Complete Thesis

University of Groningen

Coevolution in host–parasite systems Ashghali Farahani, Sajad

DOI: 10.33612/diss.128654818

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Coevolution in host–parasite systems

Behavioural strategies of native and invasive hosts

PhD thesis

to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. C. Wijmenga and in accordance with the decision by the College of Deans.

This thesis will be defended in public on

Friday 10 July 2020 at 14:30 hours

Sajad Ashghali Farahani

born on 30 June 1979 in Tehran, Iran

Supervisors Prof. J. Komdeur Prof. J. Palsbøll

Assessment Committee Prof. B. Helm Prof. M. Naguib Prof. F. Cezilly

CONTENTS

Chapter 1 General introduction: invasion and parasitism 4 Box 1 Effects of parasites on the behaviour of native and 20 invasive intermediate hosts Chapter 2 The effects of parasites upon non-host predator avoidance behaviour in ..24 native and invasive gammarids. Chapter 3 The acanthocephalan parasite Polymorphus minutus increases 37 the positive rheotaxis behavior of its gammarid hosts. Box 2 Effects of salinity and parasites on physiological changes 46 of native and invasive intermediate-hosts Chapter 4 Experimental evidence of parasitic infection increasing salinity 59 tolerance of the native gammarid hosts Chapter 5 General discussion and synthesis 62 References 74

Chapter 1 General introduction: Invasion and parasitism

It was Al-Jahiz (776-869 A.D) who first hinted that environment and migration cause changes in animal life (Shah, 2015). This means that changing environmental conditions e.g. climate change that stimulates the migration, movements of the whole populations over long distances, (Dingle and Drake, 2007; Van Buskirk et al., 2009) and invasion, movements of part of populations over the long or short distances, (Ward and Masters, 2007) of organisms. Changing life from the unconsciously mobile world, e.g. plants, to the consciously mobile world, e.g. animals, has been emphasized by scholars e.g. Nasir Aldin Tusi (1201-1274 A.D) (Alakbarli, 2001). He believed that individuals could adapt to specific environmental conditions through changes of structure and behaviour (Alakbarli, 2001). However, the role of parasites, in changing the physiology and behaviour of their hosts, during the evolution of the accidental movement and deliberate movement has been ignored. The interesting question about parasites in biological philosophy is whether they have deliberate movement and manipulate the host’s movement or whether they are submissive to their host’s movements and do not manipulate movement of their host. Parasites could migrate with their host and could select native or invasive species as new hosts, in new habitats. The fundamental question is whether parasite coevolved with new invasive host species in the same way as previous native hosts? Any influence of the parasite on its intermediate host phenotypic manipulation in order to increase trophic transmission to the definitive host is thus thought to be favoured by natural selection. This “adaptive manipulaton” hypothesis is directly related to the concept of the “extended phenotype”, introduced by Richard Dawkins (1982). As emphasized at several occassions, natural selection in a host-parasite system may not necessarily target host traits directly, but instead on the ability of parasites to alter hosts traits in a manner enhancing the trophic transmission of the parasite (Seppälä and Jokela, 2008; Seppälä et al., 2008; Cézilly and Perrot-Minnot, 2010).

More than 40% of known species are parasitic (Dobson et al., 2008). Parasites are a reflection of the local food-web structure and biodiversity in terms of the distributions of various hosts e.g. invertebrate groups, piscivorous fish, and waterfowl (Marcogliese et al., 2006). Fitness (e.g. growth, reproduction, defense, immune competence) of individuals of native host species is related to the direct or indirect effects of parasites, e.g. Pomphorhynchus laevis persuades lower immuno-competence and imposes energetic costs only in its native amphipod host Gammarus pulex; but not in invasive host Gammarus roeseli (Rigaud and Moret, 2003). However, the question is whether fitness is influenced by parasite in the same way in invasive host species as in native host species? It is important to clarify the relationship between invasive hosts and the structure and behaviour of their parasites. Invasive species as a host, often show lower parasite diversity (Keane and Crawley, 2002; Torchin et al., 2003) and higher parasite prevalence (MacNeil et al., 2003b). According to the “enemy release” hypothesis, which states that invasive species should experience a decrease in regulation by natural enemies e.g. escape from natural predators (Keane and Crawley, 2002) and escape from natural parasitism (Dick et al., 2010) to be successful in colonizing new areas.

Invasive species may introduce parasites to a new native host in a colonized area, and environmental conditions, e.g. climate change, promote future adaptations of the parasite to novel host species (Bacela-Spychalska et al., 2012) or may threat native species through being a competent host for a native parasite (“parasite spillback” recommended by Kelly et al. (2009)). Parasite spillback impacts on invasive species may increase the infection load of native parasites in native hosts and regulate native host populations through multiple-host shared-parasite systems (Kelly et al., 2009). The viability of invasive and native hosts (i.e. their survivorship, fecundity, dispersal ability, or geographic distribution) is regulated host switching mechanisms by parasite (either from the invasive host to native host or from native

5 host to the invasive host) (Pizzatto et al., 2012). However, with both increasing geographic distance and environmental dissimilarity between localities, decreasing of similar parasite community composition in the same host species are expected (Poulin et al., 2011). Maladaptation in a new host species through different migration rates between parasite and hosts (Moret et al., 2007) or parasite’s cryptic diversity in one location (Zittel et al., 2018) are consequences of parasites host switching mechanisms.

In this chapter, I will discuss positive and negative effect of invasive species. I will show responses of native species to invasive host-species, and the effects of parasites on native and invasive host-fitness. I will show competition between native and invasive hosts could be controlled by parasites/predators. Studies on invasive hosts and parasitism are necessary for a better understanding of the behaviour of parasites during host-parasite co-evolution (Janzen and May, 1979). Host-parasite coevolution refers to hosts’ ability to resist parasites under selective pressure, and parasites’ ability to overcome host defenses (Mostowy and Engelsta, 2011). Mutual selection triggers coevolution between parasites and hosts (Merlo et al., 2016).

1-Invasion

One of the most important ecological-global topics during the last decade is the consequences of invasion of non-indigenous species (Ruiz et al., 1997). Non-indigenous species, i.e. species introduced outside their native habitat by human activity (Kolar and Lodge, 2001) or migratory hosts (Bradley and Altizer, 2005). Invasive species are non-indigenous species that spread from the point of introduction and become abundant (Kolar and Lodge, 2001). Among others, ballast water from ships that contains species collected in other areas and then dropped in new areas (Ba et al., 2010), canal river connections (Van der Velde et al., 1998), escaped fish from aquaculture farms (Peterson et al., 2005), ornamental fish trade (Rixon et al., 2005) are considered to play a major role in invasion of new aquatic species in to aquatic ecosystems.

Invasion can affect the energy flow in pelagic-benthic pathways (Macisaac, 1996). Successful invasion means that invasive species were able to expand their population more quickly than native species by taking advantage of available resources and outcompeting local populations, e.g. through decreased competition from native species or increased resources thorough eutrophication from nutrient discharge (Davis et al., 2000; Wikström and Hillebrand, 2012). Invasive species effects on ecosystems could be positive (i.e. ecological services, water quality function) or negative (i.e. biodiversity loss, altering the structure and functioning of ecosystems, biodiversity function, social and economic problems). Furthermore, increasing temperatures that are due to global warming could facilitate the invasion and establishment of invasive species originating from warmer areas (Montserrat et al., 2013).

2-Negative effects of invasive species

Consequences of species invasions for the future of native habitats are biodiversity loss, altering the structure and functioning of ecosystems, biodiversity function and social- economic problems, which I will discuss below.

2-1 Biodiversity loss

Invasive species and climate change are the most important proximate causes of biodiversity loss worldwide (Perrings, 2002). Invasions cause biodiversity loss and changes in native food

6 web structure by decreasing species richness and destroying species interactions (Galiana et al., 2014). The occurrence of invasive species may cause extinction of native species (Clavero and Garcia-Berthou., 2005). Most native extinctions occur when the invasive species is a top predator (Galiana et al., 2014). Invasive species may cause a change in the genetic composition and behavioural patterns of native populations (Blackburn et al., 2014), causing extinction of parasites through changing native vector community structure (Dobson et al., 2008; Telfer and Bown, 2012) or niche shifts (Mooney and Cleland, 2001) that lead to extinction of native species.

2-2 Altering the structure and functioning of ecosystems

Both native and invasive species have a large impact on community structure and ecosystem functioning (Hooper et al., 2005). Invasion may cause reduction of activities of native species upon introduction to the new habitat; invasive species are often introduced at lower population densities than native species. The invasive species may not be able to compensate for the reduced activity of native species. For example, the replacement of native species of amphipods, e.g. Gammarus fossarum and G. pulex, by invasive species, e.g. Dikerogammarus villosus and Gammarus tigrinus, in a fresh water ecosystem resulted in a decrease of leaf litter recycling (Piscart et al., 2011b). As a consequence of invasion, native predators in freshwater ecosystems, e.g. rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta, lost body weight since they were feeding on the invasive New Zealand mud snail, Potamopyrgus antipodarium, in Green River, USA (Vinson and Baker, 2008).

2-3 Social and economic problems

The strong competitive abilities of invasive cause them to have uncontrolled population growth and causes environmental or economic damage (Lodge et al., 2009). Ecologists, fish farmers and fisheries managers are interested in assessing population densities of the invasive species as predators of the fish they catch. Detailed and continuous data about the location, movements, actions, and numbers of invasive species are necessary (Boonman-Berson et al., 2014). Invasive species have negative impacts on boating fouling (Lalaguna and Marco, 2008), reduce fisheries catchability through changes in fish community composition and biodiversity (Villamagna and Murphy, 2010) and reduce aquaculture productivity through pathogenic invasions (De Schryver and Vadstein, 2014). It is therefore important to exert more effort in controlling the negative impact of invasive species on fisheries and aquaculture industries. The invasive species’ impacts in aquaculture are immediately clear, because the cultivated species are controlled populations, but the impacts in fishery are not immediately clear, because the species that are caught, and their habitat, are not totally under human control. The realization that invasion exists, and tolerance, control (i.e. cost of mechanical, chemical and biological removal or hunting) and management of invasive species (i.e. cost of labor, cost of equipment and the frequency of treatment) needs to get more attention from natural scientists, the media and stakeholders (Villamagna and Murphy, 2010). The goal of invasive species management is to minimize economic costs and ecological damage (Villamagna and Murphy, 2010). Any policy about control of invasive species needs detailed information about ecological, societal and economic damage (Boonman-Berson et al., 2014). A study on the social impact of invasion, found that the media attributed more negative impacts to the invasive species than scientists did (Lavoie, 2010).

2-4 Biodiversity function: positive and negative

Hybridization between invasive species and native species, especially in small habitats like desert springs with low flow conditions and drought stressful condition, could lead to vigorous and fertile hybrids in F1 but the offspring of F1 hybrids (F2) are often weak and sterile in the wild (Rahel and Olden, 2008; Blackburn et al., 2014). For example, F1 hybrids of non-native rainbow trout O. mykiss and native west slope cutthroat trout Oncorhynchus clarkii lewisi were shown to have reproductive success nearly equivalent to or potentially greater than native fish (Muhlfeld et al., 2009). Biodiversity function in F1 is positive but F2 hybrids substantially reduce fitness and loose native species in next generation (Kovach et al., 2015).

3-Positive effects of invasive species

In some instances, invasive species can also have desirable effects on an ecosystem. Invasive species can provide shelter and habitat for native species, e.g. shells of invasive Asian horn snail Batillaria attramentaria used as habitat by native species in pacific ocean (Wonham et al., 2005). Invasive species can provide prey (i.e. invasive North American red swamp crayfish Procambarus clarkii in southwestern Spain) for threatened predator species (Tablado et al., 2010), or be a nutritional resource for them e.g. invasive green macro alga, Codium fragile, increase the recruitment of the mussel Mytilus galloprovincialis of sandy shores in the northern Adriatic Sea (Bulleri et al., 2006). In some instances, invasive species may, therefore, be useful catalysts for ecosystem restoration e.g. albizia sp. plantations for carbon sequestration in grasslands in Southeast Asia (Ewel and Putz, 2004) or as biofilters for use in water clarification e.g. zebra mussel Dreissena polymorpha (Elliott et al., 2008).

4-Response traits of native species to invasive host-species and effect of parasites on native and invasive host-fitness

How host-parasite coevolution due to different native and invasive hosts will act upon the diversity of functional responses traits? Will is cause either the host or parasite to evolve a faster growth rate? Will it lead to the host or parasite becoming more a generalist or specialist for certain traits (i.e. tolerance, competition and predation)?

4-1 Fast growth

Invasive species usually achieve higher densities and larger sizes compared to their original habitats (Torchin et al., 2003). For example, in Australia, invasive cane toads, Bufo marinus, attain higher densities compared to native South American populations (Lampo and Bayliss, 1996) or invasive European green crabs, Carcinus maenas, are larger in body size in invaded area, e.g. USA, South Africa and Australia, than their European native populations. Invasive species has also bigger brood size compared to native species e.g. brood size was also greater in invasive Gammarus tigrinus than in native Gammarus zaddachi in the northern Baltic Sea (Sareyka et al., 2011).

Invasive species are known as vectors of parasites in their new invaded ranges (Wattier et al., 2007). Parasites have an effect on population growth of both native and invasive hosts. Parasites, e.g. cestode Flamingolepis liguloides, and waterfowl predators limit the population growth of native host-species, e.g. native brine shrimps Artemia parthenogenetica, in comparison to invasive host-species, A. franciscana in Aigues-Mortes saltern, South of France (Sánchez et al., 2012). Parasite, microsporidium Fibrillanosema crangonycis, also increases the rates of population growth of its invasive host species, the North American amphipod Crangonyx pseudogracilis, in the UK through a combination of vertical transmission with host sex ratio distortion: female-biased sex ratios might lead to increased host population growth (Slothouber Galbreath et al., 2004).

4-2 Tolerating a range of abiotic conditions: global warming, increase in salinity, eutrophication and land use.

The potential value of invasive species in providing ecosystem functions will increase in a scenario of climate change. The potential to tolerate a wider range of environmental conditions is considered to be a key to successful invasion (Ricciardi and Rasmussen, 1998). The extended anthropogenic activity throughout the last centuries, and the increasing ionic concentration in freshwaters caused by pollution and its consequences on survival of animals with high environmental tolerance has created numerous opportunities for invasive species to spread rapidly (Jazdzewski et al., 2004). For example, under stressful conditions, such as increasing the salinity and temperature and decreased oxygen concentration, invasive species consistently showed more robustness in their normal respiratory performance than their native counterparts (Lenz et al., 2011). For example, the invasive amphipod G. tigrinus was more resistant to hypoxia, insufficient oxygen, and survived at higher temperatures than the native G. zaddachi (Sareyka et al., 2011).

In the course of the last decades, researches have focused on aquatic parasite ecology and invasion biology for understanding the role of parasites in the host communities structure (Hilker et al., 2005; Emde et al., 2012). Indeed, they have found that drivers (i.e. climate

9 change (Pellan et al., 2016), parasitism (Hatcher et al., 2006), and invasion (Iacarella et al., 2017)) have significant effects on prey-predator relationships (Fig. 1.1).

FIGURE 1.1 Climate change, parasitism and invasion are important drivers of biodiversity and evolution.

4-3 Competition

Competition may lead to a replacement or local extinction of one or several native species (Blackburn et al., 2014). The presence of invasive amphipods, by interference competition, can lead to an increased number of native amphipods in the water column with a high accessibility to predators. For example, extinction of native amphipods was accelerated in the upper Danube River because of sympatric and synergistic effects from invasive amphipods D. villosus. These increased the vulnerability of native G. pulex to predation by invasive round goby Neogobius melanostomus (Beggel et al., 2016).

Competition between native and invasive species can be controlled by parasites/predators through a different impact than interference competition on host fitness e.g. castration or increased predation (Prenter et al., 2004; Sánchez et al., 2012). Furthermore, researchers showed that the malaria parasite Plasmodium azurophilum reduced the competitive superiority of the native lizard Anolis gingivinus over the invasive lizard, A. wattsi and decrease blood hemoglobin (Schall, 1992). Parasites can form biological communities directly through population regulation of hosts (MacNeil et al., 2003b) and indirectly by apparent competition, e.g. high attack rate by the parasite Anagrus eposnative in the native congeneric grape leafhopper Erythroneura elegantula compared to invasive variegated leafhopper E. variabilis alters competitive balance from equals to disadvantagous for the native population in California's San Joaquin Valley (Settle and Wilson, 1990).

4-4 Predation

Predators directly (e.g. consumption) or indirectly (e.g. strong impacts on habitat choice and feeding activity of prey amphipod Echinogammarus marinus (Beermann et al., 2018)) have an effect on the replacement or local extinction of native species (Blackburn et al., 2014). The exploitation of attractive chemical signals, such as kairomones, by predators and parasitoids are an important mechanism to locate of prey and hosts (Zhang and Schlyter, 2010). But on another level, when the preys’ visual signals are limited by turbidity and darkness in an aquatic ecosystem, prey respond to chemical cues released by different predators (e.g. adjusted drifting behaviour in G. pulex (Dahl et al., 1998)) or by activity associated with predation (e.g. injury-released chemical cues from conspecific (Wisenden et al., 2001)),. However, reduced prey foraging activity and increased level of prey avoidance towards the chemical cues released by a predator reduce the predation risk (Thünken et al., 2010).

4-5-1 Prey activity

Prey activity and responding to cues from predators diet seems to be a critical factor in facilitating certain behavioural responses depending on the length of co-existence with starved and conspecific-fed predators e.g. decreasing native anuran tadpoles activity in presence of native predator dragonfly Aeshna sp. and predated conspecific (Nunes et al., 2013).

4-5-2 Predator avoidance by prey

Predator avoidance by prey is an important topic related to invasion ecology e.g. invasive amphipods E. ischnus avoided a larger range of fish predators than the native Gammarus fasciatus in Great Lakes (Pennuto and Keppler, 2008). Biological invasion contains a perfect case for studying the evolution of anti-predator phenotypic plasticity e.g. plastic responses usually occur when native Pelodytes punctatus and invasive Discoglossus pictus anurans tadpoles face recently invasive predator species, Eastern mosquitofish Gambusia holbrooki and crayfish Procambarus clarkii (Pujol-Buxó et al., 2013). Differences in antipredator behaviour of species are an important fitness component in most animals with evolutionary change in their activity level (Richardson, 2001; Nunes et al., 2013).

5 Aim of the thesis

The main aim of my thesis was to study the ability of parasites to alter the native and invasive intermediate host’s behaviour. The arrival of invasive host species in a native host population may promote local parasite maladaptation (Moret et al., 2007). In this thesis, I investigated whether parasites have the ability to alter their intermediate host’s behaviour, and whether this has evolved specifically to target sympatric invasive and native host-species or host-species in general. I focused on gammarids and their parasites. I assessed the effects of parasites on behavioural traits of intermediate hosts (i.e. avoidance from the non-host predator, salinity tolerance and rheotaxis) in native and invasive populations.

6 Study system

Test animals were gammarids, their parasites and non-host predator fish in the Paderborn plateau (East-Westphalia, Germany, Fig. 1.2), i.e. the sympatric species of G. pulex and Gammarus fossarum, native to this region, and Echinogammarus berilloni, representing the invasive species and Polymorphus minutus as a parasite and three-spined stickleback,

Gasterosteus aculeatus, as a non-host predator. We carried out our study from May to September in 2009, 2010, 2011 and 2012. Gammarids are most active during the summer months, probably because of increased sexual activity (Wallace et al., 1975). For this reason, we selected the summer season for our sampling time.

The Paderborn Plateau (Paderborner Hochfläche) in central Germany is the largest limestone and karst landscape with an area of 360 km² covering an altitudinal range from 80 to 370 m above sea level (Fig 1.2) (Meyer et al., 2004). Due to the local hydro-geological conditions, most of the streams and stream sections of the Paderborn Plateau show a temporary discharge regime (Meyer et al., 2004). The Paderborn plateau has an area of drying period during summertime (Meyer et al., 2004). In the middle part of rivers, Alme and Altenau, are also temporary discharge areas whilst the downstream sections of these two rivers are permanent discharge areas (Meyer et al., 2004).

We collected our samples from Alme, Lippe and Altenau rivers. We found the parasite P. minutus in some locations (see Fig. 1.3). We distinguished between infected and uninfected gammarids on the basis of orange-red spots present on the cuticle (Dezfuli and Giari, 1999). We transferred gammarids into a climate chamber in Dewar flasks, filled with water from the sampling sites. This was to keep the temperature constant and to minimize stress released during transport time (1.30 hours). The acclimation took place in a climate room at 16±1°C water temperature and dark: light cycle was 10:14 hours to simulate natural, seasonal conditions.

FIGURE 1.2 The temporary karstic stream system of the Paderborn Plateau and the distribution of Amphipods recorded from 2000 to 2003: upper (Alme1) and lower (Alme2) gauging stations (GS) in the river Alme (Meyer, et al., 2004).

Permanent River

Temporary River

FIGURE 1.3 Distribution of P. minutus in the Paderborn Plateau (2009-2012). Red arrows indicate locations where we have found the parasite P. minutus of gammarids during the sampling period.

7-Acanthocephalans as an important parasite in evolutionary ecology studies Acanthocephalans are parasites with complex life cycles involving amphipod intermediate hosts and vertebrate definitive hosts. G. pulex (Lingard and Crompton, 1972), G. roeseli (Bauer et al., 2005), G. fossarum (Van Maren, 1979), G. duebeni (Hynes and Nicholas, 1963), G. lacustris (Hynes and Nicholas, 1963), G. zaddachi (Itämies et al,. 1980), G. oceanicus (Itämies et al., 1980) are intermediate hosts for the acanthocephalan P. minutus. Acanthocephalans, with higher adaptation potential, are considered to exhibit specificity towards both intermediate and definitive hosts and it is argued that some species have switched from their intermediate host over evolutionary and historic time e.g. parasite Echinorhynchus truttae have used G. duebeni as an intermediate hosts initially but has switched to G. pulex in Great Britain (Lyndon and Kennedy, 2001).

The presence of P. minutus in gammarids is identified by an orange-red dot visible through the translucent cuticle of the infected gammarids (Fig 1.4) (Cezilly et al., 2000a). P. minutus is able to change the geotactic behaviour of infected amphipods e.g. preferentially locate gammarids at the air-water interface and top of the water column while uninfected gammarids tend to stay at the bottom of the water column (Cezilly et al., 2000a; Bauer et al., 2005; Médoc et al., 2009).

FIGURE 1.4 Parasitized amphipods with P. minutus: a) Echinogammarus berilloni b) Gammarus pulex c) Gammarus fossarum. Photos: Markus Schmidt and Sajad Ashghali Farahani

The mature phases of P. minutus occur in waterfowl that are the definitive host of this parasite, examples being: the domestic duck (Hynes and Nicholas, 1963), mallard, Anas platyrhynchos, tufted duck, Authya fuligula (Crompton and Harrison, 1965). P. minutus has been detected in many parts of the world (e.g. United Kingdom (Hynes and Nicholas, 1963), Germany (Zittel et al., 2018), Italy (Dezfuli and Giari, 1999), USA (Canaris et al., 1981), France (Cezilly et al., 2000a) and Iraq (Mhaisen 1994)). During the mature phases, P. minutus occupied 65 to 85% of the length of the intestine in mallards, one of its definitive host (Lingard and Crompton, 1972).

Amphipods are intermediate hosts of P. minutus, and recognized as important components of freshwater ecosystems (Neuparth et al., 2002). In Europe, populations of native amphipods have been progressively displaced by highly adaptive invasive species (Emde et al., 2012). Zittel et al. (2018) showed that native and invasive gammarids in Westphalia, Germany (Lippe, Emscher, Ruhr and Rhine rivers) served as hosts to a cryptic P. minutus species. We studied three amphipods species; two native and one invasive. Both G. pulex and G. fossarum are native intermediate hosts and E. berilloni is an invasive, intermediate host of P. minutus (Fig. 1.4).

G. pulex found in small streams and abundant in medium size streams (Siegismund and Müller, 1991) in intermediate water temperatures with a possibility of adjustment to “extreme" water temperatures (5-10, 20-27°C) (Maazouzi et al., 2011). G. fossarum is abundant in the upstream reaches of streams (Scheepmaker and Van Dalfsen, 1989; Siegismund and Müller, 1991; Müller, 1998, 2000). G. fossarum is adapted to intermediate water temperatures during the summer months, and needs sufficient supply of oxygen (Meijering, 1991). E. berilloni have been found in both a temporary and permanent karstic stream of Westphalia in Germany (Meyer et al., 2004). E. berilloni has an endemic distribution in channels and lakes of France and Spain (Meyer et al., 2004), especially in middle and lower courses of larger streams and rivers (Meyer et al., 2004). There is no reliable data about E. berilloni temperature preferences.

8-Non-host predator

It is favorable if the intermediate host infected with P. minutus is preyed upon by the definitive host predators, e.g. waterfowl, because this means the parasite can complete its’ life cycle. However, if the intermediate infected host is preyed upon by a non-host species, the parasite cannot complete its life. Another possibility is that an infected intermediate host is not eaten by any kind of predator. This also has a negative effect on the parasites’ life cycle (Fig. 1.5). It is estimated that only 2.5% of the parasite population is successfully transmitted to the definitive host whereas 17.1% is predated by non-host species (Mouritsen and Poulin, 2003). The remaining (~80%) infected intermediate hosts are not eaten by any predators.

Gammarids are predated upon by non-host predators, such as three-spined sticklebacks, Gasterosteus aculeatus (Médoc et al., 2009). Three-spined sticklebacks mainly feed on benthic invertebrates such as gammarid amphipods and isopods (Delbeek and Williams, 1988; MacNeil et al., 1999).

FIG 1.5 Final destination of P. minutus

9- Increased host abilities’ hypothesis

The “increased host abilities’ hypothesis” posits that parasites manipulate the behaviour of their intermediate hosts or improves its chances of intermediate host survival in order to enhance their transmission to the next host e.g. strong anti-predator response and highest escape speed in infected G. roeseli by P. minutus towards non-host crustacean predator D. villosus in order prevent inappropriate transmission (Medoc and Beisel, 2008; Beisel and Médoc, 2010). In this Hypothesis, both the parasite and its intermediate host benefit from increased host ability to survive (Medoc and Beisel, 2008). This hypothesis is in opposition of the “handicapped host’ hypothesis” which emphasizes the handicapping effects of parasite on infected gammrids. For instance, the parasite P. laevis reduces the growth rate and oxygen consumption in its intermediate host, G. pulex (Rumpus and Kennedy, 1974), thereby the handicapped host becomes more conspicuous to non-host predators.

According to the “increased host abilities’ hypothesis”, it is the capability of the parasite to affect the host’s phenotype which is the target of natural selection (Thomas et al., 2005). The multi-dimensionality in parasite-induced changes of the hosts’ phenotype has attracted the interest of parasitologists (Cézilly and Perrot-Minnot, 2010). Multi-dimensional host manipulation denotes the phenomenon when a single parasite alters multiple phenotypic traits of its intermediate host (Cézilly and Perrot-Minnot, 2010). Changes in the micro-distribution of intermediate hosts are often viewed as a consequence of multi-dimensional host manipulation by the parasite to increase its probability of trophic transmission (Lagrue et al., 2013). Multi-dimensional manipulation does not have to be specific to be adaptive e.g., carotenoid-based colouration of acanthocephalans has no adaptive value in terms of transmission (Jacquin et al., 2014). Also, when predation risk by non host predator is low, even highly nonspecific manipulation strategies can be adaptive. However, when initial predation risk is high, manipulation needs to be specific to increase parasite transmission success. (Seppälä and Jokela, 2008).

10- Competitive exclusion hypothesis

The “competitive exclusion hypothesis” predicts reducing local diversity during biological invasion by displacing native species (Muthukrishnan et al., 2018). This hypothesis has an important role in describing the population regulation mechanism in the native and invasive gammarids during the presence or absence of a fish predator. For example, the invasive D. villosus was the stronger competitor for shelter sites compared with native G. roeseli, which eventually resulted in an increased mortality of G. roeseli in the presence of a predatory fish (De Gelder et al., 2016). Evidence supporting the competitive exclusion hypothesis, where invasive species were able to outcompete native species for resources, driving them to elimination, were found by Pavlik (1983) who showed the biomass of invasive European dune grasses Ammophila arenaria was higher than that of native Elyrnus mollis in the Pacific coast of North America (Pavlik, 1983), and the invasive cordgrass Spartina alterniflora has larger biomass than native S. foliosa in San Francisco Bay (Callaway and Josselyn, 1992). Rapid growth, fecundity and a large size of invasive species increase competitive ability and contribute to invasion success (Van Kleunen et al., 2010). The fate of invasive species is, in part, governed by competition between native and invasive species according to the “evolution of increased competitive ability” hypothesis (proposed by Blossey and Nötzold, 1995).

Some parasites promote competitive exclusion, others promote coexistence, and others have little effect (Frainer et al., 2018). For example, in the UK, the invasive grey squirrels Sciurus carolinensis threatens the native competitor, the red squirrel S. vulgaris. Transmission of the Parapoxvirus from the grey squirrel causes lethal disease in the native species, but not in the invasive species (Strauss et al., 2012). Parasites can promote coexistence by regulating their hosts’ abundance during low infection prevalence (Dinoor and Eshed, 1984) or by reducing fitness differences between species that make coexistence more difficult (Mordecai, 2011). A parasites’ competitive abilities can be maximized by increased ability of infected intermediate host to be out of reach of a non-host predator.

11- Outline of the thesis

The aim of the current thesis is to understand how parasites manipulate their intermediate host behaviour and change intermediate host salinity tolerance to facilitate successful trophic transmission to the definitive host. Fig 1.6 gives a concise overview of which experiments were done, and in which chapters they were used. Most studies to date have not investigated the “increased host abilities’ hypothesis” in invasive hosts. There have also not been many investigations into anti-predatory behaviour in invasive species. To study the effects of parasite on behavioural change in native and invasive host population of gammarids, we performed an experimental to investigate prey avoidance behaviour of infected invasive E. berilloni and native G. pulex and G. fossarum (chapter 2). The objective of chapter 2 was to assess if P. minutus altered the infected gammarids behaviour towards non-host predators, more specifically three-spined sticklebacks, and if such a change was observed in all, or only sympatric, gammarid species. We tested whether the “increased host abilities’ hypothesis” in non-host predators, was more pronounced in native, sympatric co-evolved gammarids in comparison to sympatric invasive gammarids. Therefore, the two native gammarid species should exhibit a stronger avoidance behaviour compared to invasive E. berilloni. We assessed the non-host predator avoidance between uninfected and infected gammarids during choice experiments in a Y-maze olfactometer, using water with and without three-spined stickleback cues as non-host predator.

Most studies to date have investigated the prevalence of gammarids and their parasites in aquatic ecosystems but not experimentally in the lab, and our knowledge about the behaviour of invasive species are lacking. To further investigate the information about the effects of the parasite on the gammarids’ behaviour, we also performed a study (chapter 3) in which we investigated the effects of parasites on gammarid rheotaxis behaviour – to swim against or with the water current - in native and invasive populations. The research question in chapter 3 was how infection with parasites regulates rheotaxis in “sympatric” and “non-sympatric” gammarid species. We investigated how the rheotaxis controls the ecological distribution of natives during the invasion. We assessed rheotaxis between uninfected and infected gammarids during choice experiments in an open channel setup configured as an artificial water recycling canal. Lastly, in chapter 4, we studied whether the parasite affected the salinity tolerance of native hosts. P. minutus depends on its intermediate host’s ability to survive changing osmolality, raising the basic question whether P. minutus has evolved an ability to affect its intermediate hosts’ salinity tolerance. Most studies to date have not shown the effects of parasites in salinity tolerance of two native species populations which overlap in their ecological niche.

We tested the competitive exclusion hypotheses: First, we expect less salinity tolerance in uninfected gammarids compared to gammarids infected with P. minutus. Second, we predict changes in salinity tolerance to be more distinct in G. pulex than G. fossarum. Due to the fact, they inhabit different parts of the river. To study the structural changes of the parasite we manipulated salt concentrations to investigate the salinity tolerance of native gammarids. We also studied the effect of acanthocephalan parasites on gammarid survival in different salt concentrations. We investigated if salinization in a temporary plateau could be an inhibitor factor for native species. We assessed survival in different salinities between uninfected and infected gammarids during experiments in plastic containers which were placed in plastic basins containing 12 liters of water with five different salinities. In chapter 5 we will end this thesis with a general discussion on the results and synthesize the research findings presented in the previous chapters. We will emphasize the importance of parasite and invasive species in the habitat of the native population, especially during global soil salinization. Invasion and parasite interaction must be investigated very differently. It is expected that trophic interactions, predation, rheotaxis, an increasing salinity could lead to a variable response in different populations of native and invasive organisms, and parasites communities in aquatic ecosystems.

FIGURE 1.6 Model network graphs for major incentives for interaction

Top photo: Alme, Paderborn Plateau Bottom photo: Altenau, Paderborn Plateau

Box 1

Effects of parasites on the behaviour of native and invasive intermediate hosts Sajad Ashghali Farahani, Per J Palsbøll, Jan Komdeur

Amphipod communities’ composition can vary substantially due to predator and parasite density (Hatcher et al., 2006). Here, I discuss the mechanisms of behavioural manipulation by parasites upon their intermediate host’s behaviour in relation to non-host predators and rheotaxis – to swim against or with the current.

1. Behavioural manipulation Parasites have been shown to have the ability to alter the activity level of serotonin (5- hydroxytryptamine, 5-HT) in the brain of their intermediate hosts (Tain et al., 2006a). Serotonin is a known neuromodulator of biogenic amine (Shiratori et al., 2017) modulating stress response (Liang et al., 2006). As a neurotransmitter, serotonin relays signals between nerve cells, or neurons (Curran and Chalasani, 2012). The serotonin contributes to a variety of physiological processes, from neuroendocrine stress response to gut contraction (Barnes and Sharp, 1999; Nichols and Nichols, 2008). Serotonin plays a role in several behavioural traits, including thermotaxic behaviour - movement of an organism in response to temperature - (Li et al., 2013; Wong and Rankin, 2019), phototaxis behaviour - movement of an organism in response to light - (McPhee and Wilkens, 1989; Tain et al., 2006a; Rodriguez Moncalvo and Campos, 2009; Thamm et al., 2010) and geotaxis - swimming of an organism to the top or bottom of the water column - (Maximino et al., 2013). The role of serotonin in feeding behaviour (Alanärä et al., 1998; Ortega et al., 2013) and oxygen consumption (Srinivasan et al., 2008; Pérez-Campos et al., 2012) have been shown by many previous researchers.

The most important role of serotonin is related to regulating escape response (Painter et al., 2009) and predator avoidance (Weinberger and Klaper, 2014). Serotonin production increases with water temperature in invertebrates (Stefano et al., 1978). The metabolic rate of acanthocephalan parasites is highly dependent on temperature (Olson and Pratt, 1971; Tokeson and Holmes, 1982). The combined effects of elevated temperature and parasitic infection probably affects the performance of the gammarid brain and thus behavioural responses, such as phototaxis (Labaude et al., 2017).

2. Predator cues

Most studies have shown that chemical cues from predators, in contrast with visual signals, e.g. hydrodynamic cues (i.e. incoming wave surge and water currents), remain as long as the predator is present (McIntosh et al., 1999). Such cues inform prey about hunger state, densities and types of predators (Pettersson et al., 2000; Brown and Magnavacca, 2003; Schoeppner and Relyea, 2005; Ferrari and Chivers, 2006; Camacho and Thacker, 2013).

The mucous of fish skin contains glycosaminoglycans (GAGs) (Van De Winkel et al., 1986). The six major types of GAGs include: heparin, heparan sulphate, chondroitin, dermatan sulphate, hyaluronic acid and keratin sulphate (Rittschof and Cohen, 2004). However, only chondroitin fragments are a major component of alarm chemical cues to elicit prey behavioural response in the presence of predators (Mathuru et al., 2012; Farnsley et al., 2016). The chemical cues such as hypoxanthine3-N-oxide (H3NO), as an active component of the alarm pheromone system (Pfeiffer et al., 1985; Brown et al., 2000), and a variety of polypeptides in skin (Decho et al., 1998; Wisenden et al., 2009), play an important role in prey behavioural decision-making. For prey to reach maximum fitness they must have the ability to detect alarm cues of predators at an early stage in the predation sequence, before predators have detected the prey or initiated an attack (Lima and Dill, 1990; Smith, 1992). Conspecific injury cues, e.g. chemical cues released from injured prey during predatory attacks, are chemical alarms for anti- predatory responses (Wisenden et al., 1999; Smith and Webster, 2015). Aggregation behaviour (Lewis et al., 2012), reduced activity (Thünken et al., 2010), spending more time in refuges and near the surface (Médoc et al., 2009) are anti- predatory responses of gammarids after receiving chemical cues by fish or injured conspecific. Parasites alter this behaviour of gammarids, in order to enhance predation of intermediate hosts by 21

the definitive host predators (Jacquin et al., 2014) and decrease infected intermediate host vulnerability to non-host predators (Médoc et al., 2009).

3. Drifting, swimming with the current, rheotaxis

Rheotaxis is multi-sensory behaviour in which aquatic organisms tend to hold the position in the direction of flow and avoid being swept downstream by the current (Lyon, 1904; Arnold, 1974; Olive et al., 2016; Oteiza et al., 2017). This form of taxis is generally positive (orienting upstream and swimming against the water flow), but can also be drifting or negative (orientating downstream and swimming or being swept in the water flow) (Bureau Du Colombier et al., 2009). Rheotaxis is an important behaviour for the survival of many aquatic prey species. The benefits of rheotaxis in prey, include swimming away from predator cues (Bureau Du Colombier et al., 2009).

Rheotaxis is one mechanism that could facilitate the trophic transmission of parasites to their definitive hosts (Lafferty, 1999). Macneil et al.( 2003 a,b) found parasites have the ability to alter distribution of gammarids through shifting rheotaxis. They showed a higher prevalence of parasitism in the faster and shallower water bodies (upstream) compared to slower and deeper areas (downstream).

Photo: Gammarus pulex, one of the intermediate hosts of the parasite thorny-headed worm Polymorphus minutus. Photographers: Markus Schmidt and Sajad Ashghali Farahani

Chapter 2

The effects of parasites upon non-host predator avoidance behaviour in native and invasive gammarids.

Sajad A. Farahani, Per J. Palsbøll, Ido R. Pen and Jan Komdeur Under review at Parasitology

Abstract The life cycle of many parasites depends upon the successful transmission between successive hosts. Consequently, parasites are subject to selection upon traits that facilitate transmission between hosts. One such example is Polymorphus minutus, an acanthocephalan parasite, which alters the behaviour of their intermediate hosts, gammarids, to facilitate the transmission to its definitive hosts; water fowl. A fundamental question is whether this ability of P. minutus to alter its intermediate host’s behaviour has evolved specifically to target sympatric gammarids, or gammarids in general? The non-host predator avoidance of P. minutus, towards the three-spined sticklebacks, Gasterosteus aculeatus, was assessed in three gammarid species; two native species, Gammarus pulex and Gammarus fossarum, and one invasive species, Echinogammarus berilloni. Non-host predator avoidance was assessed during choice experiments (with and without fish cues) and revealed no differences between uninfected and infected G. pulex and E. berilloni. These results suggested that infection by P. minutus altered the non-host predator avoidance behaviour in native G. fossarum in a manner that increased the probability of transmission to their definitive host. Our results suggested the possibility that P. minutus’ ability to alter the host behaviour may have evolved in sympatry with the host gammarid species. Interestingly, uninfected gammarids do not appear to avoid the non-host predator. The results may also have implications in terms of the role of parasites in the success or failure of invasive species to colonise new areas.

1. Introduction The “increased host ability hypothesis” posits that parasites manipulate the behaviour of their intermediate hosts in order to enhance their transmission to the next host (Médoc and Beisel, 2008). One manner in which the transmission probability can be increased to modulate the intermediate host’s behaviour to avoid predation by non-host species (Médoc and Beisel, 2008). The life cycle of acanthocephalan parasites includes intermediate hosts, such as amphipods, isopods, ostracods, copepods, insects, and myriapods (Near et al., 1998). The acanthocephalan parasite, Polymorphus minutus, appears capable of altering the behaviour of its intermediate hosts (amphipods) in a manner that enhances their transmission to the definitive host, water fowl (Bakker et al., 1997; Kaldonski et al., 2007; Médoc and Beisel, 2008; Perrot-Minnot et al., 2007).

Previous studies have demonstrated that parasites decrease non-host predator exposure by manipulating their host’s predator avoidance behaviour (Holomuzki et al., 1988, Rigby and Jokela 2000, Matz and Kjelleberg 2005, Friman et al., 2009). Parasites modify the host’s behaviour by regulating the host’s neurotransmitter serotonin levels (Tain et al., 2006, Cézilly and Perrot-Minnot 2010, Cézilly et al., 2013). However, one fundamental question remains; has such ability to modulate the host’s behaviour evolved specifically to target sympatric hosts or more generally towards the host genus?

The aim of the present study was to assess if modulation of the host’s behaviour by P. minutus is specific to sympatric gammarids or gammarids in general. Previous work suggested that modulation of host behaviour evolved through ‘adaptation’ (Baldauf et al., 2007) and ‘exaptation’ (Combes, 2005; Beisel and Médoc, 2010) by parasites or both (Cézilly and Perrot-Minnot, 2005). Exaptation describes the case when a behavioural trait (e.g. negative geotaxis) in P. minutus, that evolved in response to predation, ultimately facilitates trophic transmission to the definitive host, i.e. due to an elevated level of avoidance towards non-host predators (Rohde, 1994; Beisel and Médoc, 2010).

The native gammarids, Gammarus pulex, and, Gammarus fossarum, are abundant in Central European upper and middle freshwater tributaries (Siegismund and Müller, 1991). In contrast, the gammarid Echinogammarus berilloni is native to the Atlantic regions of France and Spain (Médoc et al., 2015), but an invasive species in western and Central Europe (Schmidt- Drewello et al., 2016). The gammarids are predated upon by water fowl, which are P. minutus’ definitive hosts, but also by non-host predators, such as three-spined sticklebacks, Gasterosteus aculeatus (Médoc et al., 2009). Consequently, avoiding predation of gammarid hosts by non-host predators would increase the probability of predation of gammarids by water fowl and hence their transmission to the definitive host.

The objective of the present study was to assess if P. minutus infected gammarids altered the gammarids behaviour towards non-host predators, more specifically three-spined sticklebacks, and if such a change was observed in all, or only sympatric, gammarid species. We tested whether the “increased host ability hypothesis” in non-host predators, was more pronounced in native, sympatric co-evolved gammarids in comparison to sympatric invasive gammarids.

2. Materials and Methods

2.1. Sampling Specimens of G. pulex (GP) from the Altenau river (51°35ʹ56.73ʺN;8°46ʹ 56.92ʺE) and specimens of G. fossarum (GF) and E. berilloni (EB) were collected in the upper and lower parts of the Alme river, respectively (51°32ʹ50.05ʺN;8°32ʹ43.61ʺE) where three-spined sticklebacks are known to occur in both tributaries (Schmidt-Drewello et al., 2016). Both tributaries are located on the Paderborn Plateau in Westphalia, Germany. Samples were collected during May and June in 2010. All specimens were kept in Dewar flasks containing water from the sampling site and housed in two aquaria (infected vs. uninfected gammarids, each with dimensions 50×50×25cm) at a water temperature at 16±1°C (the water temperature during the sampling). Infected gammarids were identified from the number of orange-red spots (cystacanth, the developmental stage at which the parasite is able to infect its definitive hosts) present on the cuticle (Dezfuli and Giari, 1999). A dark:light cycle of 10:14 hours was maintained in order to simulate seasonal conditions. Gammarids were fed beech leaves, Fagus sylvatica, until 48 hours preceding the choice experiments. The 48-hour starvation was introduced in order to induce foraging behaviour during the experiment. The three-spined sticklebacks were F1 offspring of wild-caught individuals and were kept in an aquarium (62×81×30cm). In order to reduce potential unwarranted effects from the presence of fish feces and residual food, the fish were fed frozen chironomids (lake flies) larvae up to 72 h before the experiments were initiated. The water in all aquaria for both fish and gammarids included tap water (pH 7.4; EC 0.5 mS cm-1).

2.2. Experimental setup Laboratory experiments were conducted in a climate chamber at 16±1°C water temperature during May, June and July in 2010. Gammarid non-host predator avoidance was assessed using a choice setup (Fig. 2.1) configured as a Y-shaped maze with a basal stem (5 × 5 × 15 cm) that split into two separate upper stems (5 × 5 × 10 cm). The floor of the maze was covered with 4-6 mm gravel. Tap water was circulated into the maze through the upper stems towards the main basal stem by gravity at a rate of ca.2.3 cm3 per second. Preliminary tests with ink and salt, respectively, revealed that a flow rate at 2.3 cm3/second ensured mixing of two aquaria water in the basal stem, but prevented mixing of water in the two upper stems. The inflow water originated from one of two aquaria (each 32×17.5×19 cm). One aquarium contained tap water with ten three-spined sticklebacks introduced into the aquarium 48 hours before the initiation of the experiment. The other aquarium contained tap water only. During control experiments both aquaria contained only tap water. The control experiment conducted immediately after the test experiment. Non-host predator avoidance was assessed in uninfected and P. minutus infected gammarids. Experiments were categorized into either “I” (P. minutus infected gammarids), or “U” (uninfected gammarids). Control (i.e. no three- spined sticklebacks in either aquarium) and test (i.e. one aquarium without and the other with three-spined sticklebacks) experiments were denoted “C” and “T”, respectively. For instance, the combination UT denoted an experiment with uninfected gammarids where one aquarium contained tap water and the other ten three-spined sticklebacks. The length and the number of cystacanths of each individual gammarid were measured from imageJ (version 1.35, https://imagej.nih.gov/ij/index.html). Gammarids shorter than <8 mm and with more than one cystacanth were excluded as recommended by Bauer (2005) and Medoc and Beisel (2008) in order to avoid possible effects due to differences in size or degree of infection. Our experiment was conducted using “fresh” cues, which presumably alerted the test individuals that a non-host predator was nearby.

2.3. Data collection All experiments were conducted with single gammarids. All test individuals were used only once for the experiments. The test specimen was released into the area at the base of the main stem (denoted area C, Fig. 2.1). Movement was restricted with a plastic barrier for 300 seconds after which the test specimen could move freely within the maze for 300 seconds. The location of each specimen was recorded every 30th second. Specifically, the duration spent in areas C, A and B was noted (Fig. 2.1). The fish cues in area C are intermediate between the two other areas. The source of inflow water into areas A and B (i.e. aquaria with or without three-spined sticklebacks, respectively) was alternated between experiments. After the completion of an experiment, each specimen was re-checked for infection by other parasites, using a dissection microscope (Zeiss®Stemi 2000). The individuals with extra parasite species have deleted from analysing.

No No No fish fish fish fish

5cm control test

1 0 c mB A B A

15cm

C C

x x

FIGURE 2.1 Two-choice microcosm setup. A (control) = inflow from the aquarium without fish, A (test) = inflow from the aquarium with fish, B (control, test)= inflow from the aquarium without fish, C = mixing area, arrows = direction of flow, ×= release point of gammarids.

2.4. Statistical analyses

We modelled the transitions between areas A, B and C using a discrete-time Markov chain approach, i.e. an individual at location at time t makes a transition to area at time with probability

( | ) [1]

In other words, we estimated transition matrices of the form,

[ ] [2]

All three columns of M sum to one; hence, M represents six independent parameters. In the full model, M was allowed to vary simultaneously with the three predictor variables: time { }, species { } and treatment combination { }. All specimens started from area C, and hence the first transition corresponds to two parameters ( , , ) whereas each of the subsequent nine transitions corresponded to six parameters. In total, the full model was therefore comprised of ( ) parameters. Simpler models were assessed by excluding one or more predictor variables.

Given the transition probabilities , the probability ( ) of presence in area y at time t was estimated recursively: ( ) ∑ ( ) [3]

Finally, given the above time-dependent probabilities, we estimated the average probability of presence at a given area as the following:

( ) ∑ ( ) [4]

The transition probabilities in each column of M were estimated by treating the observed transition frequencies at a given area as drawn from a multinomial distribution. We fitted Bayesian Markov Chain Monte Carlo (MCMC) models with the brms interface (version 2.7.3; Bürkner 2016) to RStan (Stan Development Team 2018), using RStudio (version 1.2.1268; RStudio Team 2018) with R (version 3.5.0; R Development Team 2018). Each estimation was run with four MCMC chains of 2,500 iterations each, preceded by a burn-in period of 1,000 iterations, thus yielding a total of 10,000 samples per parameter per model. Mixing and convergence of chains was assessed from the trace plots and the Gelman-Rubin r-hat statistic (Gelman et al., 1992). In all cases r-hat was <1.001, which suggested convergence. The effective sample size for all parameter estimates were above 1,000. The mean and the 89th percentile intervals (PI) of the marginal posterior densities were reported for all parameters.

Different combinations of predictor variables were assessed using the Watanabe-Akaike Information Criterion (WAIC; Watanabe 2010). For each model m among a set of models R, we calculated the model weight according to

( )

[5] ∑ ( )

The statistic referred to the difference in WAIC value between model m and the best fitting model (i.e. the model with the lowest WAIC score; this model itself has ). In total seven models each with three predictor variables were compared. The models were: species*treatment, treatment, species, time*treatment, time*species, time*species*treatment and time.

3. Results

The ‘species * treatment’ model (Table 2.1) was the model with highest support at a weight of ca.1.0 among the seven competing models. No evidence of time-dependent transition probabilities was observed, i.e. all four models including time as a predictor variable were ranked lowest. In general, all test specimens tended to move away from the initial area C during the experiment and approached quasi-equilibrium frequencies in the three areas A, B and C (Fig. 2.2).

TABLE 2.1 Comparison of Bayesian multinomial Markov chain models. The models were ranked by their WAIC scores from low to high. ΔWAIC denotes the specific model’s difference in WAIC score relatively to the model with the lowest -WAIC model. Weight indicates the posterior model weight (eq. 5)

Model ΔWAIC Weight Species * Treatment 0.0 1.00 Treatment 92.4 0.00 Species 127.1 0.00 Time * Treatment 282.7 0.00 Time * Species 293.8 0.00 Time 301.9 0.00 Time * Species * Treatment 322.1 0.00

FIGURE 2.2 Temporal dynamics of proportional presence (Y axis) in areas A, B and C of the experimental aquarium of GP (G. pulex), GF (G. fossarum), and EB (E. berilloni) for the four treatment combinations (UC, UT, IC, IT). Smoothed graphs (thick lines) are predictions of the best supported model (predictor species*treatment; see Table 2.1) while raw data are represented by dots. The shaded areas represent the 89% highest posterior density interval (HPDI) for the predicted values. Time (X axis) indicate 10 recordings (one per in 30 seconds). UC= Uninfected Control, UT= Uninfected Test, IC =Infected Control, IT= Infected Test.

The invasive species EB showed no statistically significant preference for area A over B or vice versa in either of the four treatment categories (Fig. 2.3). In contrast the native species, GF and GP, showed a significant preference, mostly for area A, in six out of eight treatment categories (Fig. 2.3). In five out of six comparisons, between control (no non-host predator present) and test (non-host predator present) experiments, presence in area C decreased significantly when the non-host predator was present. The presence of infected GF and GP in area C was more frequent in the treatment group compared to uninfected individuals (Fig. 2.3).

FIGURE 2.3 Proportion of time present in different areas for all combinations of three species and four treatments. Dots indicate posterior means and error bars the 89% HPDI. Asterisks near the bottom indicate within-treatment significance of the difference between time spent at A and time spent at B, i.e. posterior probability of Pr(A) – Pr(B) >0 (red) or Pr(A) – Pr(B) <0 (green) (* p > 0.95, ** p > 0.99, *** p > 0.999). Lines between adjacent control and test treatments within areas indicate a non-significant difference (p ≥ 0.05), whereas absence of lines indicates a significant difference (p ≤ 0.05). UC= Uninfected Control, UT= Uninfected Test, IC =Infected Control, IT= Infected Test.

The most pronounced effect of non-host predator presence was observed in uninfected native gammarids, with an increase in preference for area A; the area closest to the non-host predator. Infected native gammarids also had a tendency towards increased presence in area A (statistically significant for GP, but not in the case of GF), although this effect was significantly lower than was the case for uninfected gammarids (posterior probabilities of Pr(A)UT – Pr(A)UC > Pr(A)IT – Pr(A)IC were 0.20, 0.99 and 1.00 for EB, GF and GP, respectively).

4. Discussion

4.1. Avoidance behaviour of uninfected and infected gammarids to non-host predator

The estimated increase in avoidance behaviour towards the non-host predator observed in GF infected with P. minutus in our study was consistent with the “increased host ability hypothesis ”, which predicts that parasites increase the avoidance towards non-host predators

(Medoc and Beisel, 2008). In contrast, native GP infected with P. minutus showed no avoidance behaviour towards non-host predator cues. The weak effects on non-host predator avoidance in infected gammarids detected in our study could be due to the fact that our experiments were conducted without the host predator cues, making it more difficult for individuals to discern between different predator cues and to behave accordingly. For example, Jacquin and co-workers (2014) showed that native E. berilloni infected with native P. minutus from the Lunain river (France) showed a tendency of increased activity near mallards, Anas platyrhynchos, (the definitive host) over non-host predatory fish. In their experiment both host predators and non-host predators were present (Jacquin et al., 2014). The realization of predator presence by prey possibly occurs indirectly through the detection of pheromones and allelochemical cues from other prey species released during predation (Dicke and Grostal, 2001) or directly via chemical cues from predator (Kats and Dill, 1998). In aquatic environments, prey detect predators mostly via chemical cues (Dodson et al., 1994; Chivers et al., 1996; Hettyey et al., 2010). For example, gammarids respond to chemical cues from predators at low visibility by reducing activity and move towards the bottom (Wisenden et al., 1999).Our study also revealed that uninfected gammarids had a preference towards non-host predators. This behaviour was especially noticeable in the native gammarids compared to the invasive gammarid. However, our results did not agree fully with our expectations, as native gammarids preferentially moved toward non-host predator cues whereas invasive gammarids reduced their activity in presence of non-host predator cues but did not appear to actively avoid non-host predator cues. Our results were in agreement with those previously reported by Schmidt-Drewello et al. (2016) who found that uninfected invasive E. berilloni appeared more capable of detecting chemical cues from three-spined stickleback compared to uninfected native gammarids, G. pulex and G. fossarum. There may be several causes for the observed absence of predator avoidance towards non-host predators by uninfected gammarids. Uninfected gammarids appear able to discern between the chemical cues emitted by inefficient predators, i.e. scavengers or predatory species are inefficient in targeting gammarids (e.g. cray fish, Pacifastacus leniusculus, (Abjornsson et al., 2000), stone loach, Barbatula barbatula, (Szokoli et al., 2015a) and three-spined sticklebacks (Jacquin et al., 2014)) and those cues emitted by efficient predators (e.g. sculpin , Cottus gobio, trout, Salmo trutta, (Abjornsson et al., 2000), perch, Perca fluviatilis, (Baldauf et al., 2007), and gudgeon, Gobio gobio, (Szokoli et al., 2015a)). Our results revealed that uninfected invasive gammarids compared to uninfected native gammarids on the Paderborn plateau limit their range to where fish predators prevail. The native G. pulex is an active and explorative amphipod (Truhlar and Aldridge, 2015). G. pulex shows local adaptation in the presence of fish predator and there is no need to limit their movements to the upstream area. It lives in shallow muddy streams where low water depth and dense benthic detritus may reduce the risk of detection by fish (Haddaway et al., 2014). Fish cues could act as presence of food resources for gammarids, which are omnivore scavengers and feed on carcasses and fish faeces (Wilhelm and Schindler, 2011; Jermacz et al., 2017). However, our results were not confounded by fish cues. The three-spined sticklebacks in our experiment, were not fed 72 hours prior to the experiments in order to remove such cues (Wisenden et al., 1999; Ward, 2012; Smith and Webster, 2015).

Our experiment employed cues from non-host predator that were present in the vicinity of the gammarid during the experiment, presumably alerting the individual gammarid of their proximity. Indeed, the presence of gammarids in the release area (C) decreased when “fresh” cues of non-host predator were present. Unwillingness to presence in the release area was a specific case and confirmed increasing prey activity after detecting the source of cues and confirmed increasing the time duration of presence of gammarids in area with non-host

predator cues. Our results were in contrast with previous studies that demonstrated a decrease in prey activity when predators were present, such as, reduction feeding activity (e.g. in tadpoles, Rana temporaria, (Van Buskirk et al., 2014) and swimming, crawling and downstream drift (e.g. in Hyalella azteca, (Stone and Moore, 2014), Gammarus lacustris, (Wudkevich et al., 1997) and Echinogammarus stammeri, (Dezfuli et al., 2003))

4.2. Intra-specific comparison between uninfected and infected gammarids No preference for areas with and without three-spined stickleback cues was observed in either uninfected or infected GP. This observation was in agreement with previous reports by Kaldonski et al. (2007) who did not detect any significant differences in the use of refuges between infected or uninfected G .pulex to cues from freshwater sculpin, Cottus gobio, a non- host predator. However, Kaldonski et al. (2008), showed that water scorpions, Nepa cinera, predated upon uninfected G. pulex more frequently than infected G. pulex.

We found that infection by P. minutus altered the non-host predator avoidance in native G. fossarum, in a manner that decreased the probability of presence in the area with three-spined stickleback cues, compared to uninfected G. fossarum. Conversely, neither infected nor uninfected E. berilloni showed a significant preference for a specific area (with or without three-spined stickleback cues) in either of the four treatment categories. Jacquin et al. (2014) found that infected E. berilloni by P. minutus reduced their activity in the presence of fish cues. However, Jacquin et al’s (2014) experiment was using native E. berilloni from the Lunain River (France) where non-host predator cues was from both three - spined stickleback and minnows, Phoxinus phoxinus.

The outcome of interactions between native and invasive hosts and their parasites are shaped by an array of complex ecological and evolutionary factors (Alexander et al., 1996; Parker and Gilbert, 2004) affecting the success and intensity of invasions by non-native species (MacNeil et al., 2003a; Prenter et al., 2004). The fate of invasive species is, in part, governed by competition between native and invasive species according to the “evolution of increased competitive ability” hypothesis (proposed by Blossey and Nötzold, 1995) as well as competition between uninfected and infected hosts during the invasion according to the “increased host ability hypothesis”. Competitive abilities can be maximized by increasing non-host predator avoidance behaviour and increased host ability to be out of reach of a non- host predator.

4.3. Conclusion and recommendation Our study revealed that parasites are capable of increasing non-host predator avoidance in G. fossarum. However, no effects in non-host predator avoidance were observed in uninfected or infected G. pulex and E. berilloni. Other studies have demonstrated modulation of host behaviour by P. minutus specifically in sympatric gammarids. For example, a more pronounced change in geotaxis was shown in native G. pulex infected by P. minutus compared to an infected invasive host, G. roeselii (Bauer et al., 2005). P. minutus appears able to exploit the plasticity of sympatric intermediate host antipredator responses to its own advantage.

Zittel et al. (2018) showed that the three gammarids hosts a cryptic P. minutus species. In Westphalia, P. minutus co-invaded the region with invasive gammarids, E. berilloni and G. roeseli from the Mediterranean area and Southeast Europe, respectively (Zittel et al., 2018). The P. minutus infected G. pulex is not native in Westphalia and transmitted by G. roeseli. However, the native, cryptic parasite P. minutus uses native G. fossarum as an intermediate

host (Zittel et al., 2018). Perhaps native P. minutus can potentially have different traits than invasive P. minutus in manipulating the intermediate host behaviour in such a way that it is increasing the chances of being transmitted to the definitive host. Accordingly, Zittel and colleagues (2018) recommended genetic identification P. minutus, in order to rule out possible differences due to infection by different P. minutus species. Indeed, different intermediate host species with different P. minutus species have different non-host predator avoidance behaviour. However, it is questionable if the “increased host ability hypothesis” would vary on the cryptic species of the parasite.

We conclude that uninfected gammarids do not appear to actively avoid non-host predators. We recommend to conduct experiments with different non-host predators in order to assess differences in predators’ efficiency and coevolution history of the prey-predator. An economic, environmental friendly strategy in farms is reducing the parasite effects on definitive host without using chemical medicine. We recommend biological control strategy to farm non-host fish species with domestic water fowls thereby decreasing the incidence of P. minutus in farmed water fowl.

Acknowledgments We are grateful to Frank Groenewoud, Sjouke Anne Kingma and Martijn Hammers for their constructive comments on the manuscript. We thank the Institute for Evolution and Biodiversity, University of Münster, Germany, for providing permission and the facilities to conduct the research, and students for their assistance during field sampling. We acknowledge funding from the Netherlands Organisation for Scientific Research grant numbers 854.11.003 and 823.01.014 (JK).

Photos: Sajad Ashghali Farahani

Chapter 3

Infection by the acanthocephalan parasite Polymorphus minutus increases positive rheotaxis of its gammarid hosts.

Sajad Ashghali Farahani, Per J Palsbøll, Jan Komdeur

Under review at Biological Invasions

Abstract: Successful transmission of parasites to their definitive host in freshwater ecosystems depends on the rheotaxis – to swim against or with the current - of their intermediate hosts. Infection with parasite has the potential to alter the direction and degree of rheotaxis in the native and invasive intermediate hosts to manage parasite ecological niche. However, it remains unknown how acanthocephalan parasites Polymorphus minutus change its intermediate gammarid hosts’ rheotaxis in order to increase successful transmission to its definitive hosts, waterfowls. We assessed the effect infection on rheotaxis in “sympatric” and “non-sympatric” gammarid species. We assessed and compared the rheotaxis of infected and uninfected gammarids in Paderborn Plateau in Westphalia, Germany: Echinogammarus berilloni (an invasive species), Gammarus pulex and Gammarus fossarum (both native species). We found that rheotaxis was significantly more negative in the invasive species compared to native host species. However, irrespective of species, the positive rheotaxis was significantly higher for infected compared to uninfected gammarids. Our results contribute to our knowledge of the relationships between rheotaxis, invasion and parasites, which may aid the management and conservation of waterfowls.

1. Introduction: Positive and negative rheotaxis describes a behaviour of active swimming by aquatic organisms to consistently swim against or with the current (Lyon 1904; Bureau du Colombier et al., 2009; Bak-Coleman and Coombs 2014; Johnson 2014; Olive et al., 2016; Oteiza et al., 2017). Behavioural “active drift” (Naman et al., 2016) or “active upstream movement” (Söderström, 1987) is mainly driven by the organisms’ behaviour, in order to avoid predators (Peckarsky, 1980). Another aspects of effects of rheotaxis, not tied to predation, are colonize new habitats (Brittain and Eikeland, 1988; Williams and Williams, 1993), increase species richness from headwater to downstream (Alther and Altermatt, 2018), improve foraging (Hughes 1970; Johnson 2014), sexual activity and mating success (Wallace et al., 1975; Miller 1984). Rheotaxis is also one mechanism for escaping from unfavorable environmental conditions (Fink et al., 2017; Sertić Perić et al., 2018). Rheotaxis is mainly influenced by positive phototaxis behaviour– to swim towards the source of light in water surface (Thünken et al., 2018; Hiroki, 1980) and current velocity (Hughes, 1970). Alike to parasites as a driver of trophic niche specialization has influence in rheotaxis e.g. alterations in habitat exploitation, foraging, and antipredator behaviours of hosts (Britton and Andreou 2016; Lagrue 2007). Here we investigate how parasites influence rheotaxis of their intermediate hosts. We focus on acanthocephalan parasites, Polymorphus minutus, and their intermediate amphipod hosts of three gammarid species.

Parasites can influence amphipods distribution through manipulation of the infected amphipods’ behaviour (Lagrue, 2007). The parasites influence amphipods’ behaviour e.g. phototaxis (Cézilly et al., 2000), geotaxis e.g. free swimming of organism against the gravity (Bauer et al., 2005). Parasites are valuable tracers reflecting morphological niche adaptations (Knudsen et al., 2014). Parasites determine to a large extent the ecological niche of infected hosts by modulating a host’s micro-ecological changes (Villalobos et al., 2019). The acanthocephalan parasite, P. minutus, is well known for altering the behaviour of gammarids, e.g. avoiding predation of gammarid hosts by non-host predators, in ways that enhance their trophic transmission to their definitive hosts, waterfowl (Bakker et al., 1997; Kaldonski et al., 2007; Perrot-Minnot et al., 2007; Medoc and Beisel, 2008). The intermediate hosts of P. minutus are gammarids. Here we investigate three gammarid species: two native species ,Gammarus pulex and Gammarus fossarum, and one invasive species, Echinogammarus berilloni in Central European freshwater (Lingard and Crompton, 1972; Van Maren, 1979; Ladewig et al., 2006; Zittel et al., 2018).

Gammarids are found in water streams, but the distribution between three species is differs. The common freshwater gammarid, G. pulex, is an abundant amphipod in the lower (Piscart et al., 2010) and middle parts (Karaman and Pinkster 1977; Siegismund and Müller 1991) of small streams with slow flows of water in central Europe. G. fossarum is the most frequent native gammarids in the upper parts of streams or in the mountainous areas (Scheepmaker and van Dalfsen, 1989; Siegismund and Müller 1991; Müller 2000; Pöckl et al., 2003) and headwater streams with fast flows (Weiss et al., 2014; Schmidt-Drewello et al., 2016). E. berilloni is a naturalized species in river Meuse in France and Belgium (Josens et al., 2005), the Viroin, Belgium (Schmit and Josens, 2004) and also invasive in temporary and permanent karstic streams of Westphalia in Germany (Meyer et al., 2004). The above mentioned gammarids prefer running waters but E. berilloni and G. pulex are also found in standing waters (Verberk et al., 2018). Invasive species, with wider physiological tolerance to different water temperature, often compete with native species (Wijnhoven et al., 2003) and hence displace niche of native species or hybridize population (Rothhaupt et al., 2014a).

Gammarids could have different rheotaxis depending on whether they are native or invasive species and whether they occur as sympatric or non-sympatric species. Invasive species with higher positive rheotaxis than native species leading to competitive exclusion or extinction of native species (Rothhaupt et al., 2014b). Our study had two aims. First, we investigated whether P. minutus changes its intermediate hosts’ rheotaxis to increase successful transmission to its definitive host, waterfowls. Second, we tested whether the positive rheotaxis, was more pronounced in native, sympatric co-evolved gammarids in comparison to sympatric invasive gammarids.

2. Methods:

Sampling Specimens of G. pulex specimens were collected in kick nets from the Altenau River (51°35ʹ56.73ʺN; 8°46ʹ 56.92ʺE ) and specimens of G. fossarum and E. berilloni in the upper and lower part, respectively, of the Alme River (51°32ʹ50.05ʺN; 8°32ʹ43.61ʺE), both located on the Paderborn Plateau in Westphalia, Germany. The sampling was conducted on 30th of June 2010. Infected from non-infected gammarids were identified visually. Infected specimens presented orange-red spots present on the cuticle (Dezfuli and Giari, 1999). After separating infected from non-infected individuals, all specimens were transferred to 10-liter Dewar flasks, containing water from the sampling site. Specimens were directly transported to the climate chamber where they were kept at a 16 ± 1°C water temperature. A dark: light cycle of 10:14 hours was maintained as a means to simulate seasonal conditions. Gammarids were fed with beech leaves (Fagus sylvatica) but starved in the 48 hours preceding experiments. The starvation period was intended to induce foraging behaviours during experiments (Knoppien et al., 2000).

Experimental setup The direction and degree of rheotaxis for each gammarid was assessed using an open channel setup configured as an artificial water recycling canal (300 × 16 × 20 cm) with transparent ground and side walls (Fig. 3.1). Tap water was recirculated into the canal at a rate of ca. 25 cm per second, which is the same as stream velocity at the sampling site. The circulatory canal was subdivided into 15 sections, 20 cm each (Fig. 3.1), and each section was numbered from -7 to +7. Individual gammarids was released in section 0. The floor of the canal was covered with gravel (4-6 mm). In addition, a stone (approx. 9 cm b-axis) was placed in each section to enable the gammarids to stay close and hide. Four lamps, equally distributed above and along the channel, provided a constant light intensity at 10,000 lux.

At the entrance of the canal a mechanical filter was placed to avoid accumulation of gammarids near residuals such as food or paper (Hughes, 1970) which may reduce the rheotaxis of gammarids during feeding. The experiment was conducted with 15 infected and 15 uninfected adult individuals, each of which were placed separately for the experiment. Seven, eight and seven experiments with uninfected G. pulex, G. fossarum and E. berilloni and two, three and five experiments with infected G. pulex, G. fossarum and E. berilloni were conducted, respectively. Gammarids of less than 8 mm in length (juvenile gammarids) and with more than one cystacanth were excluded from the experiment to avoid possible confounding effects of size (Bauer et al., 2005) or degree of infection (Medoc and Beisel 2008). Just prior the onset of the experiment, gammarids were released in the middle of the canal (section 0) and restricted in movement during 10 min to adapt to the new surroundings by positioning two plastic lids at both sides of this section. After the adaptation period both lids were removed simultaneously after which the gammarid was allowed to move freely

within the canal during a period of 60 minutes. After this period of time, we separated each section through the insertion of plastic lids, which too on average 30 sec and the location of each specimen was recorded. The drifted species were in the lower positions -7 to -1 (negative rheotaxis) and the upstream movement species were in the upper positions 0 to 7 (positive rheotaxis).

20cm A

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7

FIGURE 3.1 An artificial water recycling system (300×16×20 cm; l×w×h). Red arrow: direction of water current, grey arrow: gammarid release point (section 0), A: plastic net lids, distance between lids is 20 cm. B: big stone

Statistical analyses All statistical analyses were performed with R (version 3.5.1; R Development Team 2018). We used Generalized Linear Mixed Model. Using the “tidyverse” and “multcomp” packages, we constructed mixed-effects models to examine the influence of parasite infections on rheotaxis of three intermediate host species. In the full model, we included two predictor variables: species (native G. pulex and G. fossarum, and invasive E. berilloni) and infection (yes or no) and interaction between them. The mean values were assess by using the Tuckey’s Student Newman-Keuls test, and an -level at 0.05.

3. Results

The rate of occurrence in the lower positions, i.e. -7 to -1, was significantly higher in E. berilloni than in G. pulex (difference 1.24, SE 0.51, T = 2.44, P=0.04) and G. fossarum (difference 1.37, SE 0.48, T + 2.86, P=0.01, Table 3.1, Fig. 3.2). We did not detect a significant difference in distribution across all chamber positions between the two native species G. pulex and G. fossarum (difference -0.13, SE 0.53, T value -0.25, P=0.96). Compared to uninfected gammarids, the rate of occurrence in the upper positions, i.e. 0 to 7, was significantly more in infected gammarids (Table 3.1, Fig. 3.2). There was no significant difference in the rate of occurrence between infected species not between uninfected species (Table 3.1).

TABLE 3.1 Influence of infection with P. minutus, gammarid species (E. berilloni, G. fossarum, G. pulex) on the last position of gammarids at 60 min rheotaxis. Generalized linear mixed model using a two-way ANOVA. The degrees of freedom (df), F statistics and associated p value are shown. Significant P-values are displayed in bold. Effect Covariates df F P size Intercept 0.07

Infection 1 2.36 18.7 <0.0001

Species (E. berilloni, G. fossarum, G. 2 5.02 0.007 pulex) G. fossarum 0.34

G. pulex 1.22

Species×infection 2 0.86 0.42 G. fossarum× infection 1.40

G. pulex×infection 0.14

FIGURE 3.2 Box plots of accumulation rate of gammarids position in each section at 60 min after release in section 0; positive scores and 0 indicate positive rheotaxis (Y axis), negative scores indicate negative rheotaxis (Y axis). Red boxes indicate infected species, blue lines uninfected species (X axis). Species: E. berilloni, G. fossarum, G. pulex. For statistical analyses, see table 3.1.

4. Discussion: Our study showed a positive rheotaxis response in all gammarids except for uninfected, invasive gammarids.

Rheotaxis of invasive versus native species Our experiment showed that invasive gammarids had higher negative rheotaxis compared to native gammarids. Our results were consistent with similar previous correlative studies that also detected a high degree of negative rheotaxis in the invasive gammarid, E. berilloni (Meyer et al., 2004) as well as other invasive gammarid species (Macneil and Dick 2014). However, another study reported a higher degree of positive rheotaxis in the invasive amphipod G. tigrinus, relative to the native species, G. duebeni celticus (MacNeil et al., 2003a).

For invasive species the ability to swim with the water current (negative rheotaxis) is important for having continuous reproduction (Naman et al., 2016). Indeed, low ability to resist the current flow is common to less mobile invasive species (Marvier et al., 2004; Hänfling et al., 2011; Macneil and Dick 2014). This negative rheotaxis in invasive E. berilloni indicated that there is a self-regulatory mechanism for population adjustment in aquatic ecosystem to tolerate a wide range of environmental conditions (Marvier et al., 2004; Hänfling et al., 2011) e.g. erosional habitats because of high velocity and shear stress of gravel-bed streams (Grossman, 2014). However, the wider environmental tolerance of invasive species does not necessarily result in a wider realized niche e.g. invasive G. tigrinus has narrower niche space compared to native species G. oceanicus, G. salinus and G. zaddachi (Herkül et al., 2016).

Rheotaxis behaviour of infected versus uninfected, invasive and native species Gammarid species infected with P. minutus showed a significant higher ability to resist the water flow and even swim against the water flow (i.e. higher positive rheotaxis) compared to uninfected species. Our results are in agreement with other studies. For example, infected nymphs of Baetis bicaudatus (Ephemeroptera: Baetidae) by mermithid parasite Gasteromermis sp. (Vance 1996) and blackflies (Diptera: Simuliidae) larvae infected with protozoan Tuzetia debaisieuxi (Vance 1996) or mermithid nematodes exhibited less drifting compared to uninfected individuals (Adler et al., 1983). In agreement with this, a higher prevalence of parasitized G. pulex with Echinorhynchus truttae – with higher positive rheotaxis - were found in faster and shallower areas (upstream) compared to slower and deeper areas (downstream) (Macneil et al., 2003b). However, our results contradicted those of other studies. For example a high drifting (negative rheotaxis) was found in Echinogammarus stammeri (Maynard et al., 1998; Wellnitz et al., 2003) and G. pulex (Mccahon and Maund 1991; Lagrue et al., 2007) infected by acanthocephalan Pomphorhynchus laevis, and in G. fossarum infected by E. truttae (Schütze (cited in Lehmann 1967). Given that the definitive host of P. laevis and E. truttae are not birds but fish, this indicate that both the kind of parasite and definitive host species, e.g. terrestrial or aquatic definitive hosts, could lead to adaptation of gammarids to specific niches in the stream. Indeed, less active infected gammarids by P. minutus have a terrestrial definitive host (Thünken et al., 2018) but hyperactive infected gammarids by P. laevis with a weakness to maintaining the position have an aquatic definitive host (Maynard et al., 1998). Our results showing the presence of a positive rheotaxis in native and invasive gammarid populations provides a mechanism to compensate for the parasite lost population during passive drift of the infected gammarids in flooded areas. This behaviour and geotaxis (Cezilly et al., 2000) establishes homogeneous distribution of parasite in Paderborn plateau related to

foraging area of aquatic fowls. Although, there is a possibility that P. minutus in the native G. pulex and G. duebeni celticus made strong alterations in geotaxis behaviour (Cezilly et al., 2000b; Bauer et al., 2005) or positive phototaxis behaviour (MacNeil et al., 2003a), respectively. And also acanthocephalan E. truttae made strong alteration in the activity of G. pulex (more active) (Macneil et al., 2003b). These three behaviours (negative geotaxis, positive phototaxis and increase activity) exposed gammarids to water current and made them to have active positive rheotaxis. Even though, developmental stage of P. minutus has a strong effect on altering host behaviour (Bailly et al., 2018) to manage all these behaviours simultaneously.

Furthermore, we found that the effects of infection in rheotaxis in our experimental species are not statistically different. Although previous findings showed that this ability of the parasite to alter its intermediate host’s behaviour has evolved specifically to target sympatric gammarids. For example, parasite P. laevis had no effect on rheotaxis rate of the invasive G. roeseli but it manipulates the rehotaxis of G. pulex (Lagrue et al., 2007). Evolution with sympatric gammarids could increase the stable situation in the parasite population for increasing the trophy transition to the definitive host and could minimize the energetic costs of infection with suitable sympatric species (Lafferty, 1999). Conclusion and recommendations

Our study revealed that the parasite P. minutus has effect on rheotaxis in both native and invasive gammarids and parasite may restrict the gammarids ecological niche. Our findings will aid in our understanding of foraging area of definitive hosts, waterfowls. In the context of host-parasite coevolution, it is important to demonstrate whether rheotaxis is important for intermediate host species.

The stronger invasive species showed higher fitness (Luo et al., 2019a) and E. berilloni could connect their own population from downstream to upstream and vice versa with rheotaxis specially during the drought season of Paderborn plateau. However, it is recommended to conduct experiments with different diurnal-nocturnal time effects on the rheotaxis with different flow velocity in order to assess differences in light efficiency and coevolution history of the host-parasite.

Acknowledgements:

We are grateful to the Institute for Evolution and Biodiversity, University of Münster, Germany, for providing permission and the facilities to conduct the research, and students for their assistance during field sampling. We acknowledge funding from the Netherlands Organization for Scientific Research grant numbers 854.11.003 and 823.01.014 (JK).

Photos: Sajad Ashghali Farahani

Box 2

Effects of salinity and parasites on physiological changes of native and invasive intermediate-hosts

Sajad Ashghali Farahani, Per J Palsbøll, Jan Komdeur

Freshwater host infected by parasite varies due to environmental change, e.g. variation in salinity decreased the population density of uninfected hosts Daphnia dentifera and reduced infection prevalence by fungal parasites Metschnikowia bicuspidata (Merrick and Searle, 2019). The degree of salinity of water affects population densities of intermediate and definitive hosts. For example, previous studies found the parasite having the ability to affect the survival rates of their intermediate host Gammarus roeseli infected by Polymorphus minutus during salinization (Piscart et al., 2007). Biogenic amines e.g. dopamine (Pinoni and López Mañanes, 2004; Chiu et al., 2006; Liu et al., 2008), serotonin (Khan et al., 1998; Morris, 2003) and noradrenaline (Zatta, 1987) and steroid hormone e.g. corticosterone (Gomez‐Mestre et al., 2004) have been linked to osmoregulation in crustaceans. Changes in biogenic amines may be responsible for the alteration in infected gammarid behaviour by parasites e.g. Pomphorhynchus laevis (Tain et al., 2006a) or P. minutus (Brooks and Mills, 2011). Here, I discuss the process of salinization and the mechanisms of physiological changes, e.g. osmoregulation, induced by parasites enhancing its intermediate host’s salinity tolerance.

There is not much research about effects of parasites on host tolerance during salinization. However, previous studies demonstrated the negative effects of parasites on their hosts during pollution. For instance, zebra mussels Dreissena polymorpha infected by Echinoparyphium sp. (Trematoda), had more and smaller lysosomes compared to uninfected mussels (Minguez et al., 2009). In other words, both food uptake/digestion and detoxification/excretion processes of pollutants is weaker in infected zebra mussels compared to uninfected zebra mussels. Several studies have found that salinization is associated with reduced parasite success in completing their life cycle and increased definitive host fitness. For example, Miron et al. (2003) suggested treating infected fish with salt (instead of chemicals) to get reduce parasite load, e.g. in silver catfish, Rhamdia quelen infected with the protozoan pathogen Ichthyophthirius multifiliis. However, in high salinity environments, parasite increased their success in completing their life cycle and intermediate host survival by increasing haemolymph osmolality (e.g. mermithid nematode Thaumamermis zealandica on talitrid amphipods Bellorchestia (Talorchestia) quoyana;Williams et al., 2004) In addition I will briefly review the effects of salinization on parasites and the successful establishment of invasive host species, which, in part, are consequences of the parasites’ ability to alter their hosts’ physiology.

Salinization and osmoregulation

Osmoregulation, the process of maintaining internal osmotic and ionic homeostasis by enzymes and transporters, is an important, energy-consuming necessity for aquatic animals (Tseng and Hwang, 2008). For example, 11% of the energy budget in G. pulex, is devoted to osmoregulation (Sutcliffe, 1984). Osmoregulation affects an organism’s fitness, e.g. hyper- osmoregulation and the increased energetic cost thereof, can increase mortality rates (Felten et al., 2008). The main osmoregulatory organs in crustaceans are the gills. Freshwater amphipod survival rates decreases when the haemolymph osmolality increases above a critical point (Hart et al., 1991).

Water can be classified according to its salinity as follows: freshwater < 0.5 g L−1; oligohaline 0.5 - 4.9 g L−1; mesohaline water 5.0 – 17.9 g L−1; polyhaline water 18.0 -29.9 g L−1; euhaline water 30.0 – 40.0 g L−1; hyperhaline water > 40 g L−1. Salt modifies the expression of several genes coding for increasing ion transporters, e.g. Na+/K+-ATPase that transports sodium from

the cytosol of gill epithelial cells to the haemolymph (Brooks and Mills, 2011). In freshwater ecosystems, infection by the parasite P. minutus results in a reduction of the haemolymph volume (reduction of water volume as solvent in haemolymph) in G. pulex and consequently, a rise in haemolymph salt concentration and drinking rate, along with a decline in urination. However, rises in haemolymph salt concentration were not observed in uninfected G. pulex at the same salinity. Even though in mesohaline conditions, there is the same elevated haemolymph salt concentration above a critical point in both infected and uninfected G. pulex (Brooks and Mills, 2011). In other words, under mesohaline conditions, the osmoregulation in amphipods is challenged leading to decreased survival rates in G. pulex.

Salinization

Salinization refers to the build-up of dissolved, often detected as an increase in chloride (Cl−), an important anion of many salts (Kaushal et al., 2005). Chloride salts, such as sodium chloride (NaCl), are stable, neutral salts; an ionic compound. Water (H2O) is a polar solvent. Mixing the results in salt solution (e.g. salt water). NaCl reacts with water molecules to form sodium hydroxide (NaOH) and hydrochloric acid (HCl). Salinization, or salt pollution, with NaCl, in freshwater ecosystems has many different sources, such as; de-icing agents (Kaushal et al., 2005), sea spray (Herczeg et al., 2001), salt mines (Coring and Bäthe, 2011), reduced river discharge due to damming (Mirza, 1998), drought (Salarian et al., 2018) and wind-blown salt spray (Gholampour et al., 2015), evapotranspiration (e.g. the combination of direct evaporation and plant transpiration due to irrigation with groundwater for cultivating unsuitable plants in drought area and concentration of salt in soil and entering into rivers Pillsbury, 1981).

Salinization and invasions Invasive species with origins from oligohaline and brackish water have the ability to maintain a high reproductive output even under elevated salinity in freshwater (Dick and Platvoet, 1996). It is currently unknown whether the salinity tolerance of invasive species is any different to native species. However, most invasive amphipods have comparably wider environmental tolerances in terms of; pollution (Dror et al., 2019), temperature (Shatilina et al., 2011) and oxygen levels (Hänfling et al., 2011). For example, invasive E. berilloni on the Paderborn Plateau is described as a eurytherm (high thermotolerant) species (Shatilina et al., 2011). Another example is the invasive Ponto-Caspian amphipod Dikerogammarus villosus in the Rhine river that has adaptive capacities to variations in temperature and salinity (Bruijs et al., 2001) and has evolved some degree of salinity tolerance (Reid and Orlova, 2002). Grabowski et al. (2009) suggested that salinization has a significant impact on the distribution of native gammarids in small streams, and invasive gammarids in large canals. Where there is an overlap in the realized niches between native and invasive gammarids, the presence of a native stenohaline amphipods increases the physiological costs to maintain osmotic balance, and this causes reduced growth in native gammarids compared to euryhaline invasive gammarids (Rahel and Olden, 2008).

Salinization may be beneficial for invasive amphipods adapted to brackish water (Devin and Beisel, 2007) but may also limit invasion success. The invasive zebra amphipod Gmelinoides fasciatus, which spreads in specific salinities and temperatures in the Baltic Sea (Berezina and Panov, 2004). G. fasciatus was unable to survive at a salinity above 5 psu and temperatures at 23–26°C, and is incapable of reproducing in salinities higher than 2 psu (~2g L−1; Berezina and Panov, 2004)

Chapter 4 Experimental evidence of parasitic infection increasing salinity tolerance of the native gammarid hosts

Sajad Ashghali Farahani, Janske van de Crommenacker, Eize J. Stamhuis, Per J Palsbøll, Jan Komdeur Under review at Freshwater biology

Abstract Successful transmission of parasites to their final host in aquatic ecosystems with extreme osmolality changes has been linked to a general tolerance towards drought and salinity. Parasites have been shown to affect the survival of their intermediate hosts at increasing salt concentrations. The intermediate hosts of the thorny-headed worm parasite Polymorphus minutus are Gammarus pulex and G. fossarum, freshwater gammarids that inhabit seasonal and permanent freshwater waterways. Consequently, P. minutus depends on its intermediate host’s ability to survive changing osmolality, raising the basic question whether P. minutus has evolved the ability to affect its intermediate hosts’ tolerance to changes in osmolality. We assessed the salinity tolerance of G. pulex and G. fossarum, in infected and uninfected specimens to determine if P. minutus affected its intermediate host’s salinity tolerance. We found that increasing salinity negatively affected the survival rate in both gammarids, with no significant differences between gammarid species. However, irrespective of species, the survival rates of infected gammarids were significantly higher at elevated salinity compared to uninfected gammarids. These results contribute to the basic understanding of the parasites’ ability to affect key aspects of their hosts’ behaviour and physiology which presumably enhances the parasites’ transmission to successive hosts.

1-Introduction

Salinity promotes exclusion of species through competitive interactions as a consequence of overlapping niches in homogeneous environmental conditions (Lanuza et al., 2018). Salinity tolerance affects organisms' persistence in freshwater ecosystems (i.e. ponds and streams with strong seasonal variability in water level). Mortality in freshwater biota following an increase in salinity may occur through the disturbance of osmoregulation whereby heamolymph osmolality is changed, e.g. Na+, Ca2+and Cl- concentrations, above a critical point (Hart et al., 1991). Salinity tolerance is an important trait facilitating transition between fresh and saline water in benthic macroinvertebrates (Lee and Bell, 1999). Increases in salinity can affect the mortality and osmoregulation of benthic macroinvertebrate communities in several ways: changes in salinity can reduce survival (Taylor and Harris, 1986), reduce species diversity (Williams et al., 1990; Hart et al., 1991; Kennedy et al., 1997) and influence the community composition (Piscart et al., 2005) of benthic macroinvertebrates, and changes in osmoregulation will increase the toxic effects of heavy metals (e.g. cadmium; Wright and Frain, 1981) and induce stress (Glazier and Sparks, 1997).

Gammaridae, but not all species of this family, have a higher salinity tolerance compared to other major invertebrate taxa groups (Williams et al., 1990; Kefford et al., 2003). The freshwater gammarids, Gammarus pulex Linnaeus 1758 and Gammarus fossarum Koch 1835 are European native species in temperate freshwater ecosystems. The distribution of G. pulex is positively correlated with water salinity (Wijnhoven et al., 2003; Schröder et al., 2015), with populations occupying the more saline (middle and lower) parts of tributaries in Europe (Piscart et al., 2010). G. pulex has high adaptation to karst water conditions with intermittent runoffs on weathered soluble rocks, e.g. limestone, dolomite and gypsum (Schmidt-Drewello et al., 2016). G. fossarum is a typical inhabitant of springs and spring brooks in mountain regions (Janetzky 1994; Pöckl et al., 2003) and headwater streams (Lukančič et al., 2010; Weiss et al., 2014; Schmidt-Drewello et al., 2016) which are rich in oxygen and less saline. G. fossarum is more sensitive to pollutants, low oxygen, salinity and low pH compared to G. pulex (Alonso et al., 2010).

The two abovementioned freshwater gammarids are the intermediate hoststothe acanthocephalan thorny-headed worm parasite, Polymorphus minutus (Goeze, 1782). The final hosts of P. minutus are water fowls (Bakker et al., 1997; Kaldonski et al., 2007; Perrot- Minnot et al., 2007; Medoc and Beisel, 2008). It is essential for parasites that have aquatic intermediate hosts to develop an effective system to survive the extreme osmolality changes during different life stages: the parasites move from an aquatic environment into the body of the intermediate host and afterwards towards the final host that lives in a terrestrial environment (Huang et al., 2016a). In the intestines of the final host, the parasites produce eggs which in turn are released in the final host’s feces. The parasite’s life cycle is closed by its return to the intermediate host that consumes these feces (Kaldonski et al., 2007). Parasites increase their own fitness by affecting the spatial downstream movement (drift) of the gammarids to an environment with different salinity levels (Piscart et al., 2007). The aim of this study was to assess the salinity tolerance (as a proxy for environmental tolerance) in two native gammarids, G. pulex and G. fossarum, and the effect of infestation of P. minutus on either of the two gammarid species in relation to their salinity tolerance. In freshwater ecosystems, infection by the parasite P. minutus results in a reduction of the haemolymph volume (reduction of water volume as solvent in haemolymph) in G. pulex and consequently, a rise in haemolymph salt concentration and drinking rate, along with a decline in urination.

However, rises in haemolymph salt concentration were not observed in uninfected G. pulex at the same salinity. Even though in mesohaline conditions, there is the same elevated haemolymph salt concentration above a critical point in both infected and uninfected G. pulex (Brooks and Mills, 2011). In other words, under mesohaline conditions, the osmoregulation in amphipods is challenged leading to decreased survival rates in G. pulex.

P. minutus depends on its intermediate host’s ability to survive changing osmolality, raising the basic question whether P. minutus has evolved an ability to affect its intermediate hosts’ salinity tolerance? We assessed the salinity tolerance in G. pulex and G. fossarum, in infected and uninfected specimens to determine if P. minutus affected its intermediate hosts’ salinity tolerance. We tested the competitive exclusion hypotheses: First, we expect less salinity tolerance in uninfected gammarids compared to gammarids infected with P. minutus. Second, we predict changes in salinity tolerance to be more distinct in G. pulex than G. fossarum, due to the fact they inhabit different parts of the river.

2- Methods

Sampling Individuals of G. pulex were collected from the Altenau River and G. fossarum in the upper part of the Alme River during June in 2012. Both river tributaries are located on the Paderborn Plateau in Westphalia, Germany. Infection of gammarids was confirmed by the presence of orange-red spots (cystacanth, the developmental stage at which the parasite is able to infect its final hosts) on the cuticle, which are indicative of infection by P. minutus (Dezfuli and Giari, 1999). After separating infected from uninfected individuals, specimens were transferred to the lab in 10-litre Dewar flasks, containing water from the sampling site and kept at 18 ± 1°C water temperature and a 10:14 hours dark:light cycle to simulate seasonal conditions and fed with leaves (Fagus sylvatica) for 1 to 5 days. In order to reduce the amounts of ammonia compounds and potential side-effects in the water from gammarid feces, the gammarids were starved for 48 hours preceding experimentation.

Experimental setup The survival rates of P. minutus infected and uninfected gammarids, of both G. pulex and G. fossarum, under different salt concentrations, were estimated for 72 hours using the protocol by Kefford et al. (2003). Only gammarids larger than 8 mm in length and with zero (uninfected) or a single cystacanth (infected) were used in order to avoid possible effects due to differences in size or degree of infection (Bauer et al., 2005; Medoc and Beisel, 2008; Alonso et al., 2010; Dalhoff et al., 2018). Each gammarid was kept in its own plastic an open small dark-brown plastic container (6.5×6.0×6.0cm) with a plastic mesh at the bottom.

The experiment was conducted with 30 infected and 30 uninfected adult individuals, each of which were placed in plastic container to provide the possibility to cling and hide (Fig 4.1). Salinities were obtained by dissolving tap water with unrefined Atlantic sea salt (Danival®, Andiran, France). This provided ionic proportion similar to sea salt, with NaCl as main component (NaCl 0.95g, MgCl2 0.001g, CaCl2 0.002g, KCl 0.0005g). The experimental setup was identical for infected and uninfected G. pulex and G. fossarum, respectively. Infected and uninfected gammarids were kept separate. The salinity of the water was determined by EC (electrical conductivity in mS cm⁻1) with an EC meter (Wasser Test in der Welt, Weilheim, Germany). The 30 small containers were placed in five plastic basins (40.5×59.5×11.5cm), containing 12 liters of water with five different salinity and conductivity: 6 g/lit (11 mS cm-1), 12 g/lit (19.6 mS cm-1), 18 g/lit (32.8 mS cm-1), 24 g/lit

(40.6 mS cm-1) and a control (tap water, 0.5 mS cm-1). The depth of water in the basins was kept at ~5.0 cm.

We recorded the mortality of the gammarids every 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 42, 48, 60 and 72 hours after introducing the individuals to the small container, by checking the movements of the pleopods (swimmerets) as a sign of life. Non-moving individuals were assessed visually using a dissection microscope (Zeiss®Stemi 2000) to check for the presence or absence of vital signs. Temperature and conductivity were monitored during each of the time periods of the experiment and we made sure that water temperature was constant (18 ± 1°C). Salinity was adjusted accordingly, e.g. the plastic tank basin was regularly topped up with demineralized water to avoid an increase in salinity concentration due to evaporation (Dorgelo, 1976). After 72 hours each live specimen was re-checked under the stereo dissection microscope for signs of infections with parasites other than P. minutus.

FIGURE 4.1 Design for measuring survival rates of infected and uninfected G. fossarum and G. pulex in 6, 12, 18,24 g/L salt concentrations and control (tap water). Thirty plastic containers were placed in basins with 12L well aerated water, with one gammarid per basket. The experiment was repeated using the same set-up for infected G. pulex, uninfected G. pulex, infected G. fossarum and uninfected G. fossarum.

Statistical analyses

All statistical analyses were using with R, version 3.5.1 (R Development Team 2018). We modeled survival rates (McLachlan and McGiffin, 1994) using the Proportional Hazard (PH) model and the Kaplan-Meier estimator (Kaplan and Meier, 1958).

The CRAN-R package Olsurve was employed to calculate and plot the Kaplan–Meier survival curves. The survival model was generalized from a Cox hazard function (Cox, 1972), the hazard function for individual is given by

( ) ( ) ( ) Equation 1

Where ( ) is a baseline hazard function for the failure time t and ( ) is hazard ratio given the predictor X. are the regression coefficients (i.e. parameters). is an intercept parameter, and are weights, i.e. regression coefficients corresponding to . Where ̂ is estimated via the maximization of the following likelihood function ( ) ∏ ( ) Equation 2 ∑ ( )

The Proportional Hazard model can also be written in terms of the probability of surviving past time t. This is the Kaplan-Meier estimator. i.e. ̂ ( ) (Harrell, 2015): ̂ ( ) ̂ ( ) [ ̂( )] Equation 3 where ( ) is the predicted survival distribution and estimated with extension of Kaplan- ̂ Meier. is interpreted as changing rate of hazard when the covariate changed by . In the full model, we had three predictor variables: species, infection and Salinity.

We used the Akaike Information Criterion (AIC) to compare alternative regression models for the goodness of fit (Akaike, 1974). The model with the lowest AIC score was considered the most probable model. We compared seven models with three sets of predictor variables: infection, salinity, species and interactions (salinity*infection, species*infection, salinity*species and salinity*infection*species).

The validity of the PH assumption was assessed by using Schoenfeld’s global test (Grambsch and Ther-neau, 1994), which assess the independence between residuals and time. If the plot of Schoenfeld residuals against time showed a non-random pattern, i.e. the slope is not zero, the PH assumption was violated.

3- Results Influence of salinity and infection on survival Infection, salinity and species were three predictor variables of survival. The models with interactions did not yield a better fit than models that include only one variable (Table 4.1). There was no substantial difference between the models. Salinity, infection and interactions between salinity and infection violated the validity of the PH assumption as the zero slope changed significantly over time. Species and its interaction with salinity did not violate the validity of the PH assumption as the zero slope did not change over time (Fig 4.1 Supplementary).

TABLE 4.1 Akaike Information Criterion (AIC) of different distributions fitted to the full model. model AIC salinity+infection+species 3948.8 salinity+ infection 3947.6 salinity+ species 3948.8 species + infection 3948.8 infection 3947.7 species 3947.6 salinity 3946.4

The p-values for the three overall tests (likelihood, Wald, and logrank) were significant (p<0.001), indicating that the model shown in Table 4.2 is significant. There was no difference in survival rates between the two native gammarid species in different salt concentrations (Table 4.2). Compared to infected gammarids, uninfected gammarids had significantly higher risk of death (Table 4.2). In addition, there was a significant interaction between salinity and infection, indicating that a higher concentration of salt experienced by uninfected individuals resulted in higher mortality compared to infected individuals (Table 4.2).

TABLE 4.2 Hazard ratio (survival probability) at different salt concentrations and 95% confidence interval (lower - upper) for the proportion of individuals that survived after 72 hours. Covariates exp(coef) 95% CI P value salinity 1.25 1.22-1.29 <0.001 infection 0.35 0.17-0.70 <0.001 species 0.75 0.40-1.42 0.39 infection:species 2.30 0.88-6.00 0.08 salinity:infection 1.05 1.01-1.09 <0.001 salinity:species 1.02 0.98-1.05 0.22 salinity:infection:species 0.95 0.90-1.00 0.06

test Likelihood ratio test <0.001 Wald test <0.001 Score (logrank) test <0.001

Influence of salinity and infection on mortality Overall, the 72-hours survival rates were 5% (i.e. 95% died during the experiment), with the 50% survival probability estimate being 8 hours after the onset of the experiment (Fig 4.2a). In fact, 50% survival occurred 10 and 4 hours after the onset of the experiment for infected and uninfected individuals, respectively (Fig 4.2b), whereas 50% survival was similar for G. fossarum and G. pulex, occurring 8 hours after the onset of experimentation (Fig 4.2c).

(a) (b) (c)

FIGURE 4.2 Survival curve, Kaplan-Meier plot, with the probability of survival against time. The proportion of individuals that had survived up until 72 hours (solid black line in panel a and solid red and blue in b and c) and the 95% confidence interval (dashed lines). (b) Red

lines indicate infected, blue lines uninfected gammarids. (c) Red line: G. pulex, blue line: G. fossarum.

4- Discussion

Our study showed experimentally that increasing salinity negatively affected the survival rate in both gammarids, with no significant differences between gammarid species. This observation is consistent with the findings reported by other researchers showing the inability of G. pulex (Sutcliffe, 1971; Dick and Platvoet, 1996; Grabowski et al., 2009) and G. fossarum (Piscart et al., 2011b) to tolerate high salinities. The high sensitivity in G. pulex shows this species to be suitable as bio-indicator for salinity in freshwater ecosystems (Schröder et al., 2015) and our findings showed that G. fossarum as same as G. pulex are bio- indicators for salinity in freshwater ecosystems.

Our study failed to detect any differences among G. pulex and G. fossarum in mortality rate in relation to salt concentration. Given that both gammarid species need to avoid high salinity in order to survive and that both species have similar mortality rates in relation to salt concentrations, this indicates that if both gammarid species occur in more similar ecological conditions this lead to a lower rates of coexistence as a result of competitive displacement of one species (Vandermeer, 1975; Abrams, 1983). G. fossarum were dominant in permanent water areas but G. pulex who had prevalence in temporary areas are under pressure of invasion of other gammarids, e.g. Echinogammarus berilloni Catta 1878, in Paderborn plateau (Meyer, 2004). This competitive exclusion by G. fossarum appeared to increase their fitness and density. G. pulex might disappear as a consequence of drought and of higher densities of the invasive E. berilloni. Competitive interactions between species could be changed by environmental stressors, such as salinity (Pickens et al., 2019a). Even though, G. fossarum adapted to the adverse effects of drought (Poznańska et al., 2013) but G. pulex, as a typical inhabitant of temporary water bodies, was unable to survive the increasing salinity due to drought, as well as the total drying of the stream (Meyer, 2004). There are several reasons for the low ability of gammarids to survive in high salt concentrations, for example inability to prevent chloride from entering the tissues (Sutcliffe, 1971) and decreasing intra- and extracellular free amino acids (Jordan et al., 1999; Patrick and Bradley, 2000).

Our finding that infected gammarids have a significantly lower mortality risk than uninfected gammarids confirms the results of previous studies that found the parasite having the ability to increase salinity tolerance without change in osmoregulatory adjustment (e.g. P. minutus on invasive Gammarus roeseli; Gervais 1835; Piscart et al., 2007) or parasite ability to increase haemolymph osmolality (e.g. mermithid nematode Thaumamermis zealandica; Poinar, Latham and Poulin 2002, on talitrid amphipods Bellorchestia (Talorchestia) quoyana; Milne -Edwards 1840; Williams et al., 2004). P. minutus thus increase the survival rates of their intermediate host under different salinities and hence its own survival. Parasites and their intermediate hosts occur at lower trophic levels than the definitive hosts. Species at higher trophic levels typically have lower environmental tolerance compared to species at lower trophic levels (Montserrat et al., 2013). Moreover, compared to uninfected organisms, infected aquatic organisms tend to be more tolerant not only to elevated salinity but also to other factors related to global climate change (e.g. acidification) (MacLeod and Poulin, 2016).

It is possible that the survival of infected gammarids is influenced by the developmental stage of the parasite. In addition, the developmental stage of the parasite might influence the infected gammarid’s geotaxis behaviour (i.e. moving against or in agree with gravitational

forces) (Bailly et al., 2018). Our results suggest that the parasite plays a main role as a coexistence agent which increases the survival rate of the host with increasing salinity concentrations (proximate cause) to reach the parasite maximum fitness and transmission to final host (ultimate cause). A similar proximate cause e.g. protective mechanism of parasites, has been found for increasing gammarid survival rates in relation to the accumulation of the pesticide Pentachlorophenol (PCP) (Heinonen et al., 2001). Parasites modify the host’s behaviour by regulating the host’s serotonin levels (Tain et al., 2006, Cézilly and Perrot- Minnot 2010, Cézilly et al., 2013). Biogenic amines e.g. neurotransmitter serotonin have been linked to osmoregulation in crustacean (Khan et al., 1998; Morris, 2003).

For a better understanding of the higher survival of infected gammarids, the difference in oxygen consumption in intermediate hosts during infection with the parasite should be considered. Although there is lack of research about oxygen demand differences between infected and uninfected gammarids, some research about infected species showed that high oxygen demand causes stronger negative geotaxis and a greater tendency to surface swimming in native G. pulex infected by P. minutus compared with the invasive host, G. roeseli (Bauer et al., 2005). Negative geotaxis could conceivably be a behavioural response to hypoxia from the sustained hypoxic-like conditions induced by P. minutus cystacanths, or the release of lactate and succinate (the excretion/secretion products of parasites) in the host brain (Perrot-Minnot et al., 2016). Another significant factor for parasite distribution is higher parasite infection in freshwater populations compared with brackish water populations of the same species as host (Rückert et al., 2007). This means salinity is also a limiting factor for parasite distribution.

The results found by Piscart et al. 2007, that match our results, may also be caused by adaptation of the parasites with their intermediate host only and not with their final e.g. reducing survival of anadromous final host fish, that migrate from the sea up the rivers to spawn, because of destruction of their skin by parasites with a concomitant change in the osmoregulation system (Urawa, 1993). Some other biotic and abiotic factors could also affect survival of gammarids during increasing salt concentrations. Salinity tolerance of gammarids related to temperature (Kennedy et al., 2004), pH-value (Dunlop et al., 2005), dissolved oxygen(Meijering, 1991; Peeters and Gardeniers, 1998), gender effects (Sornom et al., 2010), invasion effects (Devin and Beisel, 2007; Piscart et al., 2010), length of exposure (Ellis and Macisaac, 2009) and lime content (Soucek and Kennedy, 2005). Future research into our species of Gammarids could include experiments that take into account these variables.

Conclusion and recommendation

We conclude that the parasite P. minutus significantly increases their native intermediate host salinity tolerance, because (1) the native host gammarids osmoregulatory ability is altered by P. minutus, and (2) increase in mortality with rising salinity levels was higher for uninfected gammarids. Our results showing low salinity tolerance of native species has been supported by other studies that showed high diversity in the native populations of aquatic ecosystems with a high range of sensitivity (Kelly and Dick, 2005; Leuven et al., 2009; Macneil and Briffa, 2009; Lenz et al., 2011) but our study was the first to experimentally show the parasitic infection to increase host salinity tolerance in G. pulex and G. fossarum. Changing the salinity tolerance of the gammarid hosts is important for the parasite, as it enables the gammarids to survive in more saline environments which are among the habitats where the final host is to be found. In this way, the parasite can be transmitted to the final host.

Some studies showed the average range of salinity preference for G. pulex to be 270–320 µS cm-1 (Dick and Platvoet, 1996) in their natural habitats, but our control group with tap water had a two times higher salt concentration than their normal habitats (0.5mS cm-1=500µS cm- 1). It should be noted, however, that these trials were relatively short-lasting and may not reflect actual conditions, e.g. invasion and drought periods, experienced by these species in a real habitat. Climate change may result in increased salinity through increased evapotranspiration, e.g. the combination of direct evaporation and plant transpiration due to irrigation with groundwater for cultivating unsuitable plants in drought area and concentration of salt in soil and entering into rivers (Pillsbury, 1981), wind-blown salt spray from drought areas (Gholampour et al., 2015) or sea spray coastal wetlands with sea-level rise (Herczeg et al., 2001). However, the two gammarids species used in our experiment inhabit continental freshwater streams where the increase in salinity due to climate change likely will be far below the NaCl concentrations during in our experiment.

Survival rates between the two native species in different salt concentration were equal; however, exclusion of G. pulex through competitive interactions with G. fossarum as a consequence of overlapping niches can be expected. Both native gammarids G. pulex and G. fossarum have equal salinity tolerance, but G. pulex occurs in salty and desiccated areas and G. fossarum does not. The competitive exclusion by G. fossarum appears to increase its fitness and abundance. G. fossarum seems to be better able to cope with changing environments: (i) our findings showed increasing salinity tolerance in G. fossarum and not in G. pulex; (ii) parental exposure induce transgenerational plasticity in G. fossarum and better acclimation to contaminants (Vigneron et al., 2019). In this regard, future research about the distribution of these two native gammarids on the Paderborn plateau in relation to salinity profiles is necessary. This could be combined with more research on the genetic differences between the two populations of gammarids on this plateau in relation to salt tolerance. Even so, this study proved parasite effects on survival of gammarids in different salinity conditions, which likely has drastic effects on gammarid population dynamics and potentially the entire food web. The influence of parasitic infection might enable both gammarids to adapt to and spread into saline or temporary ecosystems. Acknowledgements We thank the Institute for Evolution and Biodiversity, University of Münster, Germany, for providing permission and the facilities to conduct the research, and students for their assistance during field sampling. We acknowledge funding from the Netherlands Organization for Scientific Research grant numbers 854.11.003 and 823.01.014 (JK).

Data Availability Raw data from the study and R syntax to permit reproducible statistical analyses are available in the GitHub (https://github.com/limnologist/Salinity-survival).

FIGURE 4.1 Supplementary. Plots of scaled Schoenfeld residuals (circles) against time for each covariate (y-axis) in a model fit to the survival data. The solid line is a smoothing spline fit to the plot, with dashed lines representing a ± 2-standard-error confidence band around the fit. Non-zero slope is an indication of a violation of the PH assumption. For statistical analyses, see table 4.2.

Chapter 5

General discussion and synthesis

Introduction Parasites consist of approximately 50% of the total species in ecosystems (Hudson et al., 2006). In aquatic ecosystems their biomass, is greater than the biomass of their predators (Kuris et al., 2008; Lafferty et al., 2008; Preston et al., 2013). Parasites dominate the links in food webs. This means that food webs, which show the flow of energy throughout an ecosystem, include more parasite–host links, e.g. trophic transmission of the parasite from one intermediate host to the definitive host, than predator–prey links (Lafferty et al., 2006). This is true, even though the probability of parasites finishing their life cycle is controlled by several factors, which include intermediate and definitive hosts, as well as non-host predators. Parasites start their development in the intermediate host and can manipulate the physiology and behaviour of their intermediate hosts (Tain et al., 2006b). Parasites complete their life cycle in the definitive host to which the parasites are transmitted by predation of the intermediate host (Tain et al., 2006a). At the end of the life cycle, adult parasites typically mate and reproduce inside the definitive host. The resulting eggs or larvae are transmitted via defecation of the definitive host and subsequent infection of the intermediate host (de Vries and van Langevelde, 2018). The transmission of the parasites to their definitive host is facilitated by non-host predator avoidance by the intermediate host, which would otherwise prevent completion of the parasite’s life cycle (Vielma et al., 2019). Parasites could act as a digestive food source for non-host predator (Johnson et al., 2010; Thieltges et al., 2013) or as a non-digestive food source for a paratenic host predator – a vertebrate host that comes before the definitive host and does not require a certain developmental stage of the parasite (Médoc et al., 2011). Digestion by non-host predators means the end of the parasite’s life cycle and leads to no fitness for the parasite. The selection pressure on parasites to reach their definitive host predator is higher than reaching a non-host predator, because transmission to suitable definitive hosts is mandatory for the survival of the parasite (life-dinner principle, Dawkins and Krebs, 1979). The extinction or low fitness of intermediate or definitive hosts or both could lead to alternative mechanisms exhibited by parasites to increase their survival and the completion of their life cycle. For example, parasites have changed their intermediate hosts within the same family over evolutionary history (Lyndon, 2001), expanded the diversity of definitive host predators (Vanacker et al., 2012), or increased intermediate hosts abilities upon non-host predators avoidance behaviour (Médoc and Beisel, 2008) resulting in higher survival of parasites, completion of their life cycle and success in parasite colonization. Parasites have the ability to acquire both novel native and invasive intermediate hosts (Bauer et al., 2005).

I discussed in first chapter about response traits of native species to invasive host-species and effect of parasites on native and invasive host-fitness. The arrival of invasive host species in a native host population may promote local parasite maladaptation (Moret et al., 2007). The main aim of my thesis was to study the ability of parasites to alter the native and invasive intermediate host’s behavior. I tested whether the “increased host ability” hypothesis in non- host predators, was more pronounced in native, sympatric co-evolved gammarids in comparison to sympatric invasive gammarids. In this dissertation, I performed an experimental study to investigate the effect of infection by parasites on changes in behaviour of invasive and native intermediate hosts. I studied the thorny-headed worm parasite Polymorphus minutus and its native intermediate hosts Gammarus pulex, G. fossarum and invasive Echinogammarus berilloni on the Paderborn Plateau (Westphalia, Germany). P. minutus, comprises morphologically similar, but genetically divergent subspecies, in Westphalia (Grabner et al., 2020). In Westphalia, the P. minutus co-invaded the region with invasive gammarids, E. berilloni and G. roeseli from the Mediterranean area and Southeast Europe, respectively (Zittel et al., 2018). G. roeseli transmitted this invasive parasite to G. pulex. However, the native cryptic parasite P. minutus uses native G. fossarum as an

intermediate host (Zittel et al., 2018). Perhaps native P. minutus can potentially have different traits than invasive P. minutus in manipulating the intermediate host behaviour in such a way that it is increasing the chances of being transmitted to the definitive host. In chapter two, we investigated the effect of parasite infection on native and invasive intermediate hosts in non- host predator avoidance behaviour. Our findings revealed that native P. minutus were more successful in manipulating native G. fossarum behaviour to avoid the non-host predator, three-spined sticklebacks Gasterosteus aculeatus, than invasive P. minutus were in manipulating G. pulex and E. berilloni In chapter three, my coworkers and I investigated the role of the native and invasive parasite on rheotaxis – to swim against or with the current - in the intermediate hosts. The lack of differences in rheotaxis between infected native and invasive hosts may indicate that P. minutus enhances transmission to its definitive host, by making the intermediate host more stationary and hence more easily preyed upon by the definitive host. In the previous chapters, I described the importance of studies on invasive intermediate host species and its’ impact on parasite prevalence in food webs. I focused on the ecological effects in invasive gammarids in Europe. In chapter four, I assessed the effect of parasites on the survival of native intermediate hosts in water with different salinities. I showed that survival rate in infected, native gammarids was higher than that of uninfected native gammarids at higher salinities. This means that infected native gammarids who have drifted to downstream brackish waters, where the habitat of water fowl mostly is, have a chance to survive, and transmit the parasite successfully to definitive hosts. The key question in all chapters was if the ability of P. minutus to alter its intermediate host’s behaviour has evolved specifically to target sympatric gammarids, or gammarids in general.

In this chapter, I will assess the research findings presented in this thesis in an evolutionary framework, and discuss the effect of the parasites on their sympatric native and invasive intermediate host survival and behaviour. Indeed, invasive species are interesting as “real- time” systems to study the succession of “new” species’ establishment in invaded ecosystems. A fundamental question is whether parasites may play a role in the successful establishment of invasive gammarids? For example, are invasive gammarids more successful because they are able to avoid infection by native cryptic P. minutus than native gammarids (Zittel et al., 2018)? Below, I will discuss the success of infection and invasion in aquatic ecosystem.

1. Success of infection

The success of infection by parasites during the host-parasite coevolution is related to increasing a parasite’s ability to alter intermediate host behaviour, resulting in higher fitness of parasites. In the first part of this chapter, I will discuss the parasite’s ability to alter the behaviour of the intermediate host in sympatric host species. Second, I will discuss why the infected gammarids actively swim against the water current (positive rheotaxis). Positive rheotaxis will also decrease downstream drifting rate of the intermediate host, where most of the non-host drifting predators are present. Karst streams (intermittent streams with temporal and spatially varying discharge regime) on Paderborn Plateau, i.e. the upper catchment of the Lippe, are susceptible to summer droughts and consequently have elevated salinity during this period. Positive rheotaxis is one mechanism to keep permanent up streams as reservoirs of parasite during the summer time. It is vital for parasite in infected gammarids to cope with higher salinity levels, both in drought area and downstream, compared to uninfected gammarids. Third, I will discuss the differences in survival rates observed in infected gammarids in different degrees of salinity. Finally, I will discuss these observations in terms of ‘adaptation’ and ‘exaptation’ in host-parasite co-evolution.

1-1. Increased sympatric intermediate hosts’ abilities hypothesis

Previous studies have demonstrated that parasites decrease non-host predator exposure by manipulating the behaviour of their intermediate host (Holomuzki et al., 1988; Rigby and Jokela, 2000; Matz and Kjelleberg, 2005; Friman et al., 2009). To reach optimal fitness a parasite must keep their infected intermediate hosts in an optimal balance between survival - i.e. protection from non-host predator - and foraging - i.e. seeking food or mate (Dianne et al., 2011, 2014). The “increased host abilities” hypothesis posits that parasites have evolved in response to selection pressures on transmission to the next host, i.e. the ability to manipulate the behaviour of the intermediate hosts in such a way that it increases the chance of transmission (Medoc and Beisel, 2008; Beisel and Médoc, 2010).

I introduced two aspects of predation in chapter two: (i) efficiency of predation on native versus invasive intermediate host species, and (ii) parasite-induced changes in non-host predator avoidance behaviour. Native gammarids have been living for a substantially longer time in sympatry with native parasites than invasive gammarids. I tested whether the “increased host abilities” hypothesis, was higher in native, co-evolved gammarids in comparison to invasive gammarids. To test this hypothesis, I used P. minutus, an acanthocephalan parasite, two native intermediate hosts, G. pulex and G. fossarum, and one invasive host, E. berilloni.

The changes in behaviour observed in infected G. fossarum were consistent with the “increased host abilities” hypothesis. Our results indicated that infected native G. pulex and invasive E. berilloni avoided cues of three-spined stickle backs (i.e. chemical cues from mucous of fish skin or pheromone) significantly less compared to infected native G. fossarum. The results suggested that native cryptic P. minutus and native G. fossarum both are able to increase their transmission by avoiding predation by a non- definitive host species, thereby indirectly promoting transmission success to the definitive hosts.

My study revealed that invasive E. berilloni do not seem to detect or avoid fish cues, irrespective of the level of infection with the P. minutus. My findings indicated that there was a difference in avoidance behaviour of infected E. berilloni on the Paderborn Plateau in comparison to their native origin area in France. For example, native E. berilloni infected by native P. minutus from the Lunain river (France) reduced their activity in the presence of fish cues (Jacquin et al., 2014). However, native G. pulex infected by invasive, cryptic P. minutus , transmitted from G. roeseli to them (Zittel et al., 2018), had a tendency of swimming towards fish cues.

1-2. Avoidance of infected intermediate host by drifting

Focusing on the comparison of rheotaxis behaviour of infected versus uninfected gammarids of different species, my results showed a high degree of positive rheotaxis in infected species. This positive rheotaxis means that infected intermediate gammarid hosts in all species avoid drifting. My findings were consistent with those reported by Macneil et al (2003) who found a higher prevalence of parasitism in the faster and shallower water bodies (upstream) compared to slower and deeper areas (downstream). Furthermore, I found that the effect of infection on rheotaxis behaviour in all my experimental gammarids species were similar (Chapter 3). Rheotaxis and geotaxis (Cezilly et al., 2000) are both mechanisms potentially favouring the trophic transition of the parasite to its definitive host (Lafferty, 1999). Overall, infection with P. minutus appeared to increase rheotaxis.

1-3. Survival rates of infected and uninfected gammarids in relation to salinity

It is essential for parasites to develop an effective system to cope with extreme variations in osmolarity. Osmolarity means the total number of solute particles per liter. Parasites move from an aquatic environment into the body of the intermediate host and afterwards to a terrestrial definitive host (Huang et al., 2016b). Parasites increase their own fitness by avoiding environments with high variations in salinity levels (Piscart et al., 2007). I showed that gammarids infected with P. minutus survived at higher rates compared to uninfected gammarids at higher salinity (Chapter 4). P. minutus thus increase the survival rate of its intermediate host under different salinities and hence the survival of itself. Parasites and their intermediate hosts are at lower trophic levels than the definitive hosts. Species at higher trophic levels typically have lower environmental tolerances compared to species at lower trophic levels (Montserrat et al., 2013). Moreover, infected aquatic organisms not only tend to be more tolerant to elevated salinity but also to global climate changes (e.g. acidification) than uninfected organisms (MacLeod and Poulin, 2016). Parasites have been shown to alter the activity level of neuroendocrine factors (i.e. serotonin) in the brain of their intermediate hosts during developmental stage of parasites (Tain et al., 2006, Cézilly and Perrot-Minnot, 2010, Cézilly et al., 2013). Serotonin is a neurotransmitter that affects water and salt movements in crustacean gills through osmoregulatory mechanisms, which in turn may make intermediate host more tolerant to higher salinity levels (Péqueux, 1995; Liu et al., 2008). Serotonin plays a key role in several behavioral traits, including thermotaxic behavior -movement of an organism in response to temperature- (Li et al., 2013; Wong and Rankin, 2019), phototaxis behavior -movement of an organism in response to light- (McPhee and Wilkens, 1989; Tain et al., 2006a; Rodriguez Moncalvo and Campos, 2009; Thamm et al., 2010), the geotaxis -swimming of an organism to the top or bottom of the water column- (Maximino et al., 2013), feeding behavior (Alanärä et al., 1998; Ortega et al., 2013), and oxygen consumption (Srinivasan et al., 2008; Pérez-Campos et al., 2012).

I interpreted my findings in the light of an adaptive scenario. It is necessary to further investigate the effects of salinity in sympatric or allopatric, infected species in terms of physiological plasticity, e.g. parasites might alter the trade-off between survival and reproduction in gammarids (Dunn et al., 2012).

1-4. Plasticity and consistency of behaviour differs between infected and uninfected gammarids

Recent studies showed that behavioural plasticity (individual differences in behaviour) and consistency (consistent among-individual variation in behaviour) of intermediate hosts differed among both host sex and infection status. For example, the male aquatic isopod Caecidotea intermedius showed higher plasticity for refuge use in both uninfected and infected individuals that is infected by the parasite Acanthocephalus dirus, and higher consistency for refuge use only in infected individuals. In contrast, female aquatic isopod exhibited plasticity in uninfected and consistency in infected individuals, in activity (Park and Sparkes, 2017). I showed that avoidance from non-host predator’s cues was exhibited consistency in infected individuals of G. fossarum (Chapter 2). Consistency of rheotaxis and salinity tolerance was present in infected individuals, but not in uninfected individuals of gammarids (Chapter 3 and Chapter 4). My results indicate that P. minutus infection may shape personalities in sympatric gammarids by increasing consistency of mentioned traits. P.

minutus appear to be able to exploit plasticity in sympatric intermediate host antipredator responses toward its own advantage.

P. minutus adopts different strategies to exploit native and invasive intermediate gammarids hosts. One outcome of this adaptation could be that the parasites modify non-host predator avoidance behaviour in native G. fossarum but not in invasive E. berilloni. Consistent with “increased intermediate host abilities” hypothesis, infected invasive gammarids showed less drift compared to uninfected invasive species. Differences in ‘personality’ and behavioural plasticity between native and invasive species contribute to invasion success (Bierbach et al., 2016). Personality traits affect dispersal tendencies of invasive species (Chapple et al., 2012). A highly adaptable, invasive species may survive and reproduce thereby facilitating colonization and establishment (Plantamp et al., 2019). Contrary to my expectation, the invasive uninfected E. berillonoi drifted more than uninfected native G. pulex and G. fossarum.

1-5. Modulation of host behaviour evolved through both ‘adaptation’ and ‘exaptation’

The ultimate cause of “increased parasite fitness through transmission to a definitive host- predator” occurs in combination with proximate causes of the success or failure of infected intermediate hosts, such as avoidance of non-host predators (Chapter 2), positive rheotaxis (Chapter 3) and increased salinity tolerance of the intermediate hosts (Chapter 4). Evidence in support of my results suggested that modulation of intermediate host behaviour has evolved through ‘adaptation’ (Baldauf et al., 2007) and/or ‘exaptation’ by parasites (Cézilly and Perrot-Minnot, 2005; Combes, 2005; Beisel and Médoc, 2010). This trait can potentially be explained as an exaptation of a parasite manipulating it’s intermadite host behaviour in a manner that places the intermediate host in the vicinity of the definitive host. Hence, parasite evolution may be explained by a shift in function, from regulation of survival in the intermediate host to reproduction in the defenitive host. Predation of infected intermediate hosts by the definitive hosts, is crucial for the parasite to complete its life cycle (Harvey and Pagel, 1991). However, modulating the intermediate host’s behaviour is not always adaptive for the parasite (Bakker et al., 2017). For example, acanthocephalan parasite Pomphorhynchus laevis manipulates the behaviour of its native intermediate host, G. pulex, but is unable to manipulate the behaviour of the invasive G. roeseli (Moret et al., 2007). On the other hand, exaptation may also occur, when behavioural traits of infected intermediate host by parasite ultimately facilitates trophic transmission to the definitive host by natural selection (Cezilly et al., 2012).

As emphasized at several occassions, natural selection in a host-parasite system may not necessarily target host traits directly, but instead on the ability of parasites to alter hosts traits in a manner enhancing the trophic transmission of the parasite (Seppälä and Jokela, 2008; Seppälä et al., 2008; Cézilly and Perrot-Minnot, 2010).This means that the host –parasite coevolution is directly related to the concept of the “extended phenotype” introduced by Richard Dawkins in 1982 (Seppälä and Jokela, 2008; Seppälä et al., 2008; Cézilly and Perrot- Minnot, 2010).Examples of exaptation are: avoidance of non-definitive host predators, positive rheotaxis and increased salinity tolerance that evolved both in adaptation and exaptation by natural selection in response to predation, flooding and salinization.

2. Factors association with invasion success to dominate invaded communities by invasive species compared to native species There are behavioural differences between native and invasive gammarids, e.g. less activity in invasive species (Piscart et al., 2011a). Factors known to affect the invasion progress of invasive species compared to native species, resulting in a wider distribution in aquatic ecosystems compared to native species, are: parasitic infection (Comont et al., 2014; Haelewaters et al., 2017), presence of predators (Jermacz and Kobak, 2017), river type (Boets et al., 2015), physical-chemical and hydro-morphological variables such as conductivity, orthophosphate and dissolved oxygen concentration (Boets et al., 2013), artificial hard substrates (Van Riel et al., 2006), local adaptation (Dlugosch and Parker, 2008; Colautti and Lau, 2015), rapid adaptation of invasive species to new environments (Whitney and Gabler, 2008; Sotka et al., 2018; Stutz et al., 2018) and increased competitive ability compared to native species (Blossey and Nötzold, 1995). However, the behavioural traits that enhance invasion success, such as fast growth and high reproductive rates in invasive species (‘invasion syndrome’, Chapple et al., 2012), have received less attention compared to research on native species. Behavioural differences between native and invasive species may contribute to enhances in invasion success of invasive species. Successful invasion means that invasive species were able to take advantage of available resources and outcompete local populations, e.g. through decreased competition from native species or increased resources through eutrophication from nutrient discharge (Davis et al., 2000; Wikström and Hillebrand, 2012).

In the second part of current chapter, I will discuss the role of infection of intermediate host on avoidance of non-definitive host predators. Second, I will discuss the mechanism of higher negative rheotaxis in invasive species compared to native species. Third, I will discuss the negative effect of increasing salinity on survival rates in freshwater gammarids. Finally, I will discuss the competitive exclusion mechanism of related invasive and native species.

2-1. Gammarids do not appear to avoid non-efficient predators

Invasion can be facilitated if the invasive species are less susceptible to non-definitive host predators than native species (Schmidt-Drewello et al., 2016). I have tested non-definitive host predator avoidance behaviour of uninfected native and invasive gammarids from three- spined stickleback cues in an experimental setting. I showed that uninfected individuals of both species preferentially swim towards fish cues. This behaviour was especially noticeable in the native gammarids compared to the invasive gammarids. In contrast, invasive gammarids reduced their locomotion activity in the presence of fish cues (Chapter 2). The main question is why would the absence of avoidance from fish (predator) cues be higher in uninfected native gammarids compared to uninfected invasive gammarids? Uninfected gammarids appear able to discriminate between the chemical cues emitted by scavengers or inefficient predatory species (e.g. three-spined sticklebacks (Jacquin et al., 2014) and those emitted by the efficient predator, the trout, (Abjornsson et al., 2000)). Moreover, fish cues could act as indications of food resources to gammarids. The gammarids may behave as omnivore scavengers in aquatic ecosystems (Wilhelm and Schindler, 2011; Jermacz et al., 2017), whereas the three-spined sticklebacks used in my experiment, were not fed with gammarids or any commercial food in order to remove cues stemming from conspecific injuries, residual food and predator faces (Wisenden et al., 1999; Ward, 2012; Smith and Webster, 2015). It is likely that the signal produced by food is only one of many cues that predator relies upon.

Invasive species often show greater behavioural plasticity compared with native species in order to increase their ability to evade predation (Guo et al., 2019). For example, invasive species may be better at predator recognition. The ability of invasive species to avoid predators has been demonstrated in several previous studies, for example invasive gammarids reduce their time spent in open water in the presence of fish cues (Jermacz and Kobak, 2017). My results (Chapter 3) revealed that uninfected invasive gammarids compared to native gammarids on the Paderborn plateau limit their movements to the upstream area where the efficient predators prevail (e.g. sculpin, Cottus gobio and trout, Salmo trutta (Abjornsson et al., 2000)). The native G. pulex is an active and explorative amphipod (Truhlar and Aldridge, 2015). G. pulex shows local adaptation in the presence of the non-efficient predator. G. pulex lives in shallow muddy streams, where low water depth and dense benthic detritus may reduce the risk of detection by fish (Haddaway et al., 2014).

2-2. High drifting behaviour in invasive species.

My study detected higher levels of negative rheotaxis (drifting) in invasive gammarids compared to native gammarids (chapter 3). Active drifting is the primary anti-predation mechanism (Naman et al., 2016). Individuals that passively drift constitute the primary prey for drift-foraging predators, such as salmonids (Nielsen, 1992). G. pulex, is an abundant amphipod in the lower (Piscart et al., 2010) and middle parts (Karaman and Pinkster, 1977; Siegismund and Müller, 1991) of small streams with slow flow or standing waters (Verberk et al., 2018). G. pulex has evolved living in shallow muddy streams (Haddaway et al., 2014) and drought area’s (Meyer et al., 2004) where there is low water depth. It is expected that G. pulex use both positive and negative rheotaxis for leaving the areas with high degree of drought. G. pulex use mechanisms such as recruitment with flood (negative rheotaxis) in rainy seasons to compensate their lost population in dry area. Positive rheotaxis is necessary for G. fossarum to compensate their drift ed population from the upper parts of streams or in the mountainous areas (Scheepmaker and van Dalfsen, 1989; Siegismund and Müller, 1991; Müller 2000; Pöckl et al., 2003). Rheotaxis may enable invasive gammarids to avoid severe temporary changes in environmental conditions thereby facilitate the response to a wide range of environmental stressors, such as predation, pollution and erosional habitats (Meijering, 1991; Marvier et al., 2004; Hänfling et al., 2011; Macneil and Dick, 2014). The invasive species may leave the areas with high environmental stressors and search for a new foraging area with fewer environmental stressors.

Ecologists have divided rheotaxis into two categories: (1) active rheotaxis; behavioural and continuous as a form of active patch selection when gammarids’ predation risk is high and food availability is low (Naman et al., 2016), (2) passive rheotaxis; constant and catastrophic as a means of passive response to high (hydropeaking) flow conditions, when macro invertebrates, i.e. gammarids, cannot hold on to the bottom substrate as a result of floods (Bruno et al., 2016). I conducted my experiment without presence of predators or food in order to assess the effects of water velocity on rheotaxis, only. The water velocity in a circulatory canal was kept at a rate of ca. 25 cm per second, which is similar to the water velocity at the sampling site. I detected higher levels of negative rheotaxis in invasive E. berilloni compared to native gammarids. However, my experimental setup was unsuitable to discern between active or passive rheotaxis. I recommend another experiment with different water velocities to further gain insights into active or passive rheotaxis. Rheotaxis is necessary for gammarids reproduction and community structure during mating seasons during the late spring and summer. The middle part of the Paderborn Plateau was almost completely dry during the summer. Such a severe drought season could lead to high drift of E. berilloni.

E. berilloni could increase the connectivity between downstream to upstream con-specific populations via rheotaxis.

2-3. Lower survival of freshwater gammarids in saline waters

Survival rates are decreased in freshwater amphipods when osmoregulation is disturbed and the hemolymph osmolality increases above a critical point (Hart et al., 1991). Survival, locomotion and ventilation are all impacted by salinity stress in gammarids (Sronom et al., 2010). As expected, my study showed that increasing salinity had an adverse effect on the survival rates in G. pulex and G. fossarum (Chapter 4). Most gammarids are hyper-regulators of body ion concentrations at low salinity levels (Piscart et al., 2007). The lowered ability of freshwater gammarids to regulate chloride, and the decreasing intra- and extracellular free amino acids, resulted in a lower survival rate in gammarids at elevated salinity (Sutcliffe, 1971; Jordan et al., 1999; Patrick and Bradley, 2000; Pickens et al., 2019). It has also been suggested that salinity tolerance may differ with sex and age due to difference in metabolic rates (Schmidt-Nielsen, 1984).

Haemolymph volume regulation in invertebrate animals depends on the balance between water uptake via osmotic water influx, drinking, and loss via urination. For example, in the crab Cancer borealis, if the haemolymph volume during moulting rises, the drinking rate decreases and urination rate increases to compensate ion exchange (Greco et al., 1986). Although invasive gammarids have a wider salinity tolerance compared to native gammarids (Holopainen et al., 2016), it is currently unknown whether the tolerance of the invasive E. berilloni is any different from native G. pulex and G. fossarum. However, it is possible that invasive species could regulate their haemolymph at a higher salinity compared to native species.

2-4. Realized niches and survival of native gammarid species

My findings revealed no significant differences in salinity tolerance between the native gammarids sampled on the Paderborn Plateau (chapter 4). The ecological similar salinity tolerance in the two native species G. pulex and G. fossarum, which overlap in their realized niches resulted in an increased likelihood of competitive exclusion (Cardoso et al., 2018). It is clear that there are fitness differences among the two native species during drought seasons and invasion by E. berilloni (Meyer et al., 2004). The drought seasons is observed during summer and late spring. The salinity levels during a drought are far below the salinity during our experiments.There is overlap in the realized niches of G. pulex and E. berilloni ( see below; Meyer et al., 2004). Both native gammarids have equal salinity tolerance, but G. pulex occurs in high salty and dried out areas and G. fossarum not. The competitive exclusion by G. fossarum appeared to increase their fitness and abundance. G. fossarum seems to be better able to cope with changing environments: (i) my findings showed increasing salinity tolerance in G. fossarum and not in G. pulex; (ii) parental exposure induce transgenerational plasticity in G. fossarum and better acclimation to contaminants (Vigneron et al., 2019).

Several previous studies have shown an effect of temperature and salinity on the distribution of organisms in aquatic ecosystems (Einarson, 1993; Neuparth et al., 2002; Delgado et al., 2011). The drought season may change the number of available niches to both cold-adapted, native co-occurring species, and may limit the protrusion of warm-adapted invasive species. More research about genome wide data is needed, in order to detect possible signs of selective sweeps between two populations, both in native and invasive, in temporary and permanent

streams of the Paderborn plateau and could provide precise clarification about salinity tolerance of gammarids with different temperature preferences.

2- 5. Invasive species win anti-predator behavior: Competitive, but not comprehensive, exclusion

Some invasive species completely replace native species through competitive exclusion (Mooney and Cleland, 2001). My findings showed that invasive species compared to native species reduce their locomotion activity in the presence of non-efficient predators (i.e. three spined sticklebacks), resulting in a lowered predation risk (Chapter 2). Higher predation rates of native gammarids by three spined sticklebacks compared to invasive species on the Paderborn Plateau provides advantages for invasive E. berilloni from sympatric occurrence with native gammarid species, especially when refuge options, such as leaf disks, are available (Schmidt-Drewello et al., 2016). These two findings show competitive exclusion between invasive E. berilloni and native G. pulex and G. fossarum as a consequence of invasive species’ success. It is difficult to draw exact conclusions about the impacts of invasive species on native species either through direct (i.e. resource) or indirect (i.e. apparent) competition. Indirect competition is, for example, when an invasive species, as novel prey species, enhances growth rates of the predator population. Native gammarids are more heavily predated upon large predators relative to invasive species. This indirect competition could be more harmful to native species than direct competition (Noonburg and Byers, 2005). In my study system, home ranges differed among species: E. berilloni was more prevalent down streams than native species (Chapter 3). Nevertheless, Meyer et al. (2004) has demonstrated that both native and invasive gammarids on Paderborn Plateau are well adapted to share the same environment in the middle stream (i.e. the drought regions) despite similar ecological traits. It seems that the coexistence between native and invasive gammarids could be achieved through a combination of competition and habitat specialization (Baumart et al., 2015). E. berilloni was reported on Paderborn Plateau for the first time in 1925 (Schellenberg, 1925; Boecker, 1926). It could be during the initial phase of an invasion by E. berilloni in this ecosystem. Native species may share habitat with invasive species during the initial phases of an invasions, and eventually be partly of fully replaced by the invasive species. Conservation strategies and environmentally friendly cultured fish- domestic water fowls farms in Paderborn plateau (Westphalia, Germany)

In heterogeneous and temporarily highly variable environments, such as the Paderborn Plateau waterways, invasive species have higher fitness (Luo et al., 2019b) and a wider range of phenotypic plasticity compared to native species (Agrawal, 2001; Davidson et al., 2011; Szabó et al., 2018). The potential role of invasive species in providing ecosystem services will increase in a scenario of climate change. Furthermore, increasing temperatures that are due to global warming could facilitate the invasion and establishment of invasive species originating from warmer areas, e.g. E. berilloni originate from Spain and France (Montserrat et al., 2013). The probability of successful invasion is positively related to the number of introductions, and high genetic diversity of invasive species. An invasive species with high levels of genetic diversity is presumed able to adapt rapidly to environmental change (Riis et al., 2010). Conservation strategies can focus on preventing the establishment of invasive species in new locations, or reduce the expansion of already present invasive species to new locations (Drury and Rothlisberger, 2008; Stewart-Koster et al., 2015). However, there are contrasting views in

regards to the threats that invasive species have on biodiversity (Douglas et al., 1990; Wilson and Pärtel, 2003; Renne et al., 2006). For example, invasive perennial grass Agropyron cristatum (crested wheatgrass) would enhance biodiversity and increase belowground carbon storage in the northern Great Plains in USA and Canada (Wilson and Pärtel, 2003). One point of view is that biological invasions should be understood as a socio-ecological phenomenon (Carballo-Cárdenas, 2015). Negative consequences of invasive species for the future of native habitats are biodiversity loss (Perrings, 2002) or altering the structure and functioning of ecosystems (Hooper et al., 2005). In some instances, invasive species can also have positive effects on an ecosystem, e.g. invasive species can provide shelter and habitats for native species (Wonham et al., 2005). Invasive species can provide prey for endangered predators (Tablado et al., 2010). Invasive species act as catalysts for ecosystem restoration (Ewel and Putz, 2004) or as biofilters (Elliott et al., 2008).

Waterfowl migrate over large distances compared to other vertebrates (Clausen et al., 2002) acting as long-range vectors of parasite dispersal (Clausen et al., 2002). Water fowl act as a key dispersal vector for spreading two populations of native (upstream) and invasive (middle and downstream) P. minutus in Paderborn Plateau.

The karst streams, e.g. upper catchment of the Lippe in the Paderborn Plateau, include high relevant stress factors for aquatic organisms, such as inflow and seasonal droughts (Meyer et al., 2004). Drought will cause desiccation of aquatic organisms. The fitness consequences of stress factors for aquatic organisms in karst streams (e.g. reduced immune system, growth and mortality due to temperature, osmotic and hypoxic stress, losing the habitat due to hydraulic stress, disease due to bacteria, viruses, fungi and parasites as biotic stress) do not provide a suitable system for movement of gammarids, but mechanisms like rheotaxis and local and regional diversity contribute to the gammarids population connectivity, and this makes it a suitable habitat for them (Schofield et al., 2018).

P. minutus does not pose a threat to cultured fish, a non-host predator, but P. minutus parasite has been shown to result in septicemia in the intestines of domestic water fowl (Crompton and Harrison, 1965). Parasite ecology could help reduce the anthropogenic impact with a strategy similar to the conservation strategy on the Paderborn Plateau, e.g. connecting water resources through channelization where less waterfowl and parasites are present. Control of acanthocephaliasis in waterfowl farms, e.g. not having poultry activity near the P. minutus area, is necessary. P. minutus has been detected in poultry, which have been kept with ducks (Lingard and Crompton, 1972). Waste from poultry and domestic waterfowl into ecosystems housing wild population have shown to cause a high prevalence of acanthocephaliasis in wild waterfowl (Lingard and Crompton, 1972). One strategy could be to farm non-host fish species thereby decreasing the incidence of P. minutus in farmed water fowl. Considering the predation of farmed fish species and the gammarid species used for live feed will be beneficial to aquaculture. Use of behavioural traits of gammarids in aquaculture (e.g. rheotaxis and avoidance from a non-host predator), which could have economic and ecological benefits for aquaculture activities, could improve methods of feeding technology by live foods, e.g. gammarids. For example, high negative rheotaxis of invasive gammarids makes them a suitable live feed in non-intensive fish farming using -drift feeding fish, such as Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss, that feed facing the water flow. A reduced anti-predatory avoidance in native gammarids makes them more suitable as live feed in circular ponds used for intensive fish farming (e.g. salmon fish farms). Using live feed could reduce the negative environmental effects associated with feed pellets. In addition, increasing the temperature and salinity would decrease acanthocephaliasis in

farmed fish and hence reduce the need for chemical or antibiotic treatments. My findings should be considered in the development of environmentally friendly aquaculture and poultry systems in the future. Constructing artificial, small salt water ponds near water fowl freshwater habitats could reduce ectoparasite load as well as the occurrence of intermediate hosts that attach to waterfowl feathers. Salt water ponds also negatively affect P. minutus access to intermediate hosts.

Conclusions and recommendations for future research

Assessing the effect of non-host predators on avoidance behaviour of intermediate hosts showed that the type of predator (i.e. efficient and non-efficient or definitive and non- definitive predator) may play an important role in the regulation of native and invasive amphipod populations via parasite populations (Abjornsson et al., 2000; Baldauf et al., 2007; Jacquin et al., 2014; Szokoli et al., 2015b). The parasite P. minutus studied here has co- evolved with sympatric native gammarids, and not with invasive gammarids, which has resulted to a system to promote avoidance behaviour in native gammarids towards non-host predators. Furthermore, we found that the effects of infection in rheotaxis in native and invasive species is not different and there was no differences in salinity tolerance between infected native gammarids (Chapters 3 and 4). It is important to note, however that, the interaction of the parasite with non-host predators, co-inhabiting the same ecosystems, will influence infection dynamics in final hosts (Roberts and Heesterbeek, 2013).

Parasite populations are regulated by predation. My results revealed that the proximate causes of the success or failure of infected intermediate hosts on anti-predatory behaviour (Chapter 2). These proximate causes seem to point towards effects on the regulation of host predator populations through ultimate cause: increased sympatric intermediate hosts’ ability by parasites. The invasive, intermediate host also affects the availability of native prey and predators. It has been estimated that 2.5% of the parasite population in intermediate hosts with manipulated behaviour are transmitted successfully, whereas 17.1% are lost to non-host or paratenic host predators (Mouritsen and Poulin, 2003). Stomach content analyses of waterfowl in future studies could improve our knowledge of digested or not digested parasites and the relative preferences for invasive and native gammarids. Such analyses could confirm whether parasite-induced trophic transmission is successful.

The lower susceptibility to non-final host predators and increased negative rheotaxis behaviour of invasive species could lead to occupation of the whole ecological niche by invasive parasite and intermediate host species due to the competitive exclusion. Predicting invasion success without any consideration of host-parasite co-evolution is impossible. I must emphasize that invasion biology complex and highly context-dependent making generalizations and hence predictability difficult (Elliott-Graves, 2016). According to Daehler (2003), invasive species exhibit a higher degree of phenotypic plasticity compared to native species occupying the same niche. The elevated phenotypical plasticity of invasive species must be incorporated into predictive models of invasive species distribution (Clements and Ditommaso, 2011). Also, the evolutionary potential of invasive species should be considered in future research. Future studies, therefore, appear to be essential for a better prediction of the long-term consequences of biological invasions for restoration and conservation of the waterways on worldwide Plateau. Further sampling in different seasons as well as laboratory experiments of predators’ stomach would be required to describe the dietary preferences of the final host predators and non-host predators in more detail. I recommend future studies to

focus on final host behaviour in relation to infected intermediate hosts and their migration patterns as a consequence of global warming. The increased water temperatures due to global warming may, in turn, affect infected intermediate hosts’ temperature tolerance and increase acanthocephaliasis in fish or domestic waterfowl farm. Consequently, the need for chemical or antibiotic treatments is likely to increase.

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