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Lipid content of the European eel (Anguilla anguilla) when infected with the swim bladder nematode Anguillicoloides crassus

Laura Hinchliff (Queens University Belfast)

Supervisors Dr. D. Evans, Dr. D. Roberts

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A project supported by the European Union's INTERREG IVA Programme managed by the Special EU Programmes Body!

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Lipid content analysis of the European eel (Anguilla anguilla) when infected with the swim bladder nematode Anguillicoloides crassus
Laura Hinchliff 40007698

Submitted as partial completion of MSc in Sustainable Aquaculture and Inshore Fisheries

Submitted: 9th October 2012

Supervisors: Dr. Derek Evans (AFBI), Dr. Dai Roberts (QUB)

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NON-TECHNICAL SUMMARY
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LIPID CONTENT ANALYSIS OF THE EUROPEAN EEL (ANGUILLA ANGUILLA) WHEN INFECTED WITH THE SWIM BLADDER NEMATODE ANGUILLICOLOIDES CRASSUS
To inform management strategies for the European eel

BACKGROUND
The reproductive migration of European eels is largely dependent on adequate energy reserves stored as lipid. Factors that may influence the lipid content are therefore crucial to understand in order to assist the development of future management strategies. Anguillicoloides crassus, an invasive parasite native to the Japanese eel, has become problematic in Northern Ireland waters since its first discovery in Lough Erne in 1998. The strain of infection with A. crassus may result in a negative effect on the lipid content of eels. This report aimed to ascertain the extent of which A. crassus may hinder the reproductive migration of the European eel by assessing correlations between lipid content and parasitic infection between two eel fishery sites in Northern Ireland. This information may be used to identify key areas for the development of management strategies for the European eel. ! MAIN FINDINGS The lipid content of eels was positively influenced by length and mass, indicating that larger, heavier eels had a higher lipid content that smaller eels. Eels with a high infection intensity of A. crassus had a significantly lower lipid content in Lough Neagh but not in Lough Erne. A. crassus may result in a decrease in lipid content of eels due to A. anguilla having to exert more energy to function normally or A. crassus utilizes lipid from A. anguilla for their own gain. A. crassus generally do not kill their host as it is more beneficial for the host to remain alive.

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ABSTRACT

The swim bladder nematode Anguillicoloides crassus, an invasive parasite endemic to the Japanese eel (Anguilla japonica) has become a serious problem in A. anguilla fisheries since its first arrival to Europe in Germany and Italy in 1982. A. crassus causes a multitude of problems to A. anguilla such as intestinal lesions, alteration to the swim bladder wall and disrupting other organs in the process. The strain of hosting a sanguivorous parasite may result in depletion of energy reserves such as glucose and lipids. Lipid content analysis was performed on female silver eels from two sites in Northern Ireland (Lough Neagh and Lough Erne) and compared against standard morphometrics to ascertain the effects A. crassus may induce on A. anguilla prior to migration. The lipid content of eels was positively influenced by body length and mass as larger, heavier eels had a higher lipid content than smaller eels. Results showed that the differences in lipid content in Lough Neagh and Lough Erne could be attributed to differences in length between the two sites. This indicates that body length may be a key factor in influencing the effect that parasites have on lipid. Eels with a higher infection intensity of A. crassus were found to have a lower lipid content; however this result was only significant in Lough Neagh. The differences seen between the two sites may be explained by differences in fish communities and distribution of the intermediate and paratenic hosts in the two sites. Though results are indicative of A. crassus having an effect on the lipid content of A. anguilla, low sample sizes means it remains uncertain.

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ACKNOWLEDGEMENTS
I am grateful to those involved in the collection of eels from Lough Neagh and Lough Erne for providing samples for this research. Thanks to Richard McQuillan for laboratory assistance in the Medical Biology Center at Queens University of Belfast. I would also like to thank fellow MSc student Colleen Massey, whose assistance throughout this research was most welcome both for eel dissections and emotional support! Thanks to Integrated Aquatic Resources Management Between Ireland, Northern Ireland and Scotland (IBIS) for part-funding this research. I would like to express my gratitude to my supervisors: Dr. Derek Evans and Dr. Dai Roberts for the support that they provided. Finally, I would like to express sincere thanks to Team Eel at AFBI to Kenny Bodles (his replies to my endless emails were most helpful) and Dr. Derek Evans for providing me with facilities at AFBI, motivation and support which enabled completion of this project.

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TABLE OF CONTENTS 1! INTRODUCTION!.......................................................................................................!1! 1.1! UK eel fisheries!.............................................................................................................!1! 1.2! Northern Ireland eel fisheries!.....................................................................................!1! 1.2.1! Lough Neagh eel fishery!...............................................................................................!1! 1.2.2! Lough Erne eel fishery!..................................................................................................!2! 1.3! Status of the European eel!..........................................................................................!2! 1.3.1! Overfishing!......................................................................................................................!3! 1.3.2! Climate change!..............................................................................................................!3! 1.3.3! Parasitic infections and disease! ...................................................................................!4! 1.4! Anguillicoloides crassus!.............................................................................................!5! 1.5! Anguilla anguilla life history and spawning migrations!.........................................!6! 1.5.1! Spawning migration!.......................................................................................................!6! 1.5.2! Energetic reserves for migration!..................................................................................!7! 2! AIMS AND OBJECTIVES! .........................................................................................!7! 3! MATERIALS AND METHODS!.................................................................................!8! 3.1! Choice of study eels!.....................................................................................................!8! 3.2! Processing of eels!........................................................................................................!9! 3.3! Lipid content analysis!..................................................................................................!9! 3.4! Statistical analysis! .......................................................................................................! 10! 4! RESULTS!................................................................................................................!10! 4.1! Status of eels!................................................................................................................! 10! 4.2! Comparison between lipid and standard morphometrics: differences of length, mass and condition between sites!.....................................................................................! 11! 4.3! Comparison between lipid content and A. crassus: parasitic effect on lipid content between sites!..........................................................................................................! 11! 5! DISCUSSION! ...........................................................................................................!15! 5.1! Parasites and fisheries!...............................................................................................! 15! 5.2! Standard morphometrics: relationship with lipid content and effect with A. crassus infection! ...................................................................................................................! 15! 5.3! Infection with A. crassus and effect on lipid content!............................................! 16! 5.4! Infection with A. crassus between study sites!.......................................................! 17! 5.5! Conclusions!..................................................................................................................! 18! 6! REFERENCES!........................................................................................................!19! 7! COPYRIGHT! ............................................................................................................!26!
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LIST OF FIGURES AND TABLES

Page number Fig. 1. Fig. 2. Table 1. Life cycle of A. anguilla Sites of study eels The distribution of A. crassus infections in Lough Neagh and Lough Erne and lipid content of A. anguilla Fig. 3a. Plot of the lipid content (as a % of body mass) against body length (log) Fig. 3b. Plot of the lipid content (as a % of body mass) against body mass (log) Fig. 3c. Plot of the lipid content (as a % of body mass) against condition of eel Fig. 4a. Number of individual A. crassus per eel in Lough Neagh and Lough Erne Fig. 4b. Lipid content (as a % of body mass) in Lough Neagh and Lough Erne Fig. 5a. Lipid content (as a % of body mass) and the number of A. crassus Fig. 5b. Lipid content (as a % of body mass) and the number of A. crassus as infection intensity

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1
1.1

INTRODUCTION
UK eel fisheries The European eel (Anguilla anguilla L.) occurs widely throughout rivers, lakes and estuaries

in the UK. Several different types of fishery exist for this species as various life stages are exploited: (1) the elver or glass eel fishery, which captures young eels as they return to rivers. The Severn estuary between England and Wales boasts this commercial fishery, however glass eel fishing is illegal in Northern Ireland; (2) yellow eel fisheries exist across the UK, for example the Thames; (3) silver eel fisheries, for example Lough Neagh in Northern Ireland (Environment Agency 2009). Within England and Wales, eel fisheries are managed by The Environment Agency, and more stringent regulations regarding eel fishing are controlled by the National Eel Fishing Byelaws which were introduced in 2004 (Walker et al 2011). The only commercial source of eel in the UK is through these fisheries as there are no recreational fisheries or aquaculture of eel (Walker et al 2011). The majority of the UK Eel Management Plans (EMPs), which were set up in an attempt to aid recovery of the species (see section 1.3) have been set up at the River Basin District (RBD) level. England and Wales have ten EMPs and Scotland has one, with England, Wales and Scotland sharing a further EMP. The RBDs in Northern Ireland, however, are slightly different as it is a transboundary plan with the Republic of Ireland. Northern Ireland has three EMPs, one of which is shared with the Republic of Ireland (Walker et al 2011). 1.2 1.2.1 Northern Ireland eel fisheries Lough Neagh eel fishery Lough Neagh sustains the largest commercial wild eel fishery remaining in Europe which is managed by the Lough Neagh Fishermens Co-operative Society (LNFCS), to whom all fishing rights of eels in Lough Neagh belong (Walker et al 2011). The LNFCS has been in control of the eel fishery since the 1960s (Rosell et al 2005), and since then elvers (immature eel) have been trapped at the mouth of the River Bann and released into Lough Neagh, a strategy which was also carried out between 1932 1947 (Parsons et al 1977). The elver crash in the 1980s was widespread throughout Europe, prompting the LNFCS to also supplement natural glass eel recruits that immigrate up the lower River Bann by supplying the Lough with glass eels from a UK distributor (Allen et al 2006) from the Severn estuary fishery. Control of the eel fishery in Lough Neagh by the LNFCS enables the fishery to be managed in the interests of the fishermen and their families (DEFRA 2010a). There are two active fisheries in Lough Neagh; the yellow eel fishery, which are fished for during the summer months by long line and draft nets, and the silver eel fishery, which is active during the autumn and winter, and are caught using coghill nets (DEFRA 2010a). Approximately 250 350 tonnes of yellow eel and 75 100 tonnes of silver eel are caught annually (Walker et al 2011). 1! ! !

For both fisheries, restrictions put in place such as catch quotas, boat size restrictions, licensing schemes, minimum eel landing sizes and active fishing seasons permit sustainable management (DEFRA 2010a). 1.2.2 Lough Erne eel fishery The Erne Eel enhancement programme was a major European funded cross-border programme, set up in 1997 by the Northern Regional Fisheries Board in an attempt to enhance recruitment of eel to the population, allowing further exploitation and to develop a management plan for the future of the fishery (Maguire and Gibson 2005). Despite attempts to ensure sustainability, the closure of the Lough Erne eel fishery in 2010 due to non-compliance with EU legislation resulted in commercial eel fishing being discontinued in the Erne catchment. Previously, yellow eels were captured in Lough Erne by long-line fishing and fyke nets, with about 35 licenced fishermen in the 1980s. Silver eels were caught using stow nets in the rivers (Moriarty 1996) though the commercial silver eel fishery closed in 2001 (Walker et al 2011). The development of hydroelectric power stations in the lower River Erne in the 1950s caused the upstream migration route of glass eels and elvers to be blocked (DEFRA 2010b).Due to these obstacles, in 1962 the high mortality rate of elvers and yellow eels led to the capturing of eels in the lower river and transporting them to upper waters where the habitat is richer, increasing survival and marketable value. Similar strategies have been employed in Germany and Lough Neagh (Moriarty 1999). 1.3 Status of the European eel The status of A. anguilla has been under intense scrutiny during the last 30 years from both fishermen and scientists (Moriarty and Dekker 1997) due to declining fishery yields and awareness of poor health status. It has been apparent throughout Europe since the 1980s that A. anguilla populations have been diminishing (Belpaire et al 2009) with a 90% decline in recruitment (ICES 2002), and in 2010 was declared Critically Endangered by the IUCN (Freyhof and Kottelat 2010). Conservation efforts have been made in recognition of these issues, with the European Commission establishing legislation requiring all countries with A. anguilla habitats to produce Eel Management Plans to aid recovery (Bilotta et al 2011). In 2007, the Joint EIFAC/ICES Working Group on Eels initiated the set up of the European Eel Quality Database to collect data regarding disease and contaminants in the distribution area of A. anguilla, allowing eel quality on a European scale to be analysed (Belpaire et al 2011). It has been difficult to pinpoint the specific cause(s) for eel population decline, largely due to their catadromous life history which has prevented assessment of adults during their spawning migration in the open ocean. Lack of information available makes it difficult to identify sources of population decline (Dekker et al 2003). Overfishing, sensitivity to climate change, parasitic infections 2! ! !

(Wirth and Bernathchez 2003), disease (Belpaire et al 2011), habitat loss (Feunteun 2002) and an insufficient biomass of spawners (Dekker et al 2003) have all been considered as reasons for negative changes in eel populations. Despite some fisheries throughout Europe attempting to restore populations by stocking of elvers and glass eels, no significant impact has yet been made (Feunteun 2002). 1.3.1 Overfishing A. anguilla is commercially exploited at various life stages (Feunteun 2002) to unsustainable levels (strm and Dekker 2007). Lough Neagh alone produced 16% of total EU landings for eel in 2007 (Walker et al 2011). An extremely low recruitment rate has resulted in a declining population as older eels are removed from the stock (Freyhof and Kottelat 2010) causing a decline in European catches (Pedersen and Dieperink 2000). This outcome is not restricted to Europe, but is being observed in other eel populations worldwide (Haenen et al 2010). Populations of the American eel (Anguilla rostrata) have seen an 81-fold decline in numbers from 1985 1992 (Castonguay et al 1994). Habitat studies of shortfinned eels (Anguilla australis) in New Zealand during the late 1990s also revealed low recruitment rates (Jellyman and Chisnall 1999). The development of Eel Management Plans have aimed to overcome the effects of overexploitation by issuing a 40% escapement target of pristine production (EIFAAC/ICES WGEEL Report 2011); that is, allowing 40% of the adult eel biomass to escape to the sea that would have occurred in the absence of human interference (Aprahamian et al 2007, Bevacqua et al 2007) resulting in a positive effect on recruitment (Bilotta et al 2011). Contrasting results have been seen from different European regions. A Mediterranean lagoon in France show an optimistic 76.8% escapement rate from the local fishery (Charrier et al 2012), whereas the North Western International River Basin District (NWIRBD) show escapement rates of approximately 26%, with it seemingly unlikely that management measures will enable long term 40% escapement rates to be feasible (DEFRA 2010a). 1.3.2 Climate change Particularly within the last decade, climate change has been considered as a source of A. anguilla population decline. The spawning areas of silver eels in the Sargasso Sea may be affected by ocean-atmospheric changes, having a negative effect on leptocephali (larval) survival (Friedland et al 2007) both in the Sargasso Sea and during migration in the North Atlantic (Bonhommeau et al 2008b). By inhibiting spring thermocline mixing, the food source for leptocephalus larvae is affected by a negative impact on productivity (Knights 2003). Similar findings among declining glass eel populations have blamed increased oceanic temperature due to climate change and the resultant decrease in primary productivity for the declines in populations (Bonhommeau et al 2008b). 3! ! !

FAO landings and glass eel catches both show interannual fluctuations consistent with the North Atlantic Oscillation (NAO) (Kettle et al 2008), which is the major climate-driving signal in the North Atlantic (Friedland et al 2007). A significant negative correlation between glass eel catches and the NAO (Friedland et al 2007, Kettle et al 2008), reinforcing the belief that the positive NAO phase has an adverse influence on larval survival and migration from the Sargasso Sea to the continental shelf (Kettle et al 2008). Oceanic and climate change has also been to blame for declines in recruitment of A. rostrata, and the Japanese eel, Anguilla japonica (L.) (Knights 2003). In the late 1970s, a regime shift in temperature was detected which was followed by an alteration in glass eel recruitment for all three eel species, concluding that climate-driven processes are involved in the synchronous decline in eel populations worldwide (Bonhommeau et al 2008a). 1.3.3 Parasitic infections and disease The health status of eel populations has been of major concern globally. Eels may be inflicted with ailments such as cauliflower disease, freshwater eel disease and saltwater eel disease (Tesch 1977) which may lead to economic consequences by poor quality eels being landed. Poor health may decrease the chances of A. anguilla reaching their spawning grounds (Palstra et al 2007) during migration, reducing recruitment to the population. As eels have a complex catadromous life cycle and inhabit both estuarine and freshwater habitats during their growth phase (Dekker et al 2003) they are at risk from a variety of parasites and pathogens. Eels and other fishes are ideal hosts to parasites for many reasons; most fish are predatory, which encourages opportunistic infections by ingesting infected prey (intermediate host) such as molluscs and crustaceans. Fish are highly mobile, and often migratory; therefore they are exposed to a wide array of infections which can infect a diverse range of species. Farmed fish in particular are vulnerable to parasite transmission; exposure to environmental fluctuations, overcrowding and stressful conditions put these fish at greater risk to infection than wild fish. In particular, salmon louse (Lepeoptheirus salmonis) is a serious pest in marine aquaculture, affecting survivorship of farmed Atlantic salmon and also negatively impacting wild salmon populations (Gargan et al 2012). With regards to A. anguilla, the swim bladder nematode Anguillcoloides crassus is under particular scrutiny, first reported in Europe in 1982 and spreading rapidly throughout (Peters and Hartmann 1986). A. crassus reached Britain by 1987 (Kennedy and Fitch 1990) with the first documented record in Ireland in 1998 in the Erne catchment (Evans and Matthews 1999); by 2003, A. crassus had spread to Lough Neagh (EIFAAC/ICES WGEEL Report 2011). Within two years, 100% of both yellow and silver eels were infected with A. crassus; however infection levels fell to 68% by 2010 (DEFRA 2010a). A. crassus is now considered to be the most abundant helminth parasite of A. anguilla (Sures et al 1999). 4! ! !

1.4

Anguillicoloides crassus A. crassus is a parasitic swim bladder nematode endemic to the Japanese eel, A. japonica, in

East Asia (Moravec and Taraschewski 1988, Kelly et al 2000). This invasive parasite has spread rapidly throughout Europe, likely due to the live transport of eels (Koops and Hartmann 1989). Introduction to Britain is thought to have occurred through lorry water changes during transport of eels to continental Europe (Kennedy and Fitch 1990). Research suggests that the rapid successful spread of A. crassus may be due to its occupation of the swim bladder, a niche that native parasites do not inhabit (Kirk et al 2002, Martnez-Carrasco et al 2011). High reproductive potential and an ability to remain infectious after being maintained in 100% seawater (Kennedy and Fitch 1990, Kirk et al 2000) due to osmoconformation with A. anguilla blood plasma (Kirk et al 2002) indicate the strong colonization potential of A. crassus. Since the spread of A. crassus to Europe it has been observed that A. anguilla are more susceptible to infection, which has not infected A. japonica populations to the same extent (Kie 1991). The lack of pathological damage caused to the native host lies with its ability to encapsulate and kill A. crassus in the swim bladder wall (Knopf and Mahnke 2004). These effective defensive mechanisms are the result of a long period of co-evolution. The transmission of A. crassus from its native host to A. anguilla and A. rostrata has led to mortalities in populations by impairing normal swim bladder functioning combined with other stressors (ICES 2002, Kirk 2003). A. crassus larvae are ingested by the intermediate host (Cyclopoida), or by fish paratenic hosts (Kirk 2003) which are then consumed by A. anguilla. The larvae migrate through the intestinal wall to the swim bladder where they complete their life cycle and develop into adults (Peters and Hartmann 1986, De Charleroy 1990), forming intestinal lesions (Haenen et al 1989), altering the swim bladder wall (Wrtz et al 1996) and causing disruption to other organs in the process (van Banning and Haenen 1990); injury caused to the eel may be fatal (Molnr 1993). As an invasive species, A. crassus causes such damage to the non-native species (A. anguilla and A. rostrata) due to a lack of immunity and defensive mechanisms (Peeler et al 2011). Severe effects of anguillicolosis in A. anguilla populations have been documented; Lake Balaton, Hungary in 1991 experienced mass mortalities of eels infected with A. crassus but no other fish species were affected (Csaba et al 1993), and infected individuals showed no other signs of pathological changes, leading to the belief that parasitic infection was the sole cause of mortality (Molnr et al 1991). Infection with A. crassus may make eels more vulnerable to other diseases and induce stress responses in larval and young adult eels (Sures et al 2001), and may in fact be a greater stress factor than other parasites (Haenen et al 2010). Literature suggests that A. crassus may impair the spawning migration of mature eels (ICES 2002, Mnderle et al 2004) and is thought to be one of the main causes of European eel population collapses, draining the eels of energy, hence impairing spawning migrations (Palstra et al 2007). 5! ! !

1.5

Anguilla anguilla life history and spawning migrations A. anguilla have an intriguing life history in that they have both a continental and oceanic

phase (Fig. 1), subjecting them to a variety of perturbations (Durif et al 2005). Spawning occurs in the late winter and spring (Kleckner and McCleave 1988) in the Sargasso Sea, and the eggs develop into leptocephalus larvae. Post spawning, the leptocephali depart from the Sargasso Sea and are carried by warm currents (Bonhommeau et al 2008b) to the North African and European continental shelf where they metamorphose to glass eels, swim upstream and colonize inland rivers and lakes during their growth phase to yellow eel which may last several years (Durif et al 2005). The maturation of A. anguilla from yellow eel to silver eel, a process called silvering (van Ginneken et al 2007), signifies the preparation for their migration from their native rivers and lakes to the Sargasso Sea to spawn (Lowe 1952) during late Autumn and winter.

Fig. 1 Life cycle of A. anguilla Reference: cefas.defra.gov.uk 1.5.1 Spawning migration The complex life history of the eel has raised many questions about their migratory behaviours, namely the factors which induce their transatlantic spawning migration. In previous years factors such as age and size were thought to be important in inducing migration; more recent literature proposes that the lipid content acts as a key trigger (Larsson et al 1990) as well as being an essential energetic requirement for migration and reproduction (Belpaire et al 2009). During their life stage as a yellow eel, A. anguilla accumulates lipid which enables silver eels to endure long term starvation (Botius and Botius 1985); they do not feed during their spawning migration (Robinet and Feunteun 2002, Pierron et al 2007). The lipids are primarily accumulated during the yellow eel stage of the life cycle and stored in the muscle as triglycerides. Therefore, the quantity of lipid stored by the yellow eel largely determines the successful completion of their life 6! ! !

cycle (Pierron et al 2007). During the yellow eel phase, the juvenile eels greatly increase their food intake from March May (Tesch 1977) which may in turn make them more vulnerable to A. crassus infection as they are more likely to ingest the intermediate host. This may affect the quantity of lipid which they store and thus hinder the silvering process, having the potential to delay their reproductive migration (Tesch 1977). 1.5.2 Energetic reserves for migration The efficiency with which eels accumulate energy reserves for migration is vital as out of the various eel species, A. anguilla travels the farthest to reach spawning grounds (5000 6000 km) (van Ginneken et al 2005). On average, fat reserves of migrating eels hover around 20% (van Ginneken and van den Thillart 2000) though it has been estimated that the amount of lipid stored must exceed 20% in order to cover the metabolic cost of migration (Pierron et al 2007). An estimated 60% of the fat store is left available for the developing gonads at the end of migration (van Ginneken and van den Thillart 2000). Research has concluded that silver eel energy reserves are adequate to cover migration costs and achieve reproduction (Botius and Botius 1980, Pierron et al 2007). However, recent declines in the number of glass eels suggest that this may be due to a lack of available food for juveniles, resulting in diminishing fat stores in adult eels; this in turn may hinder them from reaching spawning grounds (Castonguay et al 1994, van Ginneken and van den Thillart 2000). Previous literature has hypothesized that the metabolic cost of infection with A. crassus may result in an increased glucose turnover (Gollock et al 2005) due to the strain of hosting a sanguivorous parasite, negatively affecting growth and behaviour (Gollock et al 2004). Glucose and lipid are important energy sources; in eels, up to 80% of the energy reserve comes from lipid (Botius and Botius 1985). It may be considered that infection with A. crassus could have an effect on the lipid content of A. anguilla; as a large proportion of the energy reserve in eels is stored as muscle fat, parasitized female silver eels with low fat content would be less likely to recruit to the next generation (Svedng and Wickstrm 1997). The quality of migrating spawners should be such as to reach the Sargasso Sea and produce viable offspring (Belpaire et al 2011), yet the ability of A. crassus to survive in 100% seawater indicates that they may survive the transition from freshwater to saltwater during the spawning migration of A. anguilla (Kirk et al 2000) and thus have the potential to decrease the fitness of silver eels throughout the journey to the Sargasso Sea.

AIMS AND OBJECTIVES

Lipid content is a vital component of the reproductive migration of A. anguilla to the

Sargasso Sea. Potential factors influencing lipid content which may impact on the successful completion of migration and quality of spawners are therefore vital to understand in order to assist the 7! ! !

development of future management strategies. The current lack of data regarding the influence of A. crassus on the lipid content of A. anguilla leaves a gap in our knowledge. The extent of which these parasites may impact upon the strategies which A. anguilla relies for successful continuation of their life cycle is therefore poorly understood. To this aim, the present study examined the lipid content of eels infected with A. crassus and compare this with standard morphometrics such as length and mass to ascertain the effects A. crassus may induce on A. anguilla prior to migration. This information will help to ascertain the extent of which A. crassus may hinder the spawning of A. anguilla as lipid content is a vital component to the successful completion of their reproductive migration (Pierron et al 2007). Correlations between lipid content and infection with A. crassus were examined in two different eel fishery sites in Northern Ireland, and compared with standard morphometrics. It is hypothesized that the length and mass will have no effect on the lipid content of the eel. It is also hypothesized that infection with A. crassus will result in no effect on the lipid content.

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3.1

MATERIALS AND METHODS

Choice of study eels A total of 51 female silver eels were collected by fishermen from Lough Neagh, and 30

female silver eels from Lough Erne in November 2011 (Fig. 2). These sites were chosen as Lough Neagh is the largest remaining commercial wild eel fishery in Europe, hence thorough understanding of parasite-host relationships is important as well as knowledge as to the quality of the eels in this fishery. As a previously active eel fishery, Lough Erne was chosen as a comparative site. Both sites were known to contain A. crassus. The collection period was timed so that female eels would be caught in their silver stage before they began their spawning migration; literature has shown that there appears to be no seasonality of A. crassus in A. anguilla (Thomas and Ollevier 1992a, Kelly et al 2000) enabling these samples to be representative of the general parasite prevalence.

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Fig. 2 Sites of study eels; Lough Neagh highlighted in dashed line, upper and lower Lough Erne highlighted in continuous line. Ref: Google maps 2012 3.2 Processing of eels Eels were collected and stored at -18oC. Prior to processing eels were defrosted at room temperature for 24 hours. Length (mm), weight (g) and sex were recorded. An incision made along the underside towards the pectoral fin allowed access to the swim bladder, at which point the number of A. crassus were assessed macroscopically. A section of muscle tissue posterior to the anus was removed from the left side of the eel and stored in 50ml universal tubes at -18oC. 3.3 Lipid content analysis Muscle samples were left to dry at 55OC for 14 days in labelled glass petri dishes (D:

90mm, H: 5mm). Dried samples were ground using a pestle and mortar, and approximately 1.4g of muscle was folded into filter paper and stapled together. Pieces of plastic were also stapled to the filter paper to differentiate between samples. The weight of the sample in paper was recorded and three samples were fitted into one extractor. The lipid content analysis procedure was adapted from methods as described by Folch et al (1957). 200ml of 1:1 (v/v) chloroform-methanol for homogenizing the tissue was measured into round beakers and placed onto a heater. Water ran continuously through the extractors for 6 hours to separate the lipids from non-lipid substances. Samples were then removed and dried at 55OC overnight to remove any remaining moisture. Samples were then placed in a dessicator for an hour to 9! ! !

allow them to cool in a dry environment without taking on additional moisture, and the final weight (g) was recorded. Results are expressed as a lipid percentage of body mass. 3.4 Statistical analysis Normality of data distribution was established using Kolmogorov-Smirnov tests. For comparison of standard morphometrics (log-transformed body length, log-transformed body mass and condition of eel) against lipid content regressions were plotted and R2 value taken. For comparison of the lipid content of eels between sites, a univariate analysis of covariance (ANCOVA) was performed with body length used as cofactor to remove any size effect. Results that showed p<0.05 were considered statistically significant. All tests were implemented using IBM SPSS Statistics 19 for Windows.

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4.1

RESULTS
Status of eels Eels (n=81) ranged in length between 435 and 699 mm (mean body length Lough Neagh =

568 mm; Lough Erne = 554.9mm) and body mass between 129.8 and 721.4 g (mean body mass Lough Neagh = 391.3g; Lough Erne = 352.1g).

Table 1 The distribution of A. crassus infections in Lough Neagh and Lough Erne and lipid content of A. anguilla Lough Neagh Lough Erne !

Number of eels examined Mean number of A. crassus Range of A. crassus recorded Mean lipid content (%) of body mass !

51 9 0 51 16.8%

30 6 0 - 36 17.4%

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4.2

Comparison between lipid and standard morphometrics: differences of length, The mean lipid content of eels as a percentage of body mass was higher in Lough Erne (Table

mass and condition between sites 1). A positive linear relationship between the lipid content and log-transformed data of eel body length (Lough Neagh R2 = 0.117; Lough Erne R2 = 0.032)(Fig. 3a) and the lipid content and logtransformed data of eel body mass (Lough Neagh R2 = 0.168; Lough Erne R2 = 0.014)(Fig. 3b) occurred at both sites. A positive linear relationship between the lipid content and condition of eel occurred at Lough Neagh (R2 = 0.061) but not at Lough Erne (R2 = 0.014)(Fig. 3c). ANCOVA showed that the differences in lipid content between Lough Neagh and Lough Erne were related to differences in length between the two sites (F= 6.556, df=1, p<0.012). When the effect of length was removed, a weaker significant difference in lipid content between the two sites remained (F=4.863, df=1, p<0.05). 4.3 Comparison between lipid content and A. crassus: parasitic effect on lipid The number of A. crassus found in the swim bladder was variable, ranging from 0 to 51 individual parasites (Fig. 4a). The number of parasites present in the swim bladder were grouped into intensity levels between 0 and 10+ individual A. crassus found. In both sites as the number of parasites present increased, the lipid content decreased (Fig. 4b); however infection intensity with A. crassus had a significant effect on the lipid content of eels from Lough Neagh (r=-0.346, p<0.05) but not in Lough Erne (r=0.072, p>0.05, NS)(Fig. 5b).

content between sites

11! !

Fig. 3a

Fig. 3b

Fig. 3c

Fig 3 Plot of the lipid content (as a % of body mass) against body length (log) (Fig. 3a), body mass (log) (Fig. 3b) and condition of eel (Fig. 3c) for Lough Neagh (blue) and Lough Erne (green). Regression lines show ! the R 2 linear value for both sites. 12! ! ! !

Fig. 4a

Fig. 4b

Fig. 4 Number of individual A. crassus per eel (Fig. 4a) and lipid content (as a % of body mass) (Fig. 4b) in Lough Neagh and Lough Erne. Outliers are indicated by O or *

13! !

Fig. 5a

Fig. 5b

Fig. 5 Lipid content (as a % of body mass) and the relationship between the number of A. crassus (Fig.5a) and the number of A. crassus as infection intensity (Fig. 5b)

14! !

5
5.1

DISCUSSION
Parasites and fisheries The negative effects of parasitism in commercially important fisheries are well documented

throughout the literature, for example sea lice (Lepeophtheirus salmonis and Caligus clemensi) parasitizing pink salmon (Oncorhynchus gorbuscha) and chum salmon (Oncorhynchus keta), and being capable of limiting conservation efforts (Krkoek et al 2005). Parasites are capable of interacting in such ways that they occur in nearly every population (Lafferty and Kuris 1999). As eels have a complex catadromous life cycle and inhabit both estuarine and freshwater habitats during their growth phase (Dekker et al 2003) they are at risk from a variety of parasites and pathogens. Previous research has shown that parasites are capable of affecting the lipid content of their host; Pomphorhynchus laevis, parasite to the amphipod Gammarus pulex, may result in lower lipid contents in gravid females (Plaistow et al 2001). 5.2 Standard morphometrics: relationship with lipid content and effect with A. The lipid content of eels was positively influenced by body length and mass; larger, heavier eels had a higher lipid content than smaller eels (Fig. 3a, 3b). Degani et al (1986) also found that larger eels had a higher percentage of fat than smaller eels. Parasite infection in fishes is usually associated with parameters such as the size of the individual (Poulin 2000). Previous literature has used body length as an indicator of the effect of parasitic infection in A. anguilla (Conneely and McCarthy 1986, Aguilar et al 2005, Lefebvre et al 2011) as it is a reliable measure of body size (Brooks and Dodson 1965), and also correlates to a reduction in volume of the swim bladder, a consequence of infection with A. crassus (Lefebvre et al 2011). Previous research has highlighted the adverse effects that A. crassus exhibit on the length and growth of A. anguilla. Baru and Proke (1999) in the Czech Republic have shown that infection and mortality parameters were dependant on the total length of the eel. Current results showed that the differences in lipid content in Lough Neagh and Lough Erne were related to the differences in length between the two sites. When the effect of length was removed, the lipid content of eels between the two sites still remained significantly different. This indicates that body length may be a key factor in influencing the effect that parasites have on lipid content. Differences in the relationship between infection with A. crassus and eel body length and lipid were observed between the sites; in Lough Neagh, infection intensity with A. crassus was found to have a significant effect on the lipid content. It was also noted in Lough Neagh that as eel body length increased, the number of parasites also increased (Fig.3a). Conversely, in Lough Erne, as eel body length increased, the number of parasites decreased (Fig.3a), however infection intensity did not have a significant effect on the lipid content of eels. Fig.5a shows a strong negative relationship 15! ! !

crassus infection

between lipid content and the number of A. crassus in Lough Neagh but not in Lough Erne. Mean data shows that in Lough Neagh, on average eels were larger, heavier and had higher infection levels of A. crassus, yet the mean lipid content was lower. Thomas and Ollevier (1992b) found a positive correlation between eel body length and the number of A. crassus present, similar to our findings in Lough Neagh. Potentially, this may impact on recruitment as research has shown that A. anguilla reach a critical length, at which point they begin their reproductive migration. This critical length is different between males and females, with female eels generally growing faster than males (Poole and Reynolds 1996). Body length has also been shown to influence the up-stream migration of yellow eels. Larger eels were capable of migrating throughout the seasons regardless of temperature, but the majority of smaller eels were restricted to a shorter migration period (Moriarty 1986). Conversely, Kangur et al (2010) found no apparent relationship between length of A. anguilla and prevalence of infection with A. crassus in Lake Vrtsjrv, Estonia. This is similar to findings of Wrtz et al (1996) who found no relationship between infection intensity and either the length or weight of eels in the River Rhine, Germany. Baru and Prokes (1996) found that A. crassus infection insignificantly affected the weight of A. anguilla. 5.3 Infection with A. crassus and effect on lipid content The current study found an increase in the number of parasites present had a negative effect on lipid content (Fig. 5a). However, infection intensity significantly affected the lipid content in Lough Neagh only; this is discussed in greater detail in section 5.4. The negative effect of A. crassus on lipid content may be explained by A. crassus impairing normal swim bladder functioning in eels, which has been shown to greatly reduce their swimming performance (Palstra et al 2007). The negative effect that A. crassus has on buoyancy and endurance means that A. anguilla may have to exert more energy in an attempt to feed and function normally, resulting in a decreased storing of lipids. Other research has shown A. crassus to have significant physiological influences on A. anguilla; Lefebvre et al (2004) showed how parasite pressure may cause significant increases in spleen size of A. anguilla, a possible immune reaction. A. crassus has been found to affect vital components in the blood of eels, such as decreasing the percentage of red blood cells and quantity of plasma proteins (Boon et al 1990). The consequences of heavy A. crassus infection may be considered similar to those of starvation in A. anguilla, in that components of the blood and liver can be affected; also considering that the sanguivorous feeding of A. crassus and expected negative effect on metabolism (Lefebvre et al 2004) results in a higher glucose turnover (Gollock et al 2005). This can result in the eels utilizing triglycerides as an energy source (Larsson and Lewander 1973) which are normally stored as an energy reserve for their reproductive migration (Pierron et al 2007). 16! ! !

The above reasoning reflects common thought that A. crassus may result in a decrease in lipid content due to factors which results in A. anguilla having to exert more energy to function normally pre-migration. However, it is widespread knowledge that parasites are capable of reallocating resources, such as lipids, from the host for their own needs (Plaistow et al 2001). It may be possible that A. crassus utilize lipid from A. anguilla for their own gain; under favourable natural conditions, A. crassus generally do not kill their host (Kie 1991) as it is more beneficial for their host to remain alive to supply A. crassus with food and habitat. A. crassus is known to infect many life stages of A. anguilla; Nimeth et al (2000) found in glass eels, an early life stage, that oxygen consumption when resting was higher during prolonged infection, a possibly result of compensating for increased body density. The lipid content of eels is known to change seasonally (Hopkirk et al 1975); though some European countries, such as Belgium and the Netherlands, have observed an overall drop in lipid content over the past 20 years (Belpaire et al 2009). Whether this decline in lipid is due to infection with A. crassus has previously been unknown. 5.4 Infection with A. crassus between study sites! When taking the two different sites into consideration, infection intensity with A. crassus was found to significantly influence the lipid content of eels in Lough Neagh but not in Lough Erne. This may be explained by the differences in fish communities and distribution of the intermediate and paratenic hosts in the two sites. Eels become progressively more piscivorous with age and increasing size (Conneely and McCarthy 1986), and as parasite infection levels tend to peak at certain age groups (Woolhouse 1998), these changes in diet combined with different age structures in the two populations may explain the variation seen in the results between Lough Neagh and Lough Erne; also, the two populations may have different fat deposition strategies, which can exist among the same species (Larsson and Lewander 1973). A. crassus was first documented in Lough Erne in 1998 (Evans and Matthews 1999), but there was no record of A. crassus presence in Lough Neagh until 2003 (EIFAAC/ICES WGEEL Report 2011). Parasite intensity levels tend to increase rapidly in a new area and then decline due to densitydependant mechanisms (Kirk 2003). Previous research has shown that once A. crassus colonize new waters, they spread swiftly and within just one year may reach 100% prevalence (Kennedy and Fitch 1990), though infection rates tend to vary between region and water type (Koops and Hartmann 1989). In Lough Neagh in 2005, prevalence of A. crassus reached 93% in yellow eels and 100% in silver eels, falling to 48.8% in yellow eels and 80.7% in silver eels by 2010 (Walker et al 2011). The difference in the range of numbers of A. crassus present can be seen in Fig. 4b. The much larger range seen in Lough Neagh than Lough Erne is indicative of a difference in parasite structure between the two populations. This may explain why results show that infection intensity with A. crassus had a 17! ! !

significant effect on the lipid content of eels in Lough Neagh but not in Lough Erne. Similarly, Kangur et al (2010) found that in Estonia, the mean intensity of infection with A. crassus was significantly higher in initial infection years from 1992 1998, than 1999- 2002. Previous research has shown that reinfection with A. crassus results in impaired development of the larvae of the parasite (Boon et al 1990), indicating some form of host defence. Kirk (2003) discusses the high levels of A. crassus during initial infection years levelling off, possibly due to an increased number of eels with thickened swim bladders; a defensive strategy against A. crassus. Similarly, when investigating A. anguilla phagocytes in response to infection with A. crassus, Knopf et al (2008) found that defence cells activated. During an 18 month survey of wild European eels, Kelly et al (2000) found no major differences on the physiological status of infected and non infected eels, indicating that adaptations to parasitism with A. crassus may be possible. Research has shown that greater damage may be caused by A. crassus during the warmer summer months (Lefebvre et al 2002). As eels for this study were collected during November to enable female silver eels to be captured before they began their spawning migration, it may be that results found for this study are not representative of the effects of A. crassus on the eel population. Though no seasonality of A. crassus infection in A. anguilla has been noted in other literature (Kelly et al 2000), further research of infected eels during the full year may show indication of possible seasonal effects of A. crassus on the lipid content. 5.5 Conclusions Previous literature has queried the impact of A. crassus on the successful completion of the spawning migration of A. anguilla (Kirk 2003). Research by Sjberg et al (2009) in the Baltic Sea found no effect of A. crassus on the body fat content or migration speeds of A. anguilla. However, it was observed that infected eels migrated closer to the shore; this indicates that the damage caused by A. crassus to the swim bladder may inhibit vertical migrations. In this study it was observed that the differences in lipid content in Lough Neagh and Lough Erne may be attributed to the differences in body length of eels between the two sites. The significance of the effects of infection intensity with A. crassus and lipid content in Lough Neagh but not in Lough Erne may be due to the low range of sizes of eels, or the low sample size from Lough Erne was not large enough for the data to be fully representative of the population. Defined size classes of eels may have been able to show whether eel size was a factor influencing A. crassus infection among Lough Neagh and Lough Erne eels. It remains uncertain if A. crassus have a direct effect on the lipid content of A. anguilla, though results suggest that there may be a relationship in the presence of specific population structures. There is no direct evidence to prove that A. crassus prevents successful completion of the eels spawning migration to the Sargasso Sea; however other research suggests that A. crassus may have an indirect effect on the lipid content by the extra metabolic cost of hosting a sanguivorous parasite 18! ! !

(Gollock et al 2004). Over the past 20 years, other countries have documented declines in lipid content of eels (Belpaire et al 2009); whether or not this apparent decline is due to infection with A. crassus or has any effect on their reproductive migration is for further research to decide. With worldwide declines in eel populations, the important aspect to consider is whether or not infection with A. crassus will affect successful recruitment of viable eels to the next generation.

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Figures

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Fig. 1 Life cycle of A. anguilla, cefas.defra.gov.uk. Accessed: 3rd October 2012 Fig. 2 Sites of study eels, maps.google.co.uk. Accessed: 3rd October 2012

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