leaves

during the field survey these results must be considered with care. As seen within the results from

the field survey, the variation in plant sizes within the group of plants that contained aphids as well

as the group of plants that did not contain aphids was very large and the ranges of both groups

almost completely overlapped (see figure 7). There is thus no clear indication of an optimal host

plant size. The result of a significant difference between the group means could have been the result

of a bias in the amount of smaller versus larger plants. There were 42 plants monitored with an

amount of leaves smaller than fifty versus 18 plants with an amount of leaves higher than fifty. The

largest plant individual had 236 leaves. When plants with a large amount of leaves are

underrepresented within the field survey, it could be that by chance alone the few bigger plants

within the field survey had aphids present on their leaves. The relative amount of bigger plants with

aphids encountered during the field survey could thus be much higher than is the case for the smaller

plants while this is not necessarily true for the real situation. From this, the wrong conclusion could

be drawn that the chance is higher of encountering aphids on bigger plants compared to smaller

plants. Moreover, in some respects the results that show greater encounter rates on bigger plants

are not as logical as for our “concealment from predators” hypothesis. Older and therefore also

bigger plants tend to be a less appropriate food source for S. rufula. As stated by Pettersson (1971c)

and Kennedy (1958) and supported by our own observations an optimal plant age for aphid feeding

26

is approximately five to six weeks. This is rather young for A. arenaria which is a perennial plant. Also,

in a study by Hacker and Bertness (1995) it was shown that salt-marsh aphids were even more

predated when present on larger host plants due to ladybird beetles landing on tall structures more

often than on shorter structures. Another outcome from the field survey shows however that the

bigger the host plant is, the lower the relative abundance of S. rufula becomes. This is more in line

with what is expected from the studies by Pettersson (1971c), Kennedy (1958) and Hacker and

Bertness (1995). When combining both outcomes from the field survey it is seen that although

aphids are apparently more encountered on bigger plants, their populations stay relatively small

compared to the (seemingly) larger availability of food resources. This seems to be contradictory but

can be explained by stating that bigger plants have a bigger chance to be noticed by S. rufula when it

is searching for a host plant. This is an application of the resource concentration effect (Otway et al.,

2005). The aphid will, however, experience higher predation rates or lower nutritional values when

feeding on these bigger plants. This could eventually result in the smaller relative amount of aphids

present on these plants.

There is another remarkable difference when comparing results from the plant suitability experiment

with field observations. Although aphid population growth during the experiment was rather limited

on dune species other than L. arenarius and A. arenaria, there are numerous observations in the field

of S. rufula feeding and reproducing on F. rubra and other species. Mummies were even encountered

on C. arenaria. When sampling locations were visited earlier in the year (April 2014) it seemed that

there were more aphids present on F. rubra than on A. arenaria. When looking at the outcomes of

the plant suitability experiment most plant species could still maintain a small number of aphids for a

certain period, with an average of 14.125, 14.8 and 15 days for the dune grasses F. rubra, E. farctus

and C. epigejos respectively. This shows that aphids can still retrieve to some extent a sufficient

amount of nutrients from these plants for their survival. According to the phenology of A. arenaria,

April is the month in which shoot growth and seed germination is initiated (Huiskes, 1979). Because

S. rufula prefers young shoots (Pettersson, 1971c) the aphid could be forced towards other dune

grass species when shoots of A. arenaria are not yet available, that is, earlier in the year. These other

dune grasses, like F. rubra, could thus function as a kind of reservoir from which the aphids can

emerge when shoots of the more suitable host plants, L. arenarius and A. arenaria, become more

abundant. For these plant species, however, no large impact on population dynamics is expected

because aphids can probably not reach sufficiently high population densities for this.

Although no statistically supported conclusions could be drawn from the one observed interaction

between S. rufula and a certain ant species (Formicidae), this is still an interesting sighting. Our

observation is in concordance with that of Pettersson (1971a) who also reported occasional visits by

ants to S. rufula populations. The aphid-ant interaction is a food-for-protection mutualism in which

the ants receive food in the form of honeydew produced by the aphids and in return, they protect

the aphids against predators and parasitoids (Styrsky & Eubanks, 2007). Besides this it is also shown

that even in the absence of predators and parasitoids ants can still influence aphid life history traits

such as live span, age of maturation and reproduction rate (Flatt & Weisser, 2000). Intuitively it can

be expected that the host plant should thus suffer from this interaction through the positive effect

ants have on aphid population densities. However, many studies show that plants actually benefit

indirectly. In these studies, increased predation or harassment of other more damaging herbivores

by hemipteran-tending ants resulted in decreased plant damage and/or increased plant growth and

27

reproduction (Styrsky & Eubanks, 2007). It is not expected that ants have a significant impact, be it

positive or negative, on overall aphid or dune grass populations at the three locations studied in this

master thesis because the interaction was encountered only once. It cannot be said, however, that

these kind of interactions are not important for other areas where S. rufula is present.

The influence of aphids on the vegetation in a species poor, natural system such as a yellow dune

habitat is an interesting topic. Besides giving important insights in the functioning of a dynamic

coastal dune landscape it can also be used outside the context of this coastal ecosystem. Despite

yellow dunes being a natural system, its simplicity in vegetation structure with a dominant presence

of A. arenaria, for example resembles that of an agricultural monoculture. Investigating aphid

dynamics in yellow dune habitats thus can give a view on fundamental differences between

agricultural and natural systems in general. Indeed, when the natural and agricultural ecosystem is

more similar, fundamental differences can more easily be distinguished. These differences could be

the key to knowing why aphids can develop into pest species in an agricultural system, while

populations are more balanced in natural ecosystems. Although agricultural systems were not the

main focus in this study certain differences between natural and agricultural systems can already be

shown. It was for example seen that aphids were less present on their host plant A. arenaria when it

stood in a species rich environment. Various possible underlying mechanisms were put forward.

Depending on which proposed mechanism is the true one, our results may or may not be used in

aphid pest management within an agricultural system. When the escape hypothesis is true and

aphids largely thrive in dynamic systems where plants are not negatively impacted by soil biota (Van

der Putten et al., 1988) no applications for agricultural systems can be found because these systems

do not harbor such dynamics. However, when the hypothesis holds that vegetatively richer

environments harbor a more diverse community of natural enemies it could be opted that pest

species are easier to control when agricultural fields are located near natural, more biodiverse areas.

The importance of habitat complexity for pest control was already pointed out by several studies

such as the one performed by Chaplin-Kramer and Kremen (2012), Müller & Godfray (1999) and

Altieri and Letourneau (1982).

4. Parasitoids: the importance of finding new associations

Parasitoids were found on S. rufula that were to our knowledge not yet known to be associated with

this aphid. This observation clarifies another link within the ecological dune network and opens new

doors for ecological research in European dune landscapes in general. It is indeed so that Aphidius

rhopalosiphi and A. avenae are both very widespread and found in countries across Europe (Nieto

Nafría, 2007). This makes that results from this study apply to a geographically wide range of dune

landscapes. Interestingly, A. rhopalosiphi and A. avenae are not usually linked to dune ecosystems.

Aphidius rhopalosiphi is known from grass infesting aphids like Diuraphis noxia, Rhopalosiphum padi

and Sitobion avenae in agricultural systems (Turpeau et al., 2011b). This could however be a

reflection of the fact that parasitoids are mainly studied in agricultural systems due to their

economical importance. Actual habitat range could be much wider. Because species like S. avenae

can also develop on rushes (Juncaceae) and sedges (Cyperaceae), it is also expected that natural

habitats containing these plant species have potential of harboring A. rhopalosiphi populations

(Blackman & Eastop, 2006). Aphidius avenae also parasitizes on S. avenae, making it already known

that distributions of both parasitoid species show overlap (Turpeau et al., 2011a). Aphidius avenae is

28

however also seen to associate with aphid species belonging to the genus Dysaphis sp. which has as

primary hosts apple and pear species (Rosaceae: Pyrus sp. & Malus sp. respectively) (Turpeau et al.,

2010b). This makes its range of suitable habitats very divers. It can thus be stated that both species

are widely distributed which makes that these species are most likely also simultaneously associated

with S. rufula beyond Belgian borders.

Although very widespread, parasitoids were found only occasionally in Ter Yde and Zwin. This could

be due to the fact that these nature reserves are more accessible for tourists, while in the Westhoek

accessibility is restricted to a few hiking trails. This is interesting because this means that there could

be an indication of habitat deterioration within the way aphids and parasitoids interact in a dune

ecosystem. Jane Memmott (2009) is a fierce proponent of using species interactions and ecological

networks in general as a habitat quality assessment tool. Next to more traditional interactions like

predator-prey and pollinator-plant interactions, she also elucidated on parasitoid-host interactions.

Parasitoids are remarkably common in food webs (Lafferty et al., 2006) and their loss could have a

profound effect on community structure and function (Lafferty et al., 2008). This, linked to the wide

distribution both host species S. rufula and parasitoids A. rhopalosiphi and A. avenae have, gives

them the potential of being used as a standard for comparing the quality of similar dune habitats

across a wide geographical range.

When coming back on the Ammophila problem, factors influencing A. arenaria and indirectly S.

rufula may also influence parasitoids through their interactions with these aphids (Harvey et al.,

2003; Godfray, 1994). Bezemer et al. (2005) found that parasitoid mortality and the proportion of

males were significantly lower when nematode and/or microorganisms communities were

introduced to the soil and in a study by Masters et al. (2001) it was found that root feeding insects

can increase the parasitism rate of seed feeding insects. Other studies with varying identities of soil

biota and the organisms they influence can be found on this subject where some proved to have

found an impact (Gange et al., 2003) and others not (Wurst & Jones, 2003). This indicates that

observed patterns are highly species dependent (Bezemer et al., 2005). Studying gradients within

parasitism rate or efficiency within a dune ecosystem has potential to give some interesting results

when linking them to gradients within S. rufula population dynamics and vegetation composition.

Schizaphis rufula is more found in yellow dune habitats, but are they also more often parasitized in

this environment? In other words do the aphids in these environments have a different impact on

parasitoid fitness and population densities than in more fixated areas? It has already been shown

that bottom-up control is important for parasitoid food webs (Petermann et al., 2010; Bukovinszky et

al., 2008; Hawkins, 1992). Host fitness can for example influence parasitoid oögenesis (Cicero et al.,

2012) or the rate of encapsulation of the parasitoid egg inside the host which is a defense

mechanism against parasitism (Klemola et al., 2008). It would be interesting to see in what way plant

fitness impacted by soil biota or abiotic factors influences higher trophic levels. This would give us a

more complete image of how S. rufula interacts with and influences its environment.

When looking at the aphid-parasitoid relationship in reverse way, that is, when considering the fact

that a new aphid host species was found for A. rhopalosiphi and A. avenae, another interesting

aspect of our study results is shown. New host discovery is important for assessing the effectiveness

of biological control by parasitoids in agricultural systems. Highly specialized predators and

parasitoids usually have the largest impact on herbivores compared to generalists (Müller & Godfray,

1999). However, the presence of other parasitoids and hyperparasitoids of the targeted pest species,

29

be it from natural populations or multiple introductions, can affect the effectiveness of an introduced

biocontrol agent (Mills, 2002). Knowing parasitoid ecology and the complete host range aids in

finding the optimal (combination of) parasitoid species for a certain agricultural system.

5. Overall conclusions

It is concluded that studying the dune aphid S. rufula and its interactions with both lower trophic

levels (the dune grasses such as A. arenaria and L. arenarius) as higher trophic levels (parasitoids)

resulted in a wider knowledge of species interactions and composition within a dune landscape. This

knowledge has the potential of being used in various study domains such as dune management and

pest control. Knowing that what has already been described regarding dune community composition

could only be the tip of the dune biodiversity iceberg (Bonte & Provoost, 2004), still many interesting

and useful facts about dune community structure are to be discovered.

Summary The ecology of a species and ecological interactions with other species tells us much about its habitat

and habitat functioning. This knowledge can help us in managing certain ecosystems. European dune

landscapes belong to the ecosystems that can benefit from this knowledge. Still, little is known about

dune biota and their interactions. Dune biota are highly influenced by their environment because of

the very specific and harsh conditions that prevail in dune habitats. This makes them more

vulnerable to habitat changes. In the light of the recent deteriorations of European sand dune

landscapes, the urge of protecting and managing these systems is particularly clear.

Schizaphis rufula is an aphid which feeds on important dune grasses like Leymus arenarius and

Ammophila arenaria and is abundantly present in European dune landscapes. Although this species

has potential to significantly impact the dune ecosystem through its influence on primary dune

fixating grasses, little is known about its ecology and natural enemies. In previous studies, parasitized

aphids called mummies were found. The identity of these parasitoids was however not known. This

master thesis has as its main aims to determine which biotic factors impact population dynamics of

the dune aphid S. rufula and under which conditions this aphid’s populations do best. More

specifically we tried to determine 1) the influence of the surrounding plant community on population

dynamics of S. rufula associated with A. arenaria in the field, 2) the specificity of the aphid’s host

choice within and between plant species and 3) the parasitoid community associated with S. rufula.

By doing so a better view can be made on what impact the aphid has on its environment.

Two different research strategies were used. First of all a more observational strategy was conducted

during the course of a field survey in dune complexes of nature reserves the Westhoek, Ter Yde and

Zwin. Secondly, field data was complemented with an experimental setup during a host suitability

experiment.

In the field survey aphid population dynamics on A. arenaria were studied. It was seen that the

presence of aphids on A. arenaria is significantly negatively influenced by the percentage of F. rubra

situated within a one meter radius from the A. arenaria host plant and also by plant species richness

R within that same area. These two factors covary along a dune fixation gradient with a lower chance

of finding S. rufula on A. arenaria when dunes become more fixated (amount of F. rubra and plant

species richness is higher). What the underlying mechanism of this decline in encounter rate is, could

30

not be determined. It is however remarkably similar to the decline in growth of A. arenaria when

going towards more fixated dunes. This is often called the Ammophila problem and the underlying

cause is still largely debated. Both abiotic as biotic factors like soil biota have been suggested.

Whatever the cause may be, it seems to also influence S. rufula population dynamics and places the

highest potential of influence of S. rufula on dune dynamics in yellow and mobile dune habitats.

Regarding intrinsic plant characteristics of the A. arenaria host plant it is found that the more leaves

the host plant had the more chance there was to encounter S. rufula feeding on the plant. This

observation must however be considered with care because of a bias towards a larger amount of

smaller plants during the sampling for host plants. It was previously shown that aphids feed most

optimally on younger plants and therefore smaller plants (Pettersson, 1971c). However, because it is

only the presence of the aphid on the host plant that was significantly influenced and not the

population density, this result could still be explained by the resource concentration effect (Otway et

al., 2005) which states that specialist insects are more likely to find, remain and reproduce on their

hosts when these plants grow in dense patches in pure stands. Bigger plants thus have a bigger

chance to be noticed by S. rufula when it is searching for a host plant. The aphid will, however,

experience difficulties when feeding on these bigger plants. This could eventually result in the

smaller relative amount of aphids.

In the plant suitability experiment it was found that aphid populations grew largest on the primary

dune fixating grasses L. arenarius and A. arenaria. Also the persistence (amount of days that S. rufula

was present on the plant individual) was longest. These species suffered the most from the presence

of S. rufula. This result is interesting because it is found independent of any other factor that could

have had an influence on population dynamics of S. rufula in the field. Consequently, both the field

survey and the plant suitability experiment indicate independent from one another that the aphid

has the potential of influencing dune dynamics.

Combining the outcomes of the field survey and the plant suitability experiment and especially when

focusing on the differences between the two can give a hint of which aspects of population dynamics

are affected by plant species alone and what the effect is of additional environmental influences on

these population dynamics in the field. Although primary dune fixating grasses seem to suffer the

most from the presence of the aphid within the plant suitability experiment, these observations were

not yet seen in the field. Consequently, there must be an additional factor in the field that limits

proliferation of aphid populations. This additional factor can be predation, parasitism, physical stress

by the overblowing sand, interactions with soil biota or a combination of these. The exact cause

could however not be determined.

During the field survey, two Aphidius species could be identified, namely A. rhopalosiphi and A.

avenae (Hymenoptera: Braconidae), and three hyperparasitoids, Apoanagyrus sp. (Hymenoptera:

Encyrtidae), Dendrocerus sp. (Hymenoptera: Megaspilidae) and Pachyneuron sp (Hymenoptera:

Pteromalidae). These species were to our knowledge not yet known to parasitize on the dune aphid

S. rufula. The importance of finding new associations cannot be underestimated and the knowledge

that comes from it can be applied in a wide array of research fields.

Jane Memmott (2009) is a fierce proponent of using species interactions and ecological networks in

general as a habitat quality assessment tool. Considering the fact that almost all parasitoids were

found in nature reserve Westhoek, which is the most monitored and the least accessible to the

broader public, it seems that a diverse parasitoid community associated with S. rufula indicates a

31

better quality habitat. Because both the aphid as its parasitoids are widespread in Europe, this gives

a high potential of using this interaction for habitat quality assessment.

Finding a new host species associated with A. rhopalosiphi and A. avenae could also benefit

agriculture. These species are often used as biocontrol agents. Knowing parasitoid ecology and the

complete host range aids in finding the optimal (combination of) parasitoid species for a certain

agricultural system.

Studying the dune aphid S. rufula and its interactions with both lower trophic levels (the dune

grasses such as A. arenaria and L. arenarius) as higher trophic levels (parasitoids) resulted in a wider

knowledge of species interactions and composition within a dune landscape. Knowing that what has

already been described regarding dune community composition could only be the tip of the dune

biodiversity iceberg (Bonte & Provoost, 2004), still many interesting and useful facts about dune

community structure are to be discovered.

Acknowledgements I want to sincerely thank Eduardo de la Peña who aided me in finding my own way of accomplishing

this master thesis without withholding guidance and clear counseling. I also want to thank Viki

Vandomme for getting me through my struggle with the PCR-machine and for staying positive when

another week ended with the diagnosis “no bands”. I want to thank Maria Njo and Tom Beeckman

from the VIB for giving me the opportunity of working with the electron microscope and Joachim

Moens and Patrick De Clercq for providing keys and advice for the identification of the parasitoids. I

also want to thank Floris Van Laere for editing the electron microscopy images, Wendy Vrydag and

Steven Goossens for reviewing my thesis and fellow thesis buddies at TEREC Eline Vermote, Karen

Bisschop, Jelle, Emily Veltjen, Margaux Boeraeve, Alexander Boffin, Stefan Vandamme, Rieneke

Vanhulle, Willem Proesmans, Hannah Volckaert and Judith for their support and company.

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37

Appendix 1. Tables

Pearson Correlation Coefficients

Prob > |r| under H0: Rho=0 Number of Observations

Diameter at

bottom Diameter at

top Length of

longest leave No. of leaves Sand A. arenaria F. rubra E. farctus C. arenaria

Species richness (R)

No. of S. rufula No. of

parasitoids Parasitism rate

Diameter at 1 0.8215 0.35492 0.70354 0.22604 -0.1968 0.06584 -0.05841 -0.01983 0.13617 0.10434 -0.05374 -0.0507

bottom <.0001 <.0001 <.0001 0.0005 0.0024 0.3139 0.3717 0.7618 0.0366 0.1099 0.4112 0.4382

236 236 236 236 236 236 236 236 236 236 236 236 236

Diameter at 0.8215 1 0.58741 0.80309 0.25783 -0.24804 -0.05274 0.05439 0.02029 0.04049 0.11732 -0.08293 -0.04845

top <.0001 <.0001 <.0001 <.0001 0.0001 0.416 0.4015 0.7544 0.5324 0.0696 0.2004 0.455

236 240 240 240 240 240 240 240 240 240 240 240 240

Length of 0.35492 0.58741 1 0.54562 0.09196 -0.02863 -0.18615 0.12319 0.15539 0.05038 0.1127 -0.02072 -0.03807

longest <.0001 <.0001 <.0001 0.1555 0.659 0.0038 0.0567 0.016 0.4373 0.0814 0.7495 0.5572

leave 236 240 240 240 240 240 240 240 240 240 240 240 240

No. of leaves 0.70354 0.80309 0.54562 1 0.20204 -0.17696 -0.12244 0.15915 0.03077 -0.04745 0.18089 -0.07328 -0.07183

<.0001 <.0001 <.0001 0.0017 0.006 0.0582 0.0136 0.6353 0.4644 0.0049 0.2581 0.2677

236 240 240 240 240 240 240 240 240 240 240 240 240

Sand 0.22604 0.25783 0.09196 0.20204 1 -0.60439 -0.33943 -0.09786 -0.18503 -0.23983 0.20283 0.11921 0.0538

0.0005 <.0001 0.1555 0.0017 <.0001 <.0001 0.1306 0.004 0.0002 0.0016 0.0652 0.4067

236 240 240 240 240 240 240 240 240 240 240 240 240

A. arenaria -0.1968 -0.24804 -0.02863 -0.17696 -0.60439 1 -0.04644 -0.1278 -0.14985 -0.3276 -0.06562 -0.0707 0.01999

0.0024 0.0001 0.659 0.006 <.0001 0.4739 0.048 0.0202 <.0001 0.3113 0.2753 0.758

236 240 240 240 240 240 240 240 240 240 240 240 240

Table 1: Matrix that shows the Pearson correlation coefficients for all pairwise comparisons between the variables from the field survey. For each pairwise comparison three values are given. The first value represents thePearson correlation coefficient itself. The second value is the p-value and the third value is the number of observations. The first ten columns represent the independent variables. The variables “Diameter at bottom” to“No. of leaves” are intrinsic plant characteristics. The unit of all measurements is centimeter. The variables “Sand” to “Species richness (R)” represent the composition of the vegetation within a one meter radius. In the fieldeach of these variables was measured in percent. The last three columns are the dependent variables. The number of S. rufula individuals and parasitoids were counted per plant. Parasitism rate is the number of parasitizedaphids or mummies per plant devided by the total number of S. rufula individuals (including mummies) on that plant.

38

F. rubra 0.06584 -0.05274 -0.18615 -0.12244 -0.33943 -0.04644 1 -0.17389 -0.03162 0.53895 -0.15852 -0.04498 -0.10331

0.3139 0.416 0.0038 0.0582 <.0001 0.4739 0.0069 0.6259 <.0001 0.014 0.488 0.1104

236 240 240 240 240 240 240 240 240 240 240 240 240

E. farctus -0.05841 0.05439 0.12319 0.15915 -0.09786 -0.1278 -0.17389 1 -0.06175 0.06733 0.02934 -0.02453 -0.01988

0.3717 0.4015 0.0567 0.0136 0.1306 0.048 0.0069 0.3408 0.2989 0.6511 0.7053 0.7593

236 240 240 240 240 240 240 240 240 240 240 240 240

C. arenaria -0.01983 0.02029 0.15539 0.03077 -0.18503 -0.14985 -0.03162 -0.06175 1 0.25135 -0.0685 -0.02452 -0.0359

0.7618 0.7544 0.016 0.6353 0.004 0.0202 0.6259 0.3408 <.0001 0.2906 0.7054 0.5799

236 240 240 240 240 240 240 240 240 240 240 240 240

Species 0.13617 0.04049 0.05038 -0.04745 -0.23983 -0.3276 0.53895 0.06733 0.25135 1 -0.2632 -0.06676 -0.11289

richness (R) 0.0366 0.5324 0.4373 0.4644 0.0002 <.0001 <.0001 0.2989 <.0001 <.0001 0.303 0.0809

236 240 240 240 240 240 240 240 240 240 240 240 240

No. of 0.10434 0.11732 0.1127 0.18089 0.20283 -0.06562 -0.15852 0.02934 -0.0685 -0.2632 1 0.65481 0.14432

S. rufula 0.1099 0.0696 0.0814 0.0049 0.0016 0.3113 0.014 0.6511 0.2906 <.0001 <.0001 0.0254

236 240 240 240 240 240 240 240 240 240 240 240 240

No. of -0.05374 -0.08293 -0.02072 -0.07328 0.11921 -0.0707 -0.04498 -0.02453 -0.02452 -0.06676 0.65481 1 0.48204

parasitoids 0.4112 0.2004 0.7495 0.2581 0.0652 0.2753 0.488 0.7053 0.7054 0.303 <.0001 <.0001

236 240 240 240 240 240 240 240 240 240 240 240 240

Parasitism -0.0507 -0.04845 -0.03807 -0.07183 0.0538 0.01999 -0.10331 -0.01988 -0.0359 -0.11289 0.14432 0.48204 1

rate 0.4382 0.455 0.5572 0.2677 0.4067 0.758 0.1104 0.7593 0.5799 0.0809 0.0254 <.0001

236 240 240 240 240 240 240 240 240 240 240 240 240

39

Morphological identification Specimen

code Primers

Sequence length

(bp) Hits indicated by BLAST

Query cover

Similarity Felsenstein-Tajima-Nei

distance

A. avenae WH472 16S 415 A. avenae (JQ240492.1) 0.93 0.99 0.0078

WH472 LWRh 754 A. avenae (JN620702.1) 0.52 1 0

A. avenae WH985 16S-R 264 A. avenae (JQ240492.1) 0.94 0.99 0.00407

A. avenae WH1581 16S 419 A. avenae (JQ240492.1) 0.92 1 0

A. rhopalosiphi WH271 16S 416 A. rhopalosiphi (JQ240518.1) 0.93 1 0

WH271 LWRh 554 A. rhopalosiphi (JN620727.1) 0.58 0.99 0.00626

A. rhopalosiphi WH273 16S-R 195 A. rhopalosiphi (JQ240518.1) 0.9 0.93 0.01809

WH273 LWRh-F 406 A. rhopalosiphi (JN620727.1) 0.79 0.98 0.0095

A. rosae (JN620738.1) 0.96 0.98 0.01596

A. rhopalosiphi WH474 16S 416 A. rhopalosiphi (JQ240518.1) 0.93 1 0

A. rhopalosiphi or A. urticae WH371 LWRh-F 464 A. urticae (JN620747.1) 0.84 0.99 0.01071

A. sonchi (JN620745.1) 0.6 0.98 0.03639

A. rhopalosiphi (JN620727.1) 0.69 0.97 0.03269

A. rhopalosiphi or A. urticae WH473 16S-F 200 A. rhopalosiphi (JQ240518.1) 0.98 0.99 0.01016

A. uzbekistanicus (JQ240542.1) 0.98 0.99 0.01016

A. funebris (JQ240506.1) 0.98 0.99 0.01016

A. urticae (JQ240540.1) 0.98 0.98 0.02052

WH473 LWRh-F 329 A. urticae (JN620747.1) 0.98 0.98 0.01213

A. eadyi (JN620707.1) 0.98 0.98 0.01626

A. rhopalosiphi (JN620727.1) 0.77 0.95 0.03297

larvae WH47para1 16S 390 A. rhopalosiphi (JQ240518.1) 0.99 1 0

WH47para1 LWRh 563 A. rhopalosiphi (JN620727.1) 0.57 0.98 0.02535

A. urticae (JN620747.1) 0.67 0.98 2.54471

larvae WH47para2 16S 393 A. rhopalosiphi (JQ240518.1) 0.98 0.99 0

A. sonchi (JQ240538.1) 0.99 0.99 0.00287

A. ervi (AF174310.1) 0.99 0.99 0.00575

A. microlophii (JQ240514.1) 0.99 0.99 0.00575

A. funebris (JQ240506.1) 0.99 0.99 0.00575

A. matricariae (JQ240510.1) 0.99 0.99 0.00867

A. urticae (JQ240540.1) 0.98 0.99 0.00867

A. uzbekistanicus (JQ240542.1) 0.98 0.99 0.00865

WH47para2 LWRh 562 A. rhopalosiphi (JN620727.1) 0.57 0.97 0.0287

A. sonchi (JN620745.1) 0.5 0.97 0.03652

A. microlophii ( JN620725.1) 0.69 0.98 0.01258

A. urticae (JN620747.1) 0.69 0.98 0.01256

larvae WH475 LWRh 550 A. urticae (JN620747.1) 0.71 0.98 0.00955

A. rhopalosiphi (JN620727.1) 0.58 0.97 0.01278

Table 2: This table shows a list of all parasitoid specimens belonging to the genus Aphidius (Hymenoptera: Braconidae) for which DNA has beensuccessfully amplified and sequenced. The table shows the results of the morphological identification which was done with the key from Tomanovićet al. (2012) and information from the website Encyclop’Aphid (http://www4.inra.fr/encyclopedie-pucerons). A unique specimen code was given toall specimens found during the study. The letters represent sampling site (WH= Westhoek, TY = Ter Yde, R= Retranchement) following two or threenumbers are the date and the last number represents the number of the specimen found on that date. Primers that were used to amplify andsequence the DNA are given in the third column. –F indicates a forward primer and –R the reverse. When neither is indicated, both were of such aquality that a contig could be made. Sequence length is given in number of base pares (bp) and most plausible hits in BLAST are shown with thequery cover, similarity and Felsenstein-Tajima-Nei distances.

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larvae R774 16S-F 192 A. rhopalosiphi (JQ240518.1) 1 0.99 0

A. uzbekistanicus (JQ240542.1) 1 0.99 0

A. microlophii (JQ240514.1) 1 0.99 0

A. funebris (JQ240506.1) 1 0.99 0

A. ervi (JQ240500.1) 1 0.99 0

larvae R775 16S-F 191 A. rhopalosiphi (JQ240518.1) 1 0.99 0

A. uzbekistanicus (JQ240542.1) 1 0.99 0

A. microlophii (JQ240514.1) 1 0.99 0

A. funebris (JQ240506.1) 1 0.99 0

A. ervi (JQ240500.1) 1 0.99 0

R775 LWRh-F 466 A. urticae (JN620747.1) 0.84 0.98 0.01888

A. eadyi (JN620707.1) 0.84 0.98 0.0189

A. microlophii (JN620725.1) 0.84 0.98 0.01893

A. ervi (JN620710.1) 0.84 0.98 0.02214

A. uzbekistanicus (JN620749.1) 0.84 0.98 0.02865

A. funebris (JN620715.1) 0.84 0.98 0.02856

A. rhopalosiphi (JN620727.1) 0.69 0.98 0.02535

egg WH47_ei1 LWRh 531 A. urticae (JN620747.1) 0.72 0.98 0.02209

A. eadyi (JN620707.1) 0.72 0.98 0.02213

A. microlophii (JN620725.1) 0.72 0.98 0.02214

A. ervi (JN620710.1) 0.72 0.98 0.02538

A. rhopalosiphi (JN620727.1) 0.61 0.97 0.02862

undevelloped due to WH47onb1 LWRh 522 A. rhopalosiphi (JN620727.1) 0.61 0.99 0.00626

hyperparasitoid A. ervi (JN620710.1) 0.73 0.99 0.0094

A. rosae (JN620738.1) 0.73 0.99 0.01261

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2. Glossary

figures and definitions for terms used in section “Results” under “Identification of parasitoids”. The wing venationis illustrated according to that of Dolichurus sp. (Apocrita: Ampulicidae). All definitions and figures are taken fromGoulet & Huber (1993).

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