Zoology Moderatorship Thesis

2016

The Effects of the Acanthocephalan Polymorphus minutus on the Cannibalistic

Behaviour of Gammarus duebeni

Laura Matthews

Declaration I, Laura Matthews declare that this thesis is my own work except where stated through

references or in the Acknowledgements and that it is 7010 words in length.

Signed:

Date:

Abstract Parasites may often mediate behavioural changes in their hosts to better serve

themselves, often at a cost to the hosts’ fitness or survivorship. Cannibalism of juvenile

or smaller conspecifics is one such behaviour that has previously been shown to be

altered by parasitic infection in many parasites and hosts. Here, the effects of the

acanthocephalan parasite Polymorphus minutus on the cannibalistic activity of its

intermediate host, Gammarus duebeni, are investigated. Individual infected and

uninfected adults were isolated and offered juvenile same-species prey, and average

amount of cannibalistic activity and cannibalistic individuals per group were assessed.

Infection was shown to alter the cannibalistic behaviour by creating a more uniform

behaviour in their hosts, whereas uninfected individuals displayed a wide range of

behavioural habits.

Acknowledgements I would first like to thank Professor Celia Holland, thank you so very much for your

advice, your guidance and for being nothing less than a fantastic supervisor. You

always seemed happy to see or hear from me, and always had a solution to any issue I

had. From hyphenating words to kick-sampling, you were greatly appreciated and

instrumental in my finishing of this project in one piece. You are one of the kindest and

most pragmatic people I have ever met, and working with you was a delight.

Maureen Williams, thank you so much for listening to every silly problem I ever had, for

being patient, and for turning up at 7 in the morning so frequently. You made the

experience so much simpler and more fun. Best of luck with your PhD, I will miss

working with you!

Thank you to Lauren Redmond and Regan Drennan for coming with me to collect my

samples. Your kicking skills were greatly appreciated, along with your friendship this

year. Thank you to Regan again, and also Tom Murphy, Dan McDermott and anyone

else I’ve forgotten for always giving me a hand when R was being temperamental.

Thank you to Paula Tierney, for helping me sort, for taking my extra Gammarus off my

hands and for just generally being the person I could moan about my experiments to,

you’re the only one that really got it. Best of luck with your sweet Gamms.

Thank you to Sam, Jack, Siki, Chloe and Caoimhe, Celia’s other supervisees, for

always being optimistic, for keeping me interested in our group meetings, and for

sharing the experience this year. Best of luck to all of you with your own projects!

Special thanks to my mum, who, while not understanding a thing about what I was

doing, was always eager to ask me how my ‘Grammadus’ were doing.

Finally, and with sincerity, thank you to all of my Gammarus who gave their lives for this

study, particularly to Wilbert (Inf16, shown in plate 5). Even in death you were the best-

behaved little friend.

Table of Contents Declaration ..................................................................................................................i Abstract ......................................................................................................................ii Acknowledgements ..................................................................................................iii Table of Contents .....................................................................................................iv Index of Figures .........................................................................................................v Index of Tables ........................................................................................................vi Index of Plates .........................................................................................................vii 1. Introduction ............................................................................................................1 2. Materials and Methods ..........................................................................................7

2.1 Site Selection ............................................................................................7 2.2 Pilot Study .................................................................................................8

2.2.1 Collection of Hosts ........................................................................8

2.2.2 Processing of animals for experimentation ...................................8 2.2.3 Experimental design .....................................................................9 2.2.4 Data Analysis ..............................................................................10

2.3 Main Experiment .....................................................................................11 2.3.1 Collection of hosts .......................................................................11

2.3.2 Processing of animals for experimentation .................................11

2.3.3 Experimental design ...................................................................12

2.3.4 Dissection and parasitological procedures .................................13

2.3.5 Statistical Analysis ......................................................................14

3. Results ..................................................................................................................15 3.1 Pilot Study ...............................................................................................15 3.2 Main Experiment .....................................................................................16 3.3 Cystacanth identification .......................................................................21

4. Discussion .......................................................................................................... 23 5. References ...........................................................................................................27 Appendix ..................................................................................................................33

Index of Figures: Figure 1: Life cycle of Polymorphus spp, adapted from Mehlhorn and Aspöck, 2007......3

Figure 2: Map of Lough Lene. Sampling site is represented by the red star....................7

Figure 3: Boxplot of the relationship between infection status and log 10 transformed

number of juveniles consumed over a period of 24 hours..............................................16

Figure 4: Boxplot of the relationship between infection status and the Log 10

transformed number of juveniles consumed over a 48-hour period...............................18

Figure 5: Plot of the cumulative mean juveniles dead in each group at each time

interval............................................................................................................................19

Figure 6: Mean juveniles eaten at each 12-hour interval................................................19

Figure 7: Scatter plot of weight of adults (g) against number of juveniles consumed in

a 48-hour period.............................................................................................................20

Figure 8: Scatter plot of lengths of adults (mm) against number of juveniles consumed

in the 48-hour experimental period.................................................................................21

Index of Tables: Table 1: Summary statistics of juvenile mortality in the pilot study...................................5

Table 2: Comparison of length and weights between the two groups of Gammarus.......8

Table 3: Summary statistics of juvenile mortality in the main experiment........................8

Index of Plates:

Plate 1: Gammarus duebeni infected with a cystacanth of Polymorphus minutus, as

indicated by the orange dot in the body cavity. ............................................................... 5

 Plate 2: Collection site, Lough Lene, County Westmeath, on day of collection for the

pilot study. ....................................................................................................................... 8

 Plate 3: Sampling Site for Main Experiment, as for pilot study. ..................................... 11

Plate 4: View of a jar from above at the 12 hour interval. One juvenile had been eaten

by an uninfected adult. Adult is shown by the arrow. ................................................... 12

 Plate 5: Infected adult prepared for dissection. Cystacanth can be seen as an orange

dot at the lower portion of the body. .............................................................................. 14

 Plate 6: Activated Polymorphus minutus viewed under the microscope. Hooks can be

seen on the proboscis, as indicated by the arrow. ........................................................ 22

 Plate 7: Closer viewing of proboscis. Characteristic hook pattern can be seen.............22

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1. Introduction: Cannibalism, or intraspecies predation (defined as the catching, killing and devouring of

an animal by a conspecific), is a widespread behaviour in the animal kingdom with at

least 3000 species, across 900 families subscribing at least partially to this method of

feeding (Fox, 1975; Polis, 1981). Cannibalism can be very advantageous. It is an

effective method of feeding, with the prey’s nutrient content matching closely what is

required of the predator, thus removing the danger of a nutritional mismatch (Bobisud,

1976). For example, arthropods tend to be limited in somatic growth by abundances of

specific rare minerals, and so, predating conspecifics eliminates the need to search for

such substances (Denno and Fagan, 2003). Cannibalism also has an indirect fitness

advantage of removing competitors, thus increasing potential resources. For example,

female three-spined sticklebacks cannibalise the eggs of conspecifics even when

alternative food is superabundant (Fitzgerald, 1992).

Cannibalism increases during periods of food scarcity for a number of reasons; the lack

of food leaves organisms weak and vulnerable to cannibalism, thus creating a ‘kill or be

killed’ scenario (Elgar and Crespi, 1992). Laws of optimal foraging theory show that

under conditions of food scarcity, individuals expand their diet into previously

unacceptable foodstuffs of high cost or low energy gain (Fox, 1975). Such predation is

common in colonial species, where the availability of same-species prey is high, or in

areas where other species to predate on are in low densities (Claessen et al., 2004).

Cannibalism is also more likely to occur in populations that have overlapping

generations in time and space, and also have notable differences in size (Wissinger

1992). It is a well-accepted fact that smaller (younger) individuals in a population are

usually cannibalized by larger (older) individuals, known as size asymmetric

cannibalism. Therefore, newborns are particularly vulnerable to attack, with such

predation by conspecifics found in over 80 species (Hardy, 1977). As such, cannibalism

can be a major source of juvenile mortality, and the danger of cannibalism decreases

with age (Polis 1981).

While cannibalism can present many advantages, and for many species may just be

considered an extension of normal predation behaviours (Hardy, 1977), it can also often

be disadvantageous. Filial cannibalism can create a potential net decrease in inclusive

fitness if the organism is likely to eat close relatives, although it may also increase the

survivorship and fitness of the remaining offspring and adult (Manica, 2002). Cannibals

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and their conspecific prey, or their preys guardians (Sherman, 1981), may be evenly

matched in fighting ability, and the likelihood of injury to the cannibal may be high

(Dawkins, 1976). Cannibalism and intraspecific necrophagy (the eating of dead

conspecifics, a behaviour closely linked with cannibalism (Rudolph, 2007)) can also

increase the likelihood of disease or parasite transmission (Pfenning et al., 1998;

Rudolph & Antonovics, 2007). For example, cannibalistic tiger salamanders

(Ambystoma tigrinium nebulosum) that ate diseased conspecifics were found less likely

to survive to metamorphosis (Pfenning et al., 1998), and that the frequency of

cannibalistic tiger salamanders is negatively correlated with the bacterial density in their

habitat, to avoid the predation of infected conspecifics (Pfenning, 1991).

Parasites are important to almost all ecosystems and individuals within them and

greatly influence ecosystem properties (Bush et al., 1997; Hatcher et al., 2012; Hatcher

et al., 2014). Parasites may also have profound effect on populations of species through

manipulation of their host’s phenotype (Dobson, 1988; Hatcher and Dunn, 2011).

Parasites may have complex life-cycles, requiring transmission through many hosts to

complete their life cycle (Parker et al., 2003; Auld and Tinsley, 2014). Manipulative

parasites can affect all aspects of their hosts’ phenotype; their behaviour, reproduction,

morphology and physiology, to further serve themselves in their survival and

transmission to the next host in their life-cycle, or to propagate throughout a particular

population of one species (Poulin, 1995; Poulin, 2010). Increased transmission of

creates an increase in fitness for the parasite (Lafferty, 1999). Specifically, the larvae of

many parasites can alter the behaviour or appearance of intermediate hosts in ways

that increase the likelihood of transmission to the next host, by increased susceptibility

to predation (Brown and Thompson, 1986). The parasite-induced change in behaviour

can increase transmission and the frequency with which the definitive host may be

infected (Lafferty, 1999).

Acanthocephalans are one such phylum of obligate intestinal parasites. Often known as

‘spiny-headed’ worms, they are recognized by their proboscis, which they use to hook

into the intestines of their hosts (Petroschenko, 1958). They have a highly conserved,

yet complex life-cycle, culminating in a definitive vertebrate host, with an intermediate

arthropod host (Near, 2002). Cystacanths (the fully developed larva of an

acanthocephalan) have the ability to manipulate the phenotypic behaviour of a wide

range of arthropod intermediate hosts (Bethel and Holmes, 1977; Cézilly et al., 2011),

and acanthocephalans use this to take advantage of trophic interactions between their

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intermediate and definitive hosts (Poulin, 1994). Polymorphus minutus is a common

acanthocephalan parasite culminating in a variety of aquatic bird species that act as

definitive hosts, with various members of the Gammarus family used as intermediate

hosts (Nicholas and Hynes, 1958).

Figure 1: Life cycle of Polymorphus spp, adapted from Mehlhorn and Aspöck, 2007.

 

Gammarus are crustaceans belonging to the order Amphipoda, a widely distributed and

speciose genus (Costa et al., 2009). The majority dwells in marine or freshwater

settings, although some species are semi-terrestrial, residing in sand or moist leaf litter

(Sexton, 1928). Gammarus are usually found in substrates that can provide both shelter

and a food source, such as in gravel or under dead vegetation (Fitter and Manuel, 1997;

McGrath et al., 2007). These freshwater amphipods are an ecologically significant

species, due to their wide distribution, the significant amount of biomass they constitute

in their habitats, and due to their role as shredders of organic material. Often, they are

the dominant macroinvertebrate in their habitat, and densities can reach up to

thousands of individuals per metre squared (Kelly and Dick 2005). Gammarus spp. are

considered detritivors, that usually feed on a thin layer of biotic material of

allochthonous leaf litter, comprised primarily of microorganisms (Nelson, 2011), and the

majority of the literature state that they form part of a herbivorous functional feeding

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group (Hatcher and Dunn, 2011). However, some studies show them to be carrion

feeders (MacNeil, Dick and Elwood 2007), and Gammarus are also often found to prey

upon conspecifics or other closely related species.

Cannibalistic species often share similar characteristics in terms of their ecology and

life-history; they live in large groups and are poor dispersers, they are continuous

breeders with overlapping generations, and live in areas with poor abundances of

alternate prey. As a result, such species often manifest cannibalism of conspecifics.

Cannibalism among Gammarus has been reported in many laboratory studies, the first

mention being by Sexton (1928). For example, Ward (1986) noted that in G. pulex,

males often eat females after copulation and once the females exoskeleton has been

shed. Size asymmetric cannibalism is particularly common in all species of amphipods,

with juveniles often acting as prey for adults (MacNeil, Dick and Elwood, 2007). Sexton

(1928) notes that individuals do not usually prey on conspecifics unless they are at

some disadvantage, namely a smaller size or are weakened from a recent moult. Many

things, such as food availability or habitat, can mediate cannibalism in Gammarus. For

example, poor quality habitats can mediate the incidence of cannibalism, with adult

Gammarus having to turn to cannibalism due to lack of food and juveniles having to

forage more widely to find food, leaving them open to predation (MacGrath et al, 2007).

While cannibalism can be disadvantageous, as mentioned above, Gammarus have

evolved methods of avoiding these negative effects. A temporal behavioural change

occurs in relation to brood stage in female G. pulex, whereby the instances of

cannibalism are significantly reduced concurrent to the time their own eggs are

hatching, to prevent the likelihood of eating their own young (Lewis et al., 2010). It is

unknown if other phenotypic recognition cues are utilized in this instance, or whether

males subscribe to a similar temporal avoidance of cannibalism. As mentioned above,

arthropods commonly subscribe to asymmetric cannibalism, avoiding potential injury in

cannibalistic attacks.

Juvenile Gammarus have developed behaviours to avoid being predated upon by

conspecifics. Like in many cannibalistic species, juveniles tend not to adhere to natal

philopatry; remaining in or returning to natal territory (Pearce, 2007), and quickly

expand outwards from their native zone, to create a separation between the vulnerable

juveniles and cannibalistic adults. Gammarus tigrinis, Gammarus mucronatus and

Gammarus lawrencianus exhibit changes in phototaxic behaviour at an age that

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correlates to a reduction in vulnerability to predation. This causes a partial separation of

adults and juveniles that reduces the likelihood of predation (Hunte and Myers, 1984).

Smaller individuals shift habitats to minimize the risk of predation by conspecifics. With

larger conspecifics in absentia, juveniles of G. pulex will use larger pores in substrates

to hide in whereas when there is predation danger, they only select smaller pores,

regardless of food availability (MacGrath et al, 2007).

Larval P. minutus lives in the body cavity of its intermediate host, Gammarus, and the

cystacanth is clearly visible through the cuticle as an orange dot. Besides this obvious

morphological change in colouration, which aids transmission of the parasite to its next

host (Bakker et al, 1997), it has other significant implications for Gammarus. Like many

parasites, acanthocephalans can negatively affect the survivorship and reproductive

abilities of their hosts; for example, Pomphorynchus laevis increases sensitivity to

heavy metal contamination in its intermediate Gammeridean host (Chen et al, 2015).

Severe infection of P. minutus in G. lacustris can cause castration of the females and

can slow down the secondary sexual characteristics development in males, leaving

them less fit than their conspecifics (Ward, 1986).

Plate 1: Gammarus duebeni infected with a cystacanth of Polymorphus minutus, as

indicated by the orange dot in the body cavity.

G. pulex infected with P. laevis showed a lower motivation to feed, and increased

refuge use than uninfected conspecifics, thus impairing optimal foraging and food intake

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(Dianne et al, 2014). Individuals are found to become highly photophilic when infected

with P.laevis (Bauer et al, 2000). Changes in behaviour tend to only occur when it is of

benefit to the parasite, i.e. when it is suitable for transmission to the next host. P.

paradoxus reverses the photonegative tendencies of infected G. lacustris, but only at

the cystacanth stage of development which is infective to the definitive host (Bethel and

Holmes, 1974).

The clear deleterious effects that parasites have on these hosts indicate that avoidance

of these parasites would be a beneficial behavioural trait of uninfected individuals. As

such, avoidance has been indicated in some studies on parasitized amphipods. In G.

lacustris, those infected with a cystacanth of P. minutus are less likely to be found in a

precopulatory mate-guarding formation. It is suggested that this is an active avoidance

of infected individuals by the uninfected individuals in the population (Ward, 1986).

As parasitism and cannibalism both play significant roles in the ecology and population

dynamics of many animals (as shown above), it is likely that infection by parasites will

influence cannibalism. Parasitism can affect the rate of foraging behaviour in many

animals, for example an increase in food intake has been recorded in G.pulex infected

with P. minutus due to a suggested increase in metabolic requirements mediated by the

parasite (Crompton 1970). It is therefore likely that it can have an effect on the rate or

propensity of cannibalism, as has been indicated in recent studies. The

acanthocephalan parasite Echinorynchus truttae mediates a reduction in intraguild

predation (the killing and consumption of closely related species, a behaviour closely

linked with cannibalism) between G. duebeni and G. pulex, and thus promotes species

coexistence (MacNeil et al., 2003). Conversely, it has been recently shown by Bunke et

al. (2015), that cannibalistic activity is increased among Gammarus duebeni celticus

infected with the microsporidian Pleistophora mulleri. However, different parasites

manipulate their hosts in a variety of ways and it is not necessarily true that this

behavioural change will be the case for all parasitized Gammarus.

As such, this project seeks to investigate the relationship between cannibalism and

infection by a macroparasite, the acanthocephalan P. minutus in Gammarus duebeni,

both host and parasite being widespread in aquatic systems. The aim of this project is

to investigate whether the incidences of cannibalism will be altered in the parasitised

individuals compared with that of the uninfected individuals.

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2. Materials and Methods 2.1 Site Selection The selected site for collecting the samples for this study was Lough Lene, a lake in the

upper Boyne catchment in County Westmeath. This site was selected due to a known

presence of Gammarus duebeni (Sutcliffe, 2010) and a comparatively high prevalence

of the acanthocephalan parasite Polymorphus minutus, found to be 6% from a

collection of Gammarus on February 12th 2015 (personal communication, Maureen

Williams). It is a limestone lake, of which the surface area is 4.16km2 (Shilland et al.,

2009). Mean depth is 4m and the maximum depth is 20m, which is considered a deep

lake (Inland Fisheries Ireland). It has been classed as a mesotrophic lake

(Environmental Protection Agency, 2008). The lake has been designated a special area

of conservation (SAC), and as such, conditions are carefully monitored. However, no

license was needed to remove a sample of Gammarus from the lake.

Figure 2: Map of Lough Lene. Sampling site is represented by the red star.

                     

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2.2 Pilot Study 2.2.1 Collection of hosts

Kick sampling for the pilot study began at approximately 8.15 a.m. at Lough Lene, on

the 3rd of November 2015. Sampling occurred in the shallow lake water close to a boat

jetty (Co-ordinates 53.660532, -7.195431), as shown in figure 2, above and plate 2,

below. Kick sampling was performed by three people using 20cm x 20cm sweep net for

twenty minutes providing one hour of sampling effort. Contents of the net were placed in

one of three 10L collection buckets filled with lake water. A 20L container of lake water

was also collected, for use later. Fallen leaves were collected as a food source. The

specimens were brought back to Trinity College, and left in the 14 degrees Celsius cold

room to acclimatize for three hours, with airlines inserted into the buckets to prevent

anoxia. The collected water was also stored in the cold room.

Plate 2: Collection site, Lough Lene, County Westmeath, on day of collection for the

pilot study.

2.2.2 Processing of animals for experimentation

The buckets were processed one at a time in the aquatic laboratory; this involved

sorting into parasitised adults, non-parasitised adults and non-parasitised juveniles.

Parasitised juveniles were not required, as cannibalistic adults have a propensity to

avoid parasitised juveniles, to avoid infection (MacNeil et al., 2003), and the use of

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parasitised individuals could be a confounding variable. Sorting was achieved by

pouring small amounts of the Gammarus and water into a white tray, picking the

selected individual up with a disposable pipette with the tip cut off and placing in the

correct labeled bucket. A second pipette was also adapted, by partially cutting off the

bulb at the back, to create a scoop that could be used to pick up the particularly large

Gammarus individuals, or Gammarus pairs in precopula formation.

Each group of animals was sorted into three separate containers full of lake water.

Juveniles were defined as being under 6mm in length, as per Bunke et al. (2015), and

suitably large adults were defined as being over 7mm in length. Adults and juveniles

were selected by eye. Parasitized individuals were recognized by their distinctive

orange dot in their body cavity and each potentially infected individual was viewed

under a dissecting microscope to confirm that they were parasitized, as some non-

parasitized Gammarus can have slightly orange colouration. Only animals with a single

dot were selected, to eliminate duel infection as a possible confounding variable. Any

parasitized juveniles found were added to the ‘discard’ bucket, as they were not

required for this experiment. Any specimens of intermediate size, between the sizes for

adult or juvenile, as judged by eye, were also put in the discard bucket, as only the

largest adult individuals were required. After looking at the parasitized adult samples, it

was decided that eight of the adults were a suitably large size and could be utilized in

the pilot study. Eight non-parasitized adults of approximately similar sizes were also

selected for experimentation. Adult were not weighed or accurately measured for the

pilot study.

2.2.3 Experimental design

Twenty-four 750ml jars were washed in an industrial glass cleaner and filled with

approximately 600mls of lake water. Jars were labeled as follows: 1-8 control (to have

no adult Gammarus in them), 9-16 non-parasitised, and 17-24 parasitised adult. Each

jar was assigned the appropriate adult, and the controls were left empty of adults. The

jars were then brought on trays to the 14 degrees Celsius cold room. These jars were

covered over with a petri dish to prevent any spillage during transport from the lab, and

to prevent any Gammarus from escaping. The placement of the jars on the trays was

randomised, to prevent any bias that may occur by placement. Over the course of the

study, the subjects were kept in an appropriate light and dark cycle. The Gammarus

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were then left to starve for a duration of 24 hours. The non-parasitized juvenile

Gammarus were returned to a large bucket with a food source for this period.

The following day, twenty-four petri dishes, each with a small amount of lake water,

were filled with 7 juveniles, using the same scoop and pipette, to be added to each of

the jars. A total of 168 juveniles (24 x 7) were used. As size discrimination was done by

eye, only the smallest of the juveniles were selected for introduction into the jars to

ensure they were in fact juveniles (below 6mm in length), and the remainders were

returned to the ‘discard’ bucket. These petri dishes were transported downstairs to the

14 degrees Celsius cold room and one petri dish of juveniles were added to each of the

jars at 4 pm, carefully ensuring that no jar had accidentally received more than one petri

dish of juveniles. The petri dish lid of each jar was quickly removed and the contents of

the petri dish were poured in. The lid was then immediately replaced.

Twenty-four hours later the jars were examined. The number of juveniles still present in

each jar was counted, by holding the jar up in front of a white piece of paper to make it

easier to see. Missing or dead juveniles were noted, and from this, the number of

cannibalistic activities was calculated.

2.2.4 Data Analysis

Data on the amount of cannibalistic activity was calculated by subtraction of the

remaining alive juveniles from the number of introduced juveniles (7). Data sets of

infection status and number of missing juveniles were created on Microsoft Excel.

Statistical analysis was done using the statistical programme R (version 3.2.3 app GUI

1.65 (6833) x86_64-apple-darwin10.8.0). A chi-squared test was used to assess the

difference in the amount of cannibalistic individuals in each infection group. Normality of

distribution of the amount of juveniles eaten was tested using a QQ plot, and the logged

amount of juveniles eaten in each group was analysed using a t-test of the log-

transformed data. Summary statistics were collated onto a table for further study if

necessary. A box plot was created showing visually the difference between the

cannibalistic activity of the infected and non-infected animals.

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2.3 Main Experiment 2.3.1 Collection of hosts

The study began on the 24th of November 2015, in the same location as the pilot study.

Conditions were slightly more windy and rainy, meaning that the organisms were

already unsettled. Four people began kick sampling at approximately 9.30 and lasted

for one hour, totaling 4 hours of sampling effort to ensure there was a more sizeable

sample of parasitized adults. Four buckets were collected in total; along with two more

buckets of lake water, and another 30-litre container of lake water. The samples were

brought back to Trinity College and each bucket was poured through a splitting funnel

into two buckets of the same type. This was done to half the amount of Gammarus in

each bucket, to prevent anoxia and overcrowding. Each bucket was then topped up with

lake water, and some detritus, leaves and stones were added to each of the buckets. A

total of eight buckets were created in this way and left in the 14 degrees Celsius cold

room to acclimatize overnight. Airlines were places in the buckets to prevent anoxia.

Plate 3: Sampling Site for Main Experiment, as for pilot study.  

2.3.2 Processing of animals for experimentation

The following day sorting of the Gammarus began. As with the pilot study, Gammarus

were sorted as follows - uninfected adults, uninfected juveniles and parasitised adults.

The pilot study indicated that there was a possible link between size of the adult and its

propensity to cannibalise juveniles, and so, it was decided to increase the size of the

suitable adults to 8mm. Once the sorting was complete, the suitably sized (now 8mm+)

parasitized adults were selected. Ten were deemed suitable. As the pilot study

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indicated that weight might have a role in the decision to cannibalise, the individuals

were weighed, to investigate this possible correlation. These were individually weighed

using a fine scale and wet weight (mass in g) was determined to four decimal places.

These individuals were returned to a jar. Using the range of these weights as a guide,

size-matched uninfected adults were selected.

A total of 4143 Gammarus were processed in this way. Of these, 135 were parasitised,

giving a prevalence of 3.3%. Adults constituted 3138 of the total amount, and juveniles

accounted for 1005. 20 adults (10 infected and 10 uninfected) were utilized in this

experiment, along with 210 uninfected juveniles.

2.3.3 Experimental design

Each adult was placed in a jar with 300 mls of lake water, providing a total of 20 jars. 10

control jars with no adult Gammarus were also used. The volume of water was

decreased from 600 to 300 mls to increase the density of the juveniles, as an increase

in density results in an increase in encounter and frequency of aggressive responses

such as cannibalism (Polis, 1981). No extra food was put in the jars, to increase the

necessity to cannibalise, and no extra detritus was added, as juveniles use these to

hide from cannibalistic adults (McGrath et al., 2007). Each jar was labeled and placed in

the 14 degrees Celsius cold room, with their positions on the trays randomised. Adult

Gammarus were starved for a period of 48 hours. During the experiment, one infected

adult died and was removed.

After 48 hours of starvation, 7 juveniles were added to each jar, as in the pilot study.

The jars were examined every 12 hours for a period of 48 hours to record any

cannibalistic activity. Remaining juveniles were counted and any moults or remnants of

juveniles were removed at 12, 24, 36 and 48 hours post-starvation, to prevent any

alternative food sources. The temperature in each jar was also noted at each time

interval. The trial ran for 48 hours. Upon completion, photographs were taking of the

remaining juveniles so they could be measured if needed.

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2.3.4 Dissection and parasitological procedures

Once the trial was complete, adult Gammarus were dissected to confirm their infection

status (parasitized or non-parasitised). Individual adults were stretched out to full length

and body length (mm) measured with an electronic vernier calipers to two decimal

places. Cysts were removed from the infected individuals by dissection.

For P. minutus the activating agent is bile salts (Lackie, 1974), so, as described by Graff

and Kitzman (1965), the activation protocol for acanthocephalan cystacanths was

carried out; the cystacanths were added to an individual labeled test tube with a

0.25mM sodium taurocholate solution (created by dissolving 0.0625g per 25 ml of

deionized water) to stimulate activation and were left in a 37 degrees Celsius incubator

for 72 hours. Both adult and juvenile Gammarus were preserved in 70% alcohol with

10% glycerol, in case further data collection was needed.

Once the cystacanths had activated, they were fixed in a 70% alcohol and 10% glycerol

solution. The parasites were mounted on microscope slides using polyvinyl lactophenol

and were left overnight in a fume cupboard to dry. Each slide was labeled and observed

under a microscope. Each parasite was identified to species level on the basis of their

proboscis morphology and numbers of rows of hooks (McDonald, 1988; Petroshenko

1958).

Plate 4: View of a jar from above at the 12 hour interval. One juvenile had been eaten

by an uninfected adult. Adult is shown by the arrow.

  14  

2.3.5 Statistical Analysis

Data sets were complied on Microsoft Word and were analyzed on R (version 3.2.3 app

GUI 1.65 (6833) x86_64-apple-darwin10.8.0). Difference in cannibalistic individuals in

each infection status was investigated using a chi-squared test. QQ normality plots

were created to determine the normality of the distribution of the lengths, of weights and

of juvenile deaths in each group. As size of the adult was suggested to be a factor in an

individual’s propensity to cannibalise, t-tests were preformed to determine whether the

weights or lengths of the infected and uninfected Gammarus were significantly different.

T-tests were conducted to investigate the difference in the overall number of

cannibalistic events in each infection group. Graphs were produced of the cumulative

mean deaths per jar of each infection status, and of the mean deaths per time period

(each 12 hour interval). Scatter plots were created to compare the infection status and

weight of the adults with the amount of juveniles eaten, and the length and infection

status with the amount of juveniles eaten.

Plate 5 Infected adult prepared for dissection. Cystacanth can be seen as an orange dot

at the lower portion of the body.

  15  

3. Results: 3.1 Pilot Study

The pilot study was conducted to ensure that there was some relationship between

infection status and cannibalism, and establish baselines of cannibalistic behaviour for

the main experiment. Only one juvenile in the control jars died, which was not attributed

to cannibalism, as the corpse was recovered from the jar at the end of the experiment.

This represents one random death; therefore it was likely that any death in the infected

or uninfected jars were from cannibalism, and not from any other cause. All the 16

adults (eight infected and eight uninfected) survived the experiment. Of the infected

individuals, 2 of the 8 adults showed cannibalistic tendencies (25%). In the uninfected

individuals, six of the 8 adults (75%) showed cannibalistic tendencies. A chi-squared

test showed that there was a significant difference in the number of cannibalistic

individuals between the two groups (X-squared = 4, df = 1, p-value = 0.0455) and that

cannibalistic individuals were more common among the uninfected than in the infected.

Summary statistics were determined (see Table 1, below).

Table 1: Summary statistics of juvenile mortality in the pilot study.

Infection Status Uninfected Infected Control

Deaths 15 3 1

Range of deaths 0-4 0-2 0-1

Mean deaths per jar 1.875 0.375 0.125

Standard Deviation 1.64 0.74 0.35

Deaths as Percentage 28.6% 5.3% 1.8%

Median 1.5 0 0

A QQ normality plot showed that the number of juveniles consumed was not distributed

normally. A t-test comparing the log transformed amount of juveniles consumed by the

infected and uninfected individuals was performed and the difference was found to be

statistically significantly different (t=-2.3977, df=12.086, p=0.03353), indicating that

uninfected adults consumed more juveniles on average than the infected did (see

Figure 4).

  16  

Figure 3: Boxplot of the relationship between infection status and log 10 transformed

number of juveniles consumed over a period of 24 hours.    

3.2 Main Experiment Of the 20 adults used, 1 died mid-experiment (jar 12, infected). This animal was

removed from the analysis. QQ normality plots indicated that the distribution of weights

and lengths of the adult Gammarus were normally distributed. A t-test compared the

weight (g) of infected and uninfected individuals post-study and the difference was not

found to be significant (t = 1.395, df=17, p=0.181). Similarly, the mean lengths (mm) of

the infected and uninfected adults were compared and not found to differ significantly (t

=1.3897 df=17 p=0.1825)(see Table 2). Therefore, the size of the individuals was not

considered to be a potentially confounding variable of cannibalistic behaviour between

infected and uninfected adults.

Infected Uninfected

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Infection Status

Log

of J

uven

iles

Con

sum

ed

  17  

Temperature was measured, and found not to have changed between jars or between

sampling times (see Appendix).

Table 2: Comparison of length and weights between the two groups of Gammarus.

Infection Status Infected Uninfected

n 9 10

Range of Length (g) 0.069-0.153 0.072-0.16

Mean Weight (g) 0.1047 0.1204

S.D of Weight 0.032 0.03

Range of length (mm) 8.65-12.92 8.9-12.23

Mean Length (mm) 10.894 10.027

S.D of Length 1.35 1.37

Two juveniles in the control jars died. The deaths in the control jars were not attributed

to cannibalism, as their bodies were recovered from the jar, and represent two random

deaths. Therefore, the death in the infected and uninfected jars was attributed to

cannibalism. Of the ten uninfected adults, 6 showed a propensity to cannibalise (60%),

whereas in the infected specimens, 7 out of 9 (78%) showed cannibalism. Difference in

amount of cannibalistic individuals was assessed using a chi-squared test and was

found to not be significant (X-squared = 2.0387, df = 1, p-value = 0.1533) (see Table 3).

A Mann-Whitney test of the unlogged data showed that there was also no significant

difference between cannibalism events in the infected and uninfected groups (W=49, P-

value= 0.764). Summary statistics were determined and are shown below (Table 3)

Table 3: Summary statistics of juvenile mortality in the main experiment.

Infection Status Uninfected Infected Control

Deaths 16 10 2

Range of deaths per jar 0-4 0-3 0-1

Mean deaths per jar 1.6 1.11 0.2

Standard Deviation 1.65 0.78 0.43

Deaths as Percentage 11.2% 7.77% 1.4%

Median 1.5 1 0

  18  

A boxplot was created to compare the difference in cannibalistic activity between the

infected and non-infected adults (figure 5, below). The boxplot shows a clear difference

in the instances of cannibalistic activity.

Figure 4: Boxplot of the relationship between infection status and the Log 10

transformed number of juveniles consumed over a 48-hour period.

 A cumulative plot of juveniles eaten over time was produced, and the average juveniles

eater per time interval was determined. Means were used, as the amount of adults used

in each infection status was not equal, and means would remove this unevenness.

Uninfected individuals were shown to eat more juveniles over the 48-hour period than

the infected individuals did, but did not eat more over every time interval. For two of the

four time-intervals, infected individuals ate more juveniles than the uninfected did.

infected uninfected

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Infection Status

Log

of J

uven

iles

Con

sum

ed

  19  

Figure 5: Plot of the cumulative mean juveniles dead in each group at each time

interval.

Figure 6: Mean juveniles eaten at each 12-hour interval.

0.0

0.5

1.0

1.5

Time (Hours)

Cum

ulat

ive

Mea

n ju

veni

les

dead

0 12 24 36 48

UninfectedInfectedControl

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Time (Hours)

Mea

n ju

veni

les

dead

0 12 24 36 48

UninfectedInfectedControl

  20  

Weight of adults was plotted against the amount of juveniles consumed (Figure 8). The

graph shows no correlation between adult weight and the propensity to cannibalise.

Figure 7: Scatter plot of weight of adults (g) against number of juveniles consumed in a

48-hour period.

 Similarly, length of adults was plotted against the amount of juveniles eaten (Figure 9)

and no obvious correlation between adult length and their propensity to cannibalise was

detected.  

0.05 0.10 0.15 0.20

01

23

45

Weight (g)of Adults

Juve

nile

s ea

ten

UninfectedInfected

  21  

Figure 8: Scatter plot of lengths of adults (mm) against number of juveniles consumed

in the 48-hour experimental period.

3.3 Cystacanth identification Of the adult Gammarus deemed to be infected at the beginning of the experiment, the

remaining nine were infected upon dissection at the end of the experiment. All had a

single cyst infection, eliminating duel infection as a possible confounding variable. All

the cysts were activated in the sodium taurochlorate solution after the 48-hour

incubation period. All the activated cystacanths looked similar upon inspection under

the microscope and were all identified by their characteristic proboscis and pattern of

hooks as the acanthocephalan parasite P. minutus.

8 9 10 11 12 13 14

01

23

45

Length (mm)of Adults

Juve

nile

s ea

ten

UninfectedInfected

  22  

Plate 7 Closer viewing of proboscis. Characteristic hook pattern can be seen

Plate 6 Activated Polymorphus minutus viewed under the microscope. Hooks can be

seen on the proboscis, as indicated by the arrow.

Plate  1Closer  view  of  proboscis  and  hooks.  

  23  

4. Discussion The pilot study indicated that there was a significant difference in the incidence of

cannibalistic activity between parasitised and unparasitised adults of G. duebeni.

However, it also indicated that there was a likely confounding variable present; that size

of the adult may play a role in the propensity to cannibalise. In some cannibalistic

species, it is the largest morphs that are found to have cannibalistic tendencies, as is

the case in Tiger Salamanders (Pfenning, 1991) and largemouth bass (Clady, 1974). Of

the uninfected individuals, the two that did not cannibalize juveniles were comparatively

small in relation to the other selected Gammarus, and the two infected individuals that

did cannibalise were notably larger than their other infected conspecifics. This prompted

the decision to weigh and measure the lengths of the adults accurately, and more

carefully size match the adults being used in the main experiment. Lengths and weights

were measured in the main experiment, but neither parameter over the range of the

adults selected was found to be a significant factor in their propensity to cannibalise.

However, the selected infected adults were larger in the main experiment than they

were in the pilot study, as there were more suitable adults to select from. The

percentage of those infected that showed cannibalistic activity rose from 25% in the

pilot study to 78% in the main experiment.

There was also a decrease in the amount of juveniles eaten by the uninfected adults,

which may also have been mediated by the change in size range. Many of the

uninfected adults used in the main experiment were smaller than those used in the pilot

study, as instead of using the largest ones, they were size matched to the range of the

infected adults. Cannibalistic individuals fell slightly, from 75% to 60% between the pilot

study and the main experiment. There is an indication therefore, that there is some link

with size in an individuals propensity to cannibalise. However the range of sizes

selected was very narrow in this experiment, and therefore it was not possible to

conclusively determine the relationship between size and cannibalistic behaviour.

There was uncertainty as to whether the absolute size of the individual adult, or the

relative size compared to its prey is the factor that mediates cannibalistic activity.

Studies of cannibalistic tendencies in a wide range of other species indicate that there is

a ‘cannibalism threshold’ i.e. that an individual should be a certain percentage larger

than its prey to cannibalize it (Polis, 1981), for example, conspecifics are only eaten by

piscivorous fish if the predator-to-prey ratio exceeds the threshold of 80-100% (Popova,

1967). It is well known that asymmetric predation is common in amphipods (MacNeil,

  24  

Dick and Elwood, 2007) and efforts were made to ensure that there was a notable size

difference between adults and their prey, by selecting from the smallest juveniles and

largest adults. However, in some instances, the ‘juveniles’ were relatively quite large,

and some of the parasitized adults were relatively small. This could have a significant

effect on the rate of cannibalism, if the cannibalism threshold had not been reached.

Therefore, in further studies, the size of the juveniles relative to the size of the adult

should be taken into account, as this could be considered a confounding variable.

The main experiment indicated that there was a marked difference in variance of

cannibalistic activity between infected and uninfected individuals. The majority of

infected adults ate one juvenile, whereas there was a wide range of numbers (0-4) of

juveniles consumed by the uninfected adults. This clearly indicates difference between

the behaviours of infected and uninfected adults, with a wide amount of variation in the

uninfected adults, and uniformity in the infected individuals. This has been suggested in

the literature on animal behaviour. In some species it has been noted that there are

‘cannibal specialists’, who are more predisposed to cannibalism than other adult

conspecifics indicating some sort of predisposition to cannibalism (Polis, 1981). This is

a kind of behavioural polyphenism or difference in ‘animal personality’, defined as the

differences in behaviour among individuals that are consistent over time and situations.

Behavioural traits may not always be beneficial when uniformly used by a whole

population, and so, populations of species may develop multiple phenotypic traits

(Reale et al., 2010). This may have an effect on propensity to cannibalise, and an effect

on the ability of the parasite to alter behaviour (Poulin, 2013). For example, in a study of

a colony of 900 herring gulls, intraspecific oophagy (the eating of eggs) and juvenile

cannibalism are common, and 23.3% of all eggs and chicks were eaten by conspecifics.

Of this, four individuals were responsible for 2-5% of this figure (Parsons, 1971). Such

specialism in the propensity to cannibalise is also found in bass (Clady, 1974), newts

(Kaplan, 1980), chimpanzees (Goodall, 1977), and many other species. There is a

similarly evident genetic component to cannibalism in some species. For example, in

eight separate strains of laboratory mice, rates of litter cannibalism remain the same for

at least 13 generations, implying a genetic link to cannibalism preferences (Hauschka,

1952). Therefore, it may have been likely that some of the selected uninfected adults

were genetically disposed to cannibalise or exhibited some sort of behavioural

polyphenism, as indicated by some uninfected individuals expansive feeding on

conspecifics, and some who were not predisposed to cannibalism. This is evident in the

  25  

wide variation in the amount of juveniles consumed by each uninfected adult; some ate

no juveniles, and some ate more than half. Similarly, some infected individuals may be

particularly adept at avoiding the parasites manipulative abilities, as exhibited in the

infected individuals that did not cannibalise, or cannibalised more than others did.

Poulin (2012) suggests that animals personality is a barrier to parasitic infection, where

some phenotypes of a polyphenotypic animals are more easy to manipulate, and some

presenting a greater difficulty.

Alternatively, the increase in cannibalism by the infected adults may have been

mediated by altered prey density. The reduction in water from 600mls per jar to 300mls

per jar increased the density from 1.14 juveniles per 100mls to 2.28 juveniles per

100mls. Increased rates of intraspecific predation have been linked to high densities or

overcrowding in the wild, as an increased density of prey will increase the probability of

encounter and the frequency of aggressive behaviours (Fox, 1975; Polis, 1981) such as

cannibalism. Therefore, the increase in density of juveniles may have affected the

instances of cannibalism in the infected adults.

Cannibalism activity may have been reduced in some of the adults, due to the temporal

shift in cannibalism activity found in recently brooding females (Lewis et al., 2010). This

would have reduced the incidence of cannibalism in the uninfected group. While it may

be beneficial to only utilize males in further studies to remove this potentially

confounding variable, it is difficult to tell the sexes apart without extensive investigation

under the microscope. Future experiments may possibly utilize males exclusively to

avoid this.

Upon inspection of the surviving juveniles, it was discovered that 2 of the remaining

juveniles were in fact infected (0.011%), as indicated by the characteristic bright orange

cyst on their sides. One was found in an ‘uninfected’ jar and one in an ‘infected’ jar.

While there is evidence of an avoidance of infected individuals by uninfected individuals

(Ward, 1986), and that more specifically, uninfected adults avoid predating infected

juveniles (MacNeil et al., 2003), this would not have affected the results as food sources

(i.e the juveniles) were already in excess, and cannibalism had still occurred in both the

of the jars these juveniles were found in. Infected adults do not discriminate between

infected and uninfected juveniles (Bunke et al., 2015), and so this would not have had

any effect in the infected jars.

  26  

 Many parasitised animals, including humans, exhibit a reduction in food intake

compared to that of uninfected conspecifics (Crompton, 1984; Kyriazakis, 1998) and it

is possible that this macroparasite alters the individual in such a way that they only

consume the minimum necessary to survive. In many of the infected jars at each

interval, large portions of deceased juveniles were removed from the floor of the jar,

whereas in the uninfected jars, where the juvenile was cannibalised, the majority of the

body had been eaten, with the exception of some limbs.

The presentation of cannibalism in this experiment was different to that found in other

similar studies. For example, G. pulex infected with P. minutus were found to have an

increased rate of foraging for food (Crompton 1970). However, in this experiment, G.

duebeni infected with P. minutus presented with a reduced intake of same-species prey

(i.e. a reduction in rate of foraging). Bunke et. al. (2015) found that for G. duebeni

infected with P. mulleri, the incidence of cannibalism increased significantly. This is

contrary to the findings in the present study, where the variation in the infected

individuals was greatly reduced. However, P. mulleri microsporidian parasite, with a

single host-species life cycle. The propagation of P. mulleri occurs exclusively through

cannibalism (MacNeil et al., 2003), and it is likely that infection by this species may

have an effect on the rate of cannibalism. The results of this study has been mirrored by

the results found in Gammarus spp. infected with a fellow acanthocephalan parasite; E.

truttae. In this case, infection mediates a reduction in intraguild predation between G.

duebeni and G. pulex (MacNeil et al., 2003).

Overall, the alterations in cannibalistic behaviour by parasitic infection have presented

differently in this experiment than in that of comparable studies, though this is not to say

that this study has produced contrary or unhelpful information. Parasites are

exceedingly complex creatures and it is evidently not possible to have a rule of thumb

for any behavioural or morphological alteration, let alone for one so complex as

cannibalism. This project has gone some way into exploring the potentially vast and

varied effects parasites may have on different hosts foraging and cannibalistic

behaviour. In the future, further studies and larger analyses are required for this vast,

yet fascinating subject.

  27  

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Appendix Results Table 4 Temperatures of jar during experimentation period.

Jar Number

Infection Status

Temperature in Degrees Celsius 0 Hour 12 Hour 24 Hour 36 Hour 48 Hour

1 Uninfected 14.0 14.0 14.1 14.0 14.0 2 Uninfected 14.4 14.3 14.4 14.3 14.4 3 Uninfected 14.0 14.0 14.2 14.1 14.0 4 Uninfected 14.1 14.0 14.0 14.1 14.2 5 Uninfected 14.0 14.0 14.0 14.0 14.0 6 Uninfected 14.0 14.1 14.1 14.1 14.1 7 Uninfected 14.3 14.3 14.1 14.1 14.2 8 Uninfected 14.0 14.0 14.0 14.0 14.0 9 Uninfected 14.0 14.1 14.2 14.2 14.0 10 Uninfected 14.1 14.2 14.0 14.0 14.0 11 Infected 14.0 14.0 14.0 14.0 14.0 12 N/A x x x x x 13 Infected 14.2 14.1 14.1 14.1 14.1 14 Infected 14.3 14.4 14.2 14.3 14.3 15 Infected 14.0 14.1 14.0 14.0 14.0 16 Infected 14.4 14.3 14.0 14.1 14.0 17 Infected 14.0 14.0 14.2 14.0 14.0 18 Infected 14.0 14.0 14.1 14.0 14.0 19 Infected 14.1 14.1 14.1 14.0 14.0 20 Infected 14.1 14.1 14.1 14.1 14.1 21 Control 14.0 14.1 14.2 14.0 14.0 22 Control 14.1 14.0 14.1 14.0 14.0 23 Control 14.1 14.1 14.3 14.1 14.0 24 Control 14.0 14.1 14.2 14.1 14.0 25 Control 14.1 14.2 14.1 14.0 14.3 26 Control 14.4 14.2 14.2 14.4 14.1 27 Control 14.0 14.2 14.0 14.0 14.2 28 Control 14.2 14.0 14.0 14.2 14.0 29 Control 14.2 14.0 14.0 14.0 14.0 30 Control 14.1 14.0 14.0 14.0 14.1

Temperature was found to not deviate significantly from 14 degrees Celsius.

Temperatures deviates the most at the 0 hour period, as the door had been opened and

closed often just previous to temperature reading, during experimental set up. Slightly

warmer jars tended to be closer to the door. The adult in jar 12 died before

experimentation began, and jar was removed.