DISSERTATION FINAL MkV

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Differential expression of hepatic proteins within Gasterosteus aculeatus upon infection with the pseudophyllian cestode Schistocephalus solidus. John Morgan

Transcript of DISSERTATION FINAL MkV

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Differential expression of hepatic proteins within

Gasterosteus aculeatus upon infection with the

pseudophyllian cestode Schistocephalus solidus.

John Morgan

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Table of Contents

Abstract ..................................................................................................................................... 3

1. Introduction .......................................................................................................................... 3

1.1 Gasterosteus aculeatus ..................................................................................................... 3

1.1.1 Distribution ................................................................................................................ 3

1.1.2 As a Model Organism ................................................................................................ 3

1.1.3 The Liver as a Histopathologic Biomarker ................................................................ 4

1.2 Schistocephalus solidus .................................................................................................... 5

1.2.1 Lifecycle .................................................................................................................... 5

1.2.2 Copepod-Stickleback transition of S. solidus ............................................................ 5

1.2.3 Mortality evasion in G. aculeatus.............................................................................. 6

1.3 Host-Parasite Interactions ................................................................................................ 6

1.3.1 Immune response of G. aculeatus upon parasitic infection, and S. solidus evasion

of the G. aculeatus immune system .................................................................................... 6

1.3.2 Behavioral resistance, and effects of S. solidus infection .......................................... 7

1.4 Proteomics ........................................................................................................................ 7

1.4.1 Proteomic Methods and Analyses ............................................................................. 8

1.4.2 Biomarker identification, and analysis ...................................................................... 8

1.4.3 Limitations, and considerations ................................................................................. 9

1.5 Key Hypotheses/Aims of Study ....................................................................................... 9

2. Materials and Methods ...................................................................................................... 10

2.1 Source of G. aculeatus, and S. solidus Species .............................................................. 10

2.2 Protein Isolation, and Quantification from Liver Samples ............................................ 10

2.3 Two-Dimensional Electrophoresis ................................................................................. 11

2.4 Statistical Analysis of Results ........................................................................................ 11

3. Results ................................................................................................................................. 12

3.1 Correlation Studies ......................................................................................................... 12

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3.2 Gel Analysis ................................................................................................................... 17

4. Discussion............................................................................................................................ 19

4.1 Size variation in G. aculeatus ........................................................................................ 19

4.2 Physiological effects of S. solidus parasitism ................................................................ 21

4.3 Differential protein expression in the G. aculeatus-S. solidus model ............................ 23

4.4 Conclusions and Further Research ................................................................................. 26

Acknowledgements ................................................................................................................ 27

Appendices .............................................................................................................................. 28

A: SPSSS Outputs ................................................................................................................ 28

(i) Percentage Body Composition Accounted for by Parasite .......................................... 28

(ii) Fish Condition and Degree of Parasitic Infection ....................................................... 28

(iii) Exponential Model: Uninfected + Infected ............................................................... 29

(iv) Exponential Model: Uninfected ................................................................................. 30

(v) Exponential Model: Infected ....................................................................................... 31

(vi) ANCOVA: No Infection vs. Single Infection ............................................................ 32

(vii) ANCOVA: Single Infection vs. Multiple Infection .................................................. 32

(viii) ANCOVA: No Infection vs. Multiple Infection ...................................................... 33

B: Gel Images ....................................................................................................................... 33

(i) No Infection ................................................................................................................. 33

(ii) Single Infection ........................................................................................................... 35

(iii) Multiple Infection ...................................................................................................... 37

References ............................................................................................................................... 40

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Abstract

Gasterosteus aculeatus and Schistocephalus solidus are two halves of a unique system used in

studying host-parasite interactions in fish. The individuality of this system stems from the

physiological and behavioural impact the pseudophyllian cestode, S. solidus has on the host.

In particular is the severe distention of the body cavity caused by the developing cestode, where

it can constitute up to 33 % of the overall mass of both host and parasite. Growing to a

proportion this large is clearly going to have a number of effects on the host organism, with

reductions in body condition of up to 0.00227 having been observed upon infection. The

potential of the liver as a histopathological biomarker is well known across a number of species,

acting as an accurate indicator of the health of an organism. Through this experiment five

potential biomarker candidates were revealed. This was achieved via use of sodium dodecyl

sulphate polyacrylamide gel electrophoresis.

1. Introduction

1.1 Gasterosteus aculeatus

1.1.1 Distribution

Gasterosteus aculeatus species solely reside within the Holarctic (Bell, 1995), with widespread

habitation of circumarctic and temperate regions (Luna & Torres, 2014), possessing a

localization to primarily freshwater bodies, estuaries, and coastal seas (Whitehead, et al.,

1989). This breadth of geographic distribution has been attributed to the association of spawn

with drifting seaweed (Safran, 1990; Safran & Omori, 1990), and furthermore has led to the

arising of three distinct, IUCN (International Union for Conservation of Nature) recognized

species; G. a. aculeatus, G. a. williamsoni, and G. a. santaeannae (Williams, et al., 1989). Of

the three distinct subspecies, within this study, the primary focus will be upon G. a. aculeatus.

1.1.2 As a Model Organism

The favorability of the three-spined stickleback as a biological research model stems from two

key attributes: an excellent suitability for laboratory study (Barber & Nettleship, 2010), and an

extensive intra-specific variation in freshwater populations (Bell & Foster, 1994). These

attributes allow for the fish to be effectively reared to full adulthood in aquaria, with little-to-

no impact on their behavioral repertoire. G. aculeatus has behaved as a model organism in a

number of studies, primarily in models studying adaptive evolution (Barrett, 2010; Chan, et

al., 2010; Jones, et al., 2012), due to the wide range of species diversity derived from the

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Gasterosteidae family (Froese & Pauly, 2014). However, as a model for physiological biology,

there has been particular focus on kidney and endocrine disruption (Bell, 2001; Hahlbeck, et

al., 2004a; Hahlbeck, et al., 2004b; Allen, et al., 2008), and health monitoring facilitated by

hepatic analysis (Holm, et al., 1993; Aniagu, et al., 2008; Orczewska, et al., 2010). The primary

interest as a model organism also stems from the parasitic infections it may contract. Its

common parasite, Schistocephalus solidus, has the ability to possess an extreme body size in

relation to its host (Wootton, 2012). This dominant feature of infection distinguishes it from

other model systems as the effects S. solidus has on its host is unique, and limited to a fraction

of other large-bodied parasite systems, such as the Lingula intestinalis-cyprinid model (Hoole,

et al., 2010), or the mammalian Spirometra mansonoides model (Siles-Lucas & Hemphill,

2002). This system is most useful when it comes to non-invasively analyzing the plerocercoid

growth in vivo by digital photography and image analysis software (Barber & Svensson, 2003).

1.1.3 The Liver as a Histopathologic Biomarker

As a general trend, the liver of an organism, and the biomarkers it produces act as a central

point of analysis when it comes to monitoring its health. This is attributed to the physiological

associations, and multi-dimensional function it possess in relation to many other organs within

an organism. This principle has been applied to a number of organisms, including humans

(Lee, 2011; Gangadharan, et al., 2012) and numerous fish (Ayas, et al., 2007; Valon, et al.,

2013), and the host in question, G. aculeatus (Handy, et al., 2002).

The study by Handy, et al., (2002) explored the application of G. aculeatus’ liver as a

biomarker, and gave insights as to how water quality affects lipid deposits within the tissues.

The observations indicated ‘mostly normal histology in the highest quality rivers’, and

increased incidence of changes in fatty tissue (from 1-2% in normal livers up to 10% in polluted

rivers), and occurrence of focal necrosis in rivers with increased levels of ionic pollutants (Ca,

Cu, K, Mg, Na, Zn). From this study it was concluded that in terms of simplicity, and

sensitivity, the liver acted as the most efficient biomarker in terms of water quality, and holistic

view of fish health within freshwater ecosystems. A further study identified a number of

biomarker subsets, including morphological indexes (condition factor and liver somatic index),

that are clearly altered in contaminated freshwater streams (Sanchez, et al., 2007).

In G. aculeatus, the liver has proved to be an effective reference point within a number of

studies analyzing the effect of S. solidus infection. Field studies have shown that wild

populations possessing parasite infections show a general trend of a lower somatic body

condition, and liver energy reserves (Barber, et al., 2008). The lower energy reserves observed

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in the liver have been shown to be associated with depleted levels of liver glycogen, and lipids

(Bagamian, et al., 2004). This reduction of lipid, and glycogen content has also been linked to

a weight reduction of the liver itself (Arme & Owen, 1967), which has been shown to increase

with multiple parasite infections (Pennycuick, 1971). The effect of parasite infection on the

liver is most apparent upon maturation, where liver size is maintained in those infected,

whereas uninfected organisms show a sharp increase in liver size during springtime maturation

(Tierny, et al., 1996). However, in a study performed by Arnott, et al. (2000), three-spined

sticklebacks were infected with the parasitic cestode, and subsequently analysed over a three

month period. The results of this study indicated that although parasite infection inhibited liver

growth, overall a parasite-associated growth enhancement was observed, maintaining “similar

or better body condition compared with uninfected fishes”.

1.2 Schistocephalus solidus

1.2.1 Lifecycle

The hermaphroditic pseudophyllidean cestode, S. solidus possesses a three-host lifecycle based

upon trophic transmission (Barber & Scharsack, 2010). Typically the definitive hosts are fish-

eating birds. However, it has been shown that other endotherms, including otters, may harbour

the parasite (Hoberg, et al., 1997). The definitive host acts as a vector for S. solidus to reach

sexual maturation, either by self- or cross-fertilisation, dependent on whether the host possesses

single or multiple infections, respectively (Schjorring, 2004). Eggs are released via fecal

excrement into water, where they proceed to develop into free-swimming coracidia. These free-

swimming coracidia are transmitted trophically into their first intermediary host, Macrocyclops

albidus (Abteilung Verhaltensökologie, Zoologisches Institut, 1997), where they develop into

procercoids. It is at this point they become infective to their obligatory specific second

intermediary host, G. aculeatus (Braten, 1966), with infection occurring trophically.

1.2.2 Copepod-Stickleback transition of S. solidus

Transfer of the parasite between its first intermediate host and second intermediate host is

initiated by the parasite causing a change in the copepod’s behavior, a common natural

occurrence (Moore, 2002). A transition occurs within the infected copepod, inducing a

behavioural shift where the organism adopts patterns of predation enhancement, opposed to

those aimed at suppression (Hammerschmidt, et al., 2009; Parker, et al., 2009). This method

acts as a means to optimize parasite fitness, increasing the probability of successful

establishment in G. aculeatus, whilst reducing the chance of mortality in the copepod host.

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1.2.3 Mortality evasion in G. aculeatus

Upon ingestion of a S. solidus infected copepod, G. aculeatus proceeds to digest the prey in

the stomach, causing procercoids to be released from the copepods’ tissues, into the digestive

tract (Marwaha, et al., 2013). These procercoids have been shown to retain their PNA-binding

sugar- saturated (D-galactose, and GalNac) outmost layer, which pertains to protecting the

parasite from enzymatic digestion in the stomach (Hammerschmidt & Kurtz, 2009). Upon

passing the stomach, S. solidus proceeds to penetrate, and anchor itself to the intestinal wall

with a hooked cercomer (Barber & Scharsack, 2010). From here the procercoid outer layer is

broken down, exposing a salicylic acid-rich tegument layer, possessing microtriches. This is

believed to act as a defense mechanism against G. aculeatus’ immune system (Hammerschmidt

& Kurtz, 2005) whilst it penetrates the hosts’ body cavity. Here the parasite settles, developing

into a plerocercoid, providing initial evasion of the hosts’ immune response to tissue injury is

successful (Wedekind & Little, 2004).

1.3 Host-Parasite Interactions

1.3.1 Immune response of G. aculeatus upon parasitic infection, and S. solidus evasion of the

G. aculeatus immune system

Providing the evasion of the initial tissue damage-facilitated immune response is successful, S.

solidus must then successfully defend against itself during the early stages of infection (1-2

weeks), where it is most vulnerable to the immune response (Barber & Scharsack, 2010).

An initial problem for the parasite is the head kidney leukocyte (HKL) respiratory burst,

releasing monocytes as an early innate response (Franke, et al., 2014). It has been shown that

S. solidus may overcome this via immune manipulation by cyclically installing surface coats

that are not immunogenic to the host (Scharsack, et al., 2007), thus causing fluctuation in levels

of monocyte proliferation. This does not immunologically compromise the host, but rather

evades parasite-specific antigens (Scharsack, et al., 2004). This immune system priming by S.

solidus has not been shown to induce resistance in the stickleback, but rather pave the way for

subsequent infections, increasing G. aculeatus’ susceptibility, allowing for subsequent

infections to outlive, and outgrow pioneer worms (Orr, et al., 1969; Jäger & Schjørring, 2000).

Once past the early infection stage S. solidus must then face the challenges brought about by

the adaptive immune response. Similarly to the monocyte response, significant B- and T-cell

proliferation is observed in organisms which successfully evaded infections (Scharsack, et al.,

2004), with a less prominent pattern of proliferation being observed among those which did

not. This indicates that a strong B- and T-cell response is key in overcoming parasitic infection.

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However, unlike mammals, there is no clear T-helper cell (Th1/Th2)-mediated immunity,

which is key in eliminating parasitic infections (Wang, et al., 2008).

1.3.2 Behavioral resistance, and effects of S. solidus infection

Unlike many organisms, G. aculeatus is unable to selectively predate on its prey due to lack of

visually identifiable characteristics on a microscopic scale. The risk of avoiding parasitized

copepods outweighs the reward, as it limits the resources available to G. aculeatus (Barber &

Scharsack, 2010). Although, with its prey residing on such a small scale, the risk of infection

is relatively low (Lafferty, 1992). However, this risk is dramatically increased by behavioral

modifications of procercoids to their copepod host, altering swimming and activity patterns

(Pasternak, et al., 1995; Wedekind & Milinski, 1996), actively approaching G. aculeatus

(Jakobsen & Wedekind, 1998).

Studies have shown that individuals harboring S. solidus parasites are less favorable to potential

partners, with females preferring mates with genes encoding parasite resistance (Keymer &

Read, 1991). This is phenotypically exhibited in males due to a brighter red nuptial coloration

(Barker & Milinski, 1993). Offspring of these males have been shown to possess a higher

resistance to parasite infection (Barber, et al., 2001).

In short, the findings suggest that although G. aculeatus has the potential to successfully clear

S. solidus infections, the parasite itself has successfully developed its own molecular response

to the means of defense exhibited by the host. This allows for the parasite to successfully evade

G. aculeatus’ immune system, and establish an infection, leaving the host more susceptible to

subsequent infection (Iqbal, 2014).

1.4 Proteomics

As an analytical technique, genomics is effective in providing an insight into predicted gene

products. However, a number of these gene products seemingly do not possess a known

function (Blackstock & Weir, 1999). Proteomics addresses this by aiming to provide a

comprehensive, quantitative protein complement of a genome (Anderson & Anderson, 1998).

This is achieved by analysis of products of transcription, i.e. proteins, and any post-translational

modifications they may possess (Mann & Jensen, 2003). In terms of host-parasite interaction

studies, proteomics has proved an invaluable resource, and possesses a lot of potential in the

elucidation of molecular mechanisms, including those that cause alterations in the hosts’

behaviour (Biron, et al., 2005). In particular, proteomic techniques have been heavily utilised

in host-parasite studies pertaining to humans, including; Toxoplasma gondii (Bradley, et al.,

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2005), Schistosoma japonicum (Liu, et al., 2007), Brugia malayi (Bennuru, et al., 2009) and

Plasmodium species (Bautista, et al., 2014), and in each the importance of proteomics has been

highlighted when it comes to identifying unique, and novel protein interactions.

1.4.1 Proteomic Methods and Analyses

When analyzing proteins, there are a number of methods, and angles that may be taken. The

first is protein-detection by immunoassay, an antibody/immunoglobulin-based technique to

quantify macromolecule concentration in solutions (Darwish, 2006). There are a number of

these techniques available, but in proteomics, ELISA (Enzyme-Linked ImmunoSorbent

Assay), SISCAPA (Stable Isotope Standard Capture with Anti-Peptide Antibodies), and MSIA

(Mass Spectrometric Immunoassay), prove to be most effective in quantitatively measuring

protein content of a small sample (Braitbard, et al., 2006; Anderson, et al., 2009; Weiss, et al.,

2014). However, in more complex mixtures, more powerful methods are required, such as;

MALDI (Matrix-Assisted Laser Desorption/Ionization), ESI (Electrospray Ionization), and

FASTpp (Fast Parallel Proteolysis). These methods have been shown to allow for a greater rate

of elucidation in complex protein mixtures (Song, et al., 2005; Yang, et al., 2007; Minde, et

al., 2012).

One of the most commonplace methods of protein analysis is by the use of two-dimensional

(2D) gel electrophoresis. By separation of complex mixtures on a two-dimensional plane, it is

possible to isolate, and determine specific post-translational protein modifications (Gygi, et al.,

2000) across a narrow pH range. The high resolving power, coupled with its large sample

loading capacity make this an effective standard for proteomic analysis, allowing for direct,

and global views of a sample proteome at any given time point (Chevalier, 2010).

1.4.2 Biomarker identification, and analysis

With proteomics becoming increasingly commonplace it has proved particularly useful in the

study of disease. It has achieved this by monitoring factors such as protein abundance,

structure, and/or function. Often referenced to as biomarkers, these three attributes behave as

useful indicators of pathological abnormalities (Hanash, 2003; Xiao, et al., 2005) within a host.

Every measurable indicator of a protein is unique, giving rise to an individual biomarker for

every possible change which may be observed in a protein (Biomarkers Definitions Working

Group, 2001). In parasitology, these biomarkers prove themselves to be invaluable in

measuring the type, and extent of an infection, as well as any physiological changes that may

be associated with it (Deckers, et al., 2008). In fish species, including G. aculeatus, these

biomarkers allow for monitoring changes in processes, for example glutathione-S-transferase

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(GST) activity (Frank, et al., 2011); fast-start performance (Blake, et al., 2006) and hepatic

activity (Katsiadaki, et al., 2010).

1.4.3 Limitations, and considerations

Although proteomics has been proved as an effective method of protein quantification, and

analysis, as with anything, it is not without its limitations. The primary limitation is the effect

of post-translational modifications on proteins. Generally these modifications have a profound

effect on the activity, and stability of a given protein, potentially producing a number of

permutations, with varying function(s) (Febbraio, et al., 2003). To identify and isolate these

proteins more specific, and complex analyses are required, such as glycoproteomics (Tissot, et

al., 2009) and phosphoproteomics (Kalume, et al., 2003).

On the transcriptomic level, issues arise in the expression levels of proteins, as once

transcribed, proteins may rapidly degrade, or be differentially expressed, resulting in

measurements which are unrepresentative of the true transcriptional output (Belle, et al.,

2006).Another impactful issue with proteomics is the reproducibility of experiments between

laboratories, with separate studies showing differing levels of proteins present in an organism.

One comparison that may be drawn is an experiment performed by Peng, et al., (2003), where

1504 yeast proteins were identified, with only 858 having been previously identified

(Washburn, et al., 2001). Of the 858 identified, 607 were not discovered by Peng, et al., (2003).

These levels of reproducibility, 57% and 59% respectively, brings to light how encompassing

proteomics studies are when mapping the proteome on a large-scale.

1.5 Key Hypotheses/Aims of Study

This project monitors the response of G. aculeatus to infection with S. solidus, on a proteomic

level. This will be achieved by comparatively assessing the presence/absence of protein

markers within the liver of G. aculeatus, signaling the presence of S. solidus infection. Two-

Dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis will be used to test

this. From this the aim is to address the following key hypotheses;

1. S. solidus infection presents clear patterns of differential/unique protein expression in

G. aculeatus species.

2. There is a relationship between the number of parasite infections, and the degree of

protein expression observed.

3. The liver acts as an accurate indicator of the health of G. aculeatus.

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2. Materials and Methods

2.1 Source of G. aculeatus, and S. solidus Species

Thirty-two G. aculeatus samples were collected from a lake, Llyn Ffrongoch in Ceredigion,

Wales. Each individual was measured for both length and weight, and subsequently dissected

and screened for presence of S. solidus infection. Those possessing an infection were recorded

(including number of parasites), and those possessing an infection had their parasite(s)

weighed. The liver was then excised under a ddH2O media, rapidly cooled by means of liquid

nitrogen, and stored at -80 °C to ensure minimal tissue degradation occurred until they were

used.

2.2 Protein Isolation, and Quantification from Liver Samples

Protein extracts were obtained by homogenization of liver samples on ice in an Eppendorf

containing a lysis buffer composed of 20 mM KHPO4, pH 7.4, 0.1% v/v Triton –X 100 and a

cocktail protease inhibitor (Roche, Complete-Mini EDTA-free). These homogenates were

subject to centrifugation at 100,000 x g for 45 minutes at 4 °C, and the resulting supernatant

was removed and retained, residual pellets were suspended in 5 µl homogenization buffer by

sonication to be used for subsequent analysis.

To the supernatant, 5 µl of 20% w/v TCA in ice-cold acetone was added, mixed well by

inversion, and stored at -20°C for 1 hour. Samples were subsequently subjected to

centrifugation at 21,000 x g for 20 minutes at 4 °C, the supernatant was discarded, and the

pellet re-suspended in 200 µl of ice-cold acetone via sonication. The pellet was then re-

subjected to centrifugation for a further 15 minutes at 4 °C and 21,000 x g, and once complete

the supernatant was discarded. For a final time, the pellet was submerged in 200 µl ice-cold

acetone, sonicated, and subjected to centrifugation for 15 minutes at 4 °C and 21,000 x g. The

supernatant was removed, and discarded, the pellet was air dried for 15 minutes at -20oC, then

stored at -20oC until needed.

The pellets were sonicated into 50 µl Buffer Z (4.8g 8 M Urea, 200 mg CHAPS, 50 mg DTT,

50 µl 0.5% ampholytes, 5 ml dd H2O), 5 µl of which were taken, and quantified using Bradford

Reagent (Bradford, 1976). The absorbance values obtained were recorded and compared

against a standardized (Bovine Serum Albumin) curve. Comparison against this standardized

curve allowed for calculation of the protein concentrations in mg/ml. For any concentration

<100 µg/µl, all of the remaining solution, 45 µl, was added to 80 µl Buffer Z, for a total volume

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of 125 µl. For values ≥ 100 µg/µl, the samples were diluted to 100 µg per 125µl, by addition

of excess Buffer Z.

2.3 Two-Dimensional Electrophoresis

Of each sample, 125 µl was loaded onto 7 cm pH 3-10L IPG strips, and in-gel rehydrated

overnight and subsequently isoelectrically focused to 10-13,000 V h, limited to 40 µA/gel at

20 °C on a Protean IEF Cell (BioRad).

After focusing, strips were equilibrated for 15 minutes in reducing equilibration buffer (50 mM

Tris-HCl, 6 m Urea, 30% v/v glycerol, 2% w/v SDS, 1% w/v DTT) followed by 15 minutes in

alkylating equilibration buffer (50 mM Tris-HCl, 6 m Urea, 30% v/v glycerol, 2% w/v SDS,

4% w/v iodoacetamide). Any strips not being run immediately were stored at -20 °C until

needed, prior to incubation with DTT and IAA

Sodium dodecyl sulphate (SDS) polyacrylamide gels (7 cm) were prepared for each of the

samples, the resolving gel consisting of; 8.85% acrylamide, running gel buffer (1.5 M Tris-

HCl, 0.4% SDS, pH 8.5), ddH2O, 10% v/v ammonium persulphate, and 10µl TEMED. The

stacking gel consisted of the same constituent ingredients at the same concentrations, but the

running gel buffer was substituted for stacking gel buffer (0.5 M Tris-HCl, 0.4% v/v SDS, pH

6.8). The IPG strips were set into the 14% gels with 6% agarose in 0.125M Tris-HCl, pH 6.8,

and run at 70 V for 30 minutes, then 150 V until completion, at 20 °C. The gels were then fixed

for 1 hour with 40% ETOH, 10% acetic acid, then followed by two 10-minute washes with

dH2O. After washing, the gels were stained for a minimum of 8 hours using Colloidal

Coomassie blue stain (80% Colloidal Coomassie to 20% Methanol). Once complete the gels

were washed 3 times for 5 minutes using 1% acetic acid.

The gels were imaged on a GS-800 calibrated densitometer (BioRad), and stored in 1% acetic

acid at 5 °C.

2.4 Statistical Analysis of Results

Analysis of variance (ANOVA) and regression analyses were performed in order to monitor

the effects of S. solidus parasitism on the mass, length, and body condition of the infected fish.

Whereas ANCOVA (analysis of covariance) was performed in order to measure the effect of

parasitism on the quantity of protein in the liver, whilst accounting for body condition.

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3. Results

3.1 Correlation Studies

Figure 1 shows a non-linear relationship between the weight and length of G. aculeatus

species, and rather presents itself as an exponential function. The average taken of uninfected

and infected samples indicates that parasitism has an impact on overall weight, and length of

the organism, reducing them by 0.078 g (0.285 g to 0.207 g) , and 0.47 mm (28.33 mm to 27.86

mm) respectively. The majority of samples resided in the region of 22-32 mm possessing

weights of 0.012-0.086 g. However, only un-parasitized organisms exceeded these growth

parameters. Regression analysis of the exponential indicates P<0.0001 for each of the three

curves, indicating that any significant amount of variation in weight is explained by length.

This is also reaffirmed by the high values obtained for R2 for each curve.

Figure 1: Correlation between Fish Mass and Fish Length, in grams and millimetres

respectively. Overall, an exponential increase in the relationship between mass and length is

observed in the species. An average of both parasitized, and un-parasitized organisms was

taken, and plotted. The regression lines are representative exponential growth curves of

Uninfected, Infected, and Total (Infected + Uninfected) organisms.

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Figure 2: Percentage of the overall mass accounted for by S. solidus upon measuring the

weight of G. aculeatus before dissection. These values account for organisms possessing single,

and multiple infections. Organisms possessing a single infection are found to the left of the

dashed line, whereas organisms possessing multiple infection are situated to the right.

Further analysis of weight indicates that up to 33% of an organisms’ weight may be accounted

for by S. solidus upon infection (Fig. 2). However, it may also be as low as 10% in other cases.

It can be seen that organisms possessing multiple infections have a greater percentage of their

overall body weight accounted for by S. solidus plerocercoid, than those organisms possessing

a single infection. Further manipulation of the data indicated that, on average, single infections

constituted for 21.67% of G. aculeatus’ overall body weight, whereas multiple infections

constituted for 23.16% of body weight on average. However, analysis of variance indicated

that the two groups do not possess means which are significantly different (F1,16= 0.308, P=

0.587)

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Figure 3: The effect of S. solidus parasitism on the body condition (a) of G. aculeatus species.

A subset of four samples were taken from each grouping as to not skew the results due to

varying sample numbers within the datasets. The LWR (Length Weight Relationship) values

for each fish were calculated by use of the equation: W= aLb, where W is the weight of the fish

in grams, L is the length of the fish from snout to tail in millimetres, b is the isometric growth

in body proportions (3.10 for G. aculeatus), and a is the parameter describing the body shape

and condition. The values obtained were then averaged for each degree of infection, and

plotted accordingly. The error bars seen are representative of the standard error within each

group.

Length and weight data was used to calculate the body condition (a) of each sample, allowing

monitoring of the effect of differing degrees of S. solidus parasitism on the body condition of

G. aculeatus (Figure 3). Overall a reduction of 0.00227 (0.01051 to 0.00824) is seen between

the ‘No Infection’ and ‘Single Infection’ sample groups, but an increase of 0.00113 (0.00824

to 0.00937) separating the ‘Single Infection’ and ‘Multiple Infection’ groups. The reduction

seen between the ‘No Infection’ and ‘Single Infection’ samples shows a statistically significant

reduction in body condition (P= 0.014), with no overlap of the standard error. The increase

seen between ‘Single Infection’ and ‘Multiple Infection’ possesses no statistical significance

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(P= 0.165), and thus is indicative that any variance observed between the two sample groups

may be attributed to standard error. The reduction in body condition between the ‘No Infection’

and ‘Multiple Infection’ also presented itself as insignificant (P= 0.165). The standard error

across the three degrees of infection are relatively small, indicating a high degree of fidelity in

the results. However, of the three, the largest degree of error is seen within the ‘No Infection’

grouping.

Figure 4: Effect of varying degrees of parasitism on protein total protein in the liver of G.

aculeatus. A total of 12 liver samples were assayed for protein, 4 for each group; ‘No

Infection’, ‘Single Infection’, and ‘Multiple Infection’. Error bars are representative of the

standard error within each group.

Figure 4 shows a trend in the average total quantity of liver protein across the three sample

groups. Overall a reduction in the concentration of liver protein is seen as the degree of S.

solidus plerocercoid infection increases. However, this trend is seemingly non-significant, due

to the nature of the error bars associated with the data. Initially it may be seen that the ‘No

Infection’ sample group possesses a significantly greater error bar in relation to the two

infection group, whereas the standard error between the ‘Infection’, and ‘No Infection’ was

relatively small. However, with the standard error for each of the sample groups encompassing

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the average liver protein concentration of the others, little significance may be drawn from the

correlation between degree of infection, and average liver protein concentration alone.

Figure 5: Profile plots resulting from

ANCOVA analysis of (A) ‘No Infection’ and

‘Single Infection’, (B) ‘No Infection’ and

‘Multiple Infection’, and (C) ‘Single

Infection’ and ‘Multiple Infection’. For each

model, the concentration of liver protein

(µg/µl) was the dependent variable, with the

body condition of G. aculeatus acting as a

covariate.

Analysis of covariance revealed no significant correlation upon analysis of the quantity of liver

protein in relation to the degree of parasitic infection whilst accounting for the effect of body

condition (Fig 5). Analysis comparing the ‘No Infection’ and ‘Single Infection’ sample groups

(Fig. 5A), generated a profile plot shown an increase of roughly 11500 µg in the estimated

marginal means of protein from ‘No Infection’ to ‘Single Infection’ (R2=-0.024, F1,8=1.821,

P=0.235). Furthermore, an increase of only approximately 3000 µg is observed from ‘No

Infection’ to ‘Multiple Infection’ (Fig. 5B), substantially less than the reduction seen when

comparing ‘No Infection’ to ‘Single Infection’, and remains unrepresentative on any

statistically significant reduction in the quantity of liver protein (R2=-0.303, F1,8=0.355,

P=0.577).

A B

C

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3.2 Gel Analysis

.

(Figure legend overleaf)

A

B

Mult

iple

Infe

ctio

n

1

1

1

2

2

2

3

3

3

4

4

4

5

5

a

b

c

Sin

gle

Infe

ctio

n

No I

nfe

ctio

n

C

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Figure 6: Representative 2D SDS-PAGE arrays for the liver proteins of G. aculeatus from A

‘No Infection’, B ‘Single Infection’ and C ‘Multiple Infection’ sample groups. The proteins

were profiled on 14 % polyacrylamide gels and stained with coomassie blue. Proteins were

isoelectric focused on 7 cm pH 3-10 IPG strips. Circled proteins appear to show significant

difference between gels. The differences observed are relatively consistent across the replicate

gels. Discrepancies are highlighted in the appendices. Proteins spots seemingly with

differences in expression are circled and numbered accordingly (1-5) across each of the three

representative gels.

Upon comparison of the ‘Single Infection’ and ‘Multiple Infection’ sample groups, a decrease

of approximately 1250 µg is seen in the estimated marginal means of protein when the degree

of parasitic infection increases (Fig. 5C). This marginal value shows no significant reduction

in the quantity of liver protein when the number of parasites present in the body cavity

increases, with little variance being accounted for by regression between the two groups,

subsequently showing no significance (R2=-0.383, F1,8=0.60, P=0.816).

Table 1: Calculated relative Mr of protein spots (in Daltons) across the three gels seen in

Figure 6. The values were ascertained through calculation of the relative motility of the ladder,

generation of a standard curve, and calculation by means of the straight line equation

(y=mx+c).

Mass of Protein Spot (Da)

Spot No Infection

(A)

Single Infection

(B)

Multiple Infection

(C)

Average

1 7535.28 9428.80 8110.58 8358.22

2a 10885.85 - - -

2b 7722.36 - - -

2c 5753.60 - - -

3 - 8994.44 - -

4 11293.76 - - -

5 - 21522.69 23861.08 22691.89

Comparison of Fig. 6A, and 6B, shows a reduction in intensity of spot 1, with further reduction

seen upon comparing Fig. 6B and 6C. Spots 2 a, b, and c, are seemingly only present in Fig.

6A, with no observable presence on 6B and 6C. However, a little streaking in that area on Fig

6B, possibly indicates the presence of some protein. Spot 3 does not seem to have a presence

in the ‘No Infection’ sample (Fig. 6A) upon comparison to the other degrees of infection (Fig.

6B and 6C). This is may also be true of Spot 5, although, the clustering of proteins in Fig. 6A

makes it difficult to distinguish. Conversely, Spot 4 only shows presence in the ‘No Infection’

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sample group, and not in the ‘Single Infection’, or ‘Multiple Infection’ groups. All masses for

the relevant spots are seen in Table 1, with averages taken for spots with differing values (Spots

1 and 5). These values are mathematical estimations, and not discrete values.

4. Discussion

4.1 Size variation in G. aculeatus

Age, size and lifespan are critical factors affecting the lifetime reproductive output of an

individual (Clutton-Brock, 1988), and subsequently evolution within wild populations

(Charlesworth, 1994). Drastic fluctuation in any of these factors has the potential to heavily

impact the effective population size and generation length, subsequently leading to a reduction

in the overall genetic diversity within a population (Waples, 2010). Generally wild adult

sticklebacks reach an age ranging from 1.8-3.6 years, with potential maximum lifespans of

three to six years, dependent on locality (DeFaveri & Merila, 2013). This results in a large

degree of variation between the body sizes of individual organisms. Furthermore, this leads to

significant differences both within, and between populations, particularly when comparing

freshwater and marine species. Marine species’ lifespans, survivability, and mass exceed that

of their freshwater counterparts (Leinonen, et al., 2006), irrespective of S. solidus parasitism.

Although no between-population variation was explored in this study, within-population

variation may possess a significant role in the results ascertained from this study.

Uninfected (‘No Infection’) organisms showed the greatest variation in both length and weight,

with sizes ranging between 19-41.5 mm, and 0.082- 0.9863 g respectively (Fig. 1). Uninfected

organisms show limited growth of the fish in the x-axis, with any further increase in body

weight residing in the y-axis, indicating that G. aculeatus species do not generally exceed 40-

50 mm in length, without having to gain significantly more mass, irrespective of parasitism.

However, ‘Infected’ (‘Single Infection + ‘Multiple Infection’) individuals did not achieve the

upper band of growth (≥36.5 mm and ≥0.517 g) seen in the unparasitised specimens, and failed

to surpass 32 mm, and 0.0086 g, suggesting that S. solidus infection negatively impacts growth

in both the 𝑥- and 𝑦-axis. This is further seen in Figure 1, with the exponential growth curve

for both the ‘Infected’, and ‘Total’ groupings residing lower than that of the ‘Uninfected’

group, whilst each is extrapolated to the length of 43 mm. Previously it has been shown that,

under laboratory conditions, S. solidus plerocercoids have a significant impact on the growth,

and morphology of G. aculeatus species. A study by Barber & Svensson (2003) showed that

over a period of 6 weeks, infected females underwent gross distention of the body cavity, and

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rapid weight gain. This rapid gain in body weight was associated with parasite growth, rather

than the host, reducing the weight and length gained by G. aculeatus. Similar distention has

also observed in males, which results in failed nest construction and further disruption of

spawning behavior (Williams & Jones, 1994). Interestingly, the mean value for length of

uninfected organisms resided at 28.3 mm in this experiment, 21.7 mm lower than the average

length of 50 mm (Scott & Crossman, 1998). Furthermore, the average length for parasitized

organisms is only 0.47 mm less than that of uninfected organisms, with a reduction of 0.078 g

in weight. The marginal difference in mass observed may be accounted for by the nutritional

requirements of the S. solidus plerocercoid, where it has been shown that there is an observable

behavioral response in G. aculeatus which results in an increased food intake, as not to harm

the host (Barber, 2005).

In terms of percentage composition of the organism, the plerocercoids generally constituted

10-33% of the overall body weight of G. aculeatus (Fig. 2), further reaffirming the findings by

Barber & Svensson (2003). The lack of significant difference in average percentage

constitution between the ‘Single Infection’ and ‘Multiple Infection’ sample groupns (P>0.05)

is indicative that individual parasite size is dependnt on parasite load. However, the differing

size of sample groups may skew the data, resulting in little variance, due to the ‘Multiple

Infection’ sample size being less than half of that of the ‘Single Infection’.

Two notable factors not accounted for in these results are age of the sticklebacks, and

temperature of their environment, as both have been shown to significantly impact growth

rates. Temperature-dependent growth models for the stickleback have revealed that growth

occurs between 5-29 oC, with estimates showing the optimal growth temperature lying around

21.7 oC (Lefebure, et al., 2011). Seasonally this effect is also pronounced, with 60% higher

growth rates being achieved in the summer compared to winter. As the samples collected in

this study were carried out in late autumn/early winter, the growth parameters measured may

be more representative of the winter model. However, irrespective of temperature, it has been

shown that over the 128 days post-hatch (ph), G. aculeatus standard length reaches 33-41 mm

(Wright, et al., 2004).

Upon comparison of the data obtained through this study, only 4 of the 32 samples collected

were within this 33-41 mm range, three of which showed no observable parasitism. With a

number of uninfected samples in this study posessing values below this range, age and water

temperature should be considered as determinant factors. However, as all but one of the

parasitized organisms lay below the 33 mm threshold, it may be stipulated that S. solidus

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parasitism, combined with the aforementioned determinant factors, further impedes the growth

of sticklebacks. The reduced organism size observed may also be explained by individuals

being immature, and having not reached the 128 ph threshold.

Effect of varying degrees of parasitism on the overall body condition of sticklebacks is

observed in this study through the means of the length-weight-relationship (LWR) (Fig. 3).

Observations have shown that as the degree of parasitism increases a decrease in body

condition is observed, with an average reduction of 16.19% in body condition being measured

for organisms possessing both single and multiple infections. This average reduction is implicit

of S. solidus having a pronounced effect on the body condition of G. aculeatus. In comparison

to published values, the body condition calculated for the ‘No Infection’ sample group (a=

0.01051) resides close to the average of 0.01023 for healthy individuals (FishBase, 2015). This

indicates that the samples ascertained in the experiment show that, whilst not possessing

average body dimensions, the conditions observed are standard. When comparing the ‘Single

Infection’ (a= 0.00822) and ‘Multiple Infection’ (a= 0.009370) sample groups to the average

range (0.00404 – 0.02593), it is apprent that body condition is maintained and does not fall

below the range for the species, but there is a notable deviation from the norm, further showing

the impact of plerocercoids on the body condition of sticklebacks. However, the body condition

of ‘Multiple Infection’ samples rested higher than organisms possessing only a single parasites,

which is most likely down to the size of the sample groups. Although the degree of standard

error in the results was relatively low, and relatively accurate of body condtion averages, a

larger sample size may have produced more pronounced differences, which may have

accounted for this discrepency, as an increase in the degree of parasitism would be expected to

further reduce or maintain the ‘Infected’ body condition, rather than improve it (Bagamian, et

al., 2004).

4.2 Physiological effects of S. solidus parasitism

As an intermediate host, G. aculeatus has a number of roles to fulfil in order to successfully

facilitate the transition of S. solidus to its definitive host. In order for this to occur, the

plerocercoid induces a number of physiological and behavioral changes within the host in order

to increase the chances of transmission. Behavioral changes, such as shoaling, and foraging,

are thought to be induced by changes in brain monoaminergic activity, with upregulation of

norepinephrine, and 5-hydroxytryptamine increasing the chronic stress experienced by the

infected organisms (Overli, et al., 2001). Physiologically, a number of changes come about

upon S. solidus infection, particularly in the (aforementioned) brain, and kidney, and liver. The

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kidney has been extensively studied, and utilized as a physiological marker in a number of

parasitism studies, with applications such as; monitoring the heterotrophy in infected nest-

building males (Rushbrook & Barber, 2006), monitoring spiggin levels in reproductive

physiology (Rushbrook, et al., 2007); and immunological granulocyte responses (Scharsack,

et al., 2004).

Morphologically, the liver of G. aculeatus shows a reduction in absolute weight in infected

individuals during all seasons of the year (Arme & Owen, 1967; Tierny, et al., 1996), but has

also conversely shown gross enlargement (via growth enhancement) under laboratory

conditions (Arnott, et al., 2000). As liver weight was not measured in this experiment, no

comment is able to be made on any morphological differences that may suggest a direct

relationship between the hepato-somatic index, and parasitic infection. Subsequently, from this

study there was no observable direct correlation between the average concentration of liver

protein, and the degree of parasitism (Fig. 4), inferring that (increased) parasitism has no effect

on the average concentration of liver proteins, and that there is no direct action of S. solidus on

the liver of its stickleback host. Although no significant decrease is observable within the

results, a decrease is seen nonetheless, and potentially, the high degree of variability within the

results may be explained by the small sample sizes, as the protein concentration was only

measured across four individuals within each degree of infection, a total of 12 samples overall.

However, small S. solidus specimens have been shown to lie below the peritoneal lining of the

body cavity, whereas in heavily infected organisms, anterior displacement of the liver and heart

has also been seen, with the gall bladder staying in place, and remaining free of liver tissues

(Arme & Owen, 1967).

When taking body condition as a cofactor in ANCOVA analysis, it is further reaffirmed that

there is no distinct correlation between increasing parasitic infection and a reduction in the

quantity of hepatic proteins (Fig. 5). Although no statistical significance is observed, there is a

reduction in the quantity of total liver proteins across the three sample groups. This still may

be due to the effect of S. solidus parasitism, as reduced energy reserves within the hepatic

structures of sticklebacks have been shown to be heavily influenced by the food rations

available to them, with low-food environments negatively impacting liver size (Wright, et al.,

2007). Although in a non-mutual symbiotic relationship, S. solidus does increase the quantity

of food G. aculeatus eats in order to maintain the needs of both itself and its host, which may

explain the similar quantities of liver protein seen across the three degrees of infection.

However, the aforementioned study by Wright et al. (2007) has shown that in parasitized

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organisms there is a substantial reduction in liver size, with compensatory growth responses

dropping by up to 20%, inferring that a lower quantity of protein would be expected.

Other studies have shown that there is a histological effect observed within G. aculeatus upon

S. solidus parasitism, with reduced energy reserves seen in the hepatic structures, as well a

general metabolic drain (Arme & Owen, 1967; Pennycuick, 1971; Bagamian, et al., 2004). The

findings implied that upon parasitism, chemical composition, and function of the liver were

hanged in response to the foreign body.

There is no statistical significance underpinning the differing levels of protein concentration

between the ‘Single Infection’ and ‘Multiple Infection’ groupings, contrary to what was

predicted. Although a slight reduction in concentration was observed between the two sample

groups, the results are indicative of multiple S. solidus infections having little effect in

decreasing the metabolic reserves available to its host. However, when multiple infections are

present, S. solidus individuals have been shown to grow in proportion to the total intraspecific

density, which subsequently resulted in smaller overall size for the individuals in comparison

to single infections (Brusca & Brusca, 2002; Michaud, et al., 2006). This growth adaptation

may give reasoning to the only slight reduction in the overall liver protein concentration, with

smaller parasites generally having lower nutritional requirements (Gunn & Pitt, 2012). As

previously stated, an increased sample size may present a more significant difference across

the three degrees of infection, particularly between uninfected and infected (single and

multiple) samples, especially due to the contradictions presented by this study in comparison

to other published works. Although, when comparing organisms possessing only a single

plerocercoid to those with multiple infections, it should be noted that morphological

adaptations by the parasite result in no significant change in the quantity of hepatic proteins.

4.3 Differential protein expression in the G. aculeatus-S. solidus model

Upon analysis by SDS-PAGE, the varying levels of protein expression was visualized across

the three degrees of infection (Fig. 6). Three representative gels were chosen for differential

analysis, providing the most defined ‘spots’ for the relative treatment groups. The other gels

produced remained relatively consistent in comparison to these representatives, with only slight

deviation in protein expression patterns observed in three of the twelve gels, the discrepancies

of which are highlighted in the appendices.

From comparative analysis, seven protein spots were identified as having potential to act as

histopathological biomarkers in S. solidus infection. Of the seven spots, one showed a reduction

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in size across the three infection groups, four seemed exclusive to organisms with plerocercoid

infections (Single + Multiple), one was exclusive to the ‘Single infection’ group, and the final

seemed unique to un-parasitized organisms. Spot 1 is a relatively small protein (in respect to

the other labelled spots) with an average calculated mass of 8358.22 Da (Table 1). Of the 12

samples screened, 10 expressed this spot (see appendix B (d) and (j)); in those that did not the

gels had under-run, resulting in distorted images. Of those with clear expression, a general

trend of reduction in size of the spot may be seen as the degree of infection increases,

suggesting that increased S. solidus parasitism reduces the overall concentration of this low Mr

protein.

Spots 2a, b, and c (Mr’s of 10885.85 Da, 7722.36 Da, and 5753.60 Da respectively) all appeared

near-exclusively in sticklebacks possessing no infection (appearing only once in an organism

possessing a single infection (see Appendix B (f)). The lack of these proteins in infected

samples indicates that parasitism by S. solidus directly influences a system(s) that near-

eliminates the expression of this particular group of proteins in infected sticklebacks. This loss

of protein may be indicative of breakdown of a stored protein in response to parasitic infection.

Most likely, it is a stored metabolic protein that is utilized as a substitute energy resource upon

infection, due to the derivation of nutrition it causes. It may also be stipulated that these protein

spots could potentially be a cascade of proteins synthesized in the liver due to the unity in their

disappearance. However, further research should be performed in order to confirm this.

Spot 3 (Mr= 8994.44 Da) seemed to be the only spot which was exclusive to the ‘Single

Infection’ sample group on the representative gels, and was also present on two of the three

other replicates. It was however also observed in a ‘Multiple Infection’ sample (see Appendix

B (i)). The seeming exclusivity of this protein in organisms possessing a single infection may

suggest that lone S. solidus plerocercoids residing in the body cavity of sticklebacks may

secrete a protein that is picked up by the liver over the course of infection. The reason it may

only be present in higher concentrations in organisms possessing a single infection may be

down to the reduced plerocercoid size in organisms possessing multiple infections. Due to the

reduced parasite size, there may be insufficient chemical signals in order to trigger expression

of enough of these host-derived proteins to be visualized on a 2D gel.

However, Spot 4 (Mr= 11293.76 Da) only resided within samples possessing no parasitic

infection, suggesting that S. solidus parasitism eradicates expression of this liver protein within

the host organism. From all the potential (identified) spots, it is the strongest candidate to act

as a histopathological biomarker to indicate parasitic infection by S. solidus.

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Spot 5 (average Mr= 22691.89 Da) appeared to be exclusive to organisms possessing parasitic

infections, thus indicating another potential secretion of hepatic proteins in response to S.

solidus parasitism. However, due to the ‘noise’ in that area on the representative gel (and gels

in appendices), the results may potentially be deemed inconclusive, and further research should

be performed to confirm that the spot is in fact exclusive to organisms infected with the

plerocercoid.

As with all gel analysis, the relative abundance, or prevalence of protein on an image is

subjective, and may not necessarily be accurate representations of the true status of the

proteome within in the organism. Further in-depth exploration via gel image analysis software,

Progenesis QI®, trypsin digests and mass spectrometry are crucial in confirming the uniqueness

and identities of the identified spots. Although the majority of gel images were relatively clear,

a number possessed large degrees of horizontal streaking, affecting the overall quality of the

image, as well as impacting the interpretation of the images. This issue may be accounted for

by potentially insufficient/excessive focusing time, or highly alkaline conditions (Gorg, et al.,

1997), with the latter of two being unlikely to be a factor in this experiment. Another issue

arose in comparative analysis of the gels, as computational gel image analysis was not

performed, and as analysis was done by eye, there was a high degree of human error involved.

This may have been improved upon by, not only using software, but also performing

fluorescent two-dimensional “Difference Gel Electrophoresis”(DiGE), allowing for the

analysis of multiple protein samples, by co-separation on the same 2DE gel (Viswanathan, et

al., 2006). 2D DiGE possesses a number of advantages over conventional 2DE, such as;

fluorescence intensity allowing for the ratios of individual proteins in separate samples to be

compared (Chevalier, 2010), eliminating the need for generation of ‘averaged’ gels in

computational analysis (Goldfarb, 2007), high throughput analysis of protein expression

profiles due to being able to load a gel with up to three separate samples by use of cyanine dyes

(Cy2, Cy3, and Cy5) (Marouga, et al., 2005), and accurate quantification of low abundance

proteins (Lilley & Friedman, 2004). However, it does not come without its disadvantages due

to the cyanine dyes increasing the hydrophobicity of, and removing charge from, the protein

via lysine binding. Although, this may be overcome by labelling free cysteines via saturation

binding, but comes at the sacrifice of partial labeling of the proteins in a sample, due to some

not possessing free cysteines (Shaw, et al., 2003).

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4.4 Conclusions and Further Research

In conclusion, parasitic infections of S. solidus bestow a number of physiological changes

within its host, G. aculeatus. A significant reduction in overall mass, and length of sticklebacks

has been observed upon plerocercoid invasion of the body cavity, with up to 33% of the overall

mass (fish + parasite) being accounted for by S. solidus in infected organisms. With percentage

compositions as high as this, the uniqueness of this model is clear to see, with the parasite

attaining its final size within the host, causing proportionally less harm than other cestodes

which require long gestation periods in the digestive tracts of their hosts (Smyth & McManus,

2007). However, there is a marginal, yet definitive observable decrease in the body condition

of infected organisms, showing that although sticklebacks may accommodate their

pseudophyllian cestode passengers, they do not escape the battle completely unscathed.

Although, discrepancies between single and multiple infections were not clearly visible, with

multiple infections not presenting a significantly greater decrease in the body condition of their

hosts over organisms only presenting a single infection, indicating that increased parasitism

does not necessarily confer greater harm being caused to the host.

It has been shown that as the degree of parasitic infection increases, there is a decrease in the

quantity of liver protein. However, the significance of these results suggests that any variation

across differing degrees of infection is justifiable by standard variance, implying that S. solidus

poses little effect on the histology of the liver. Although there is no significant effect on the

quantity of protein within the liver, the expression patterns within the structure does show

variation across differing degrees of infection, with distinct patterns of protein being expressed

in organisms possessing no infection, and those harboring a parasite.

With any experiment of this nature, there are a number of factors unaccounted for which may

explain for some of the variance seen within the results. This is particularly true in the case of

environmental factors, including seasonal variation, temperature fluctuations and quality of

habitat (presence of pollutants), all of which have been shown to contribute towards variable

growth rates within G. aculeatus species. In further experimentation an F1 generation may be

reared under laboratory conditions, with plerocercoids introduced, and environmental factors

altered artificially to see their effect upon the growth of G. aculeatus. Although differential

expression between varying levels of parasitic infection was described, the identities of the

spots isolated remain unknown, leaving an incomplete picture of host-parasite interactions on

a proteomic level. Further work should be done to extract and isolate these potential

histopathological biomarkers, and explore the roles they have to play in the system.

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Acknowledgements

I would like to thank my supervisor Russell Morphew, for his continual support, advice, and

enthusiasm in this project, Rebekah Stuart and for her patience, knowledge and laboratory

expertise.

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Appendices

A: SPSSS Outputs

(i) Percentage Body Composition Accounted for by Parasite

(ii) Fish Condition and Degree of Parasitic Infection

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(iii) Exponential Model: Uninfected + Infected

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(iv) Exponential Model: Uninfected

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(v) Exponential Model: Infected

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(vi) ANCOVA: No Infection vs. Single Infection

(vii) ANCOVA: Single Infection vs. Multiple Infection

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(viii) ANCOVA: No Infection vs. Multiple Infection

B: Gel Images

(i) No Infection

(a) G. aculeatus Liver sample 4. Quantity of liver protein: 2480.63 µg. VH: 10721. Fish

length: 19 mm. Fish weight: 0.082 g

Of the four ‘No Infection’ samples, Liver 4 showed the lowest levels of protein, producing a

gel possessing very faint spots. This lead to some difficulty in analyzing the expression patterns

within the image. The low levels of protein are likely to be attributed to the dimensions of the

sample. There was a large degree of clustering towards the bottom of gel which may have been

more efficiently resolved if the gels were run for longer.

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(b) G. aculeatus Liver sample 9. Quantity of liver protein: 53341.88 µg. VH: 12541. Fish

length: 41.5 mm. Fish weight: 0.983 g

This sample was chosen as a representative gel as it appeared as one of the most clearly

resolved gels, with relatively discrete patterns of expression observable. There was a slight

degree of streaking observed, especially towards the right hand side of the gel. However, not

to the extent where distinguishing between spots was not possible. This sample was also the

one possessing the highest quantity of liver protein. This is likely attributed to the dimensions

of the fish.

(c) G. aculeatus Liver sample 10. Quantity of liver protein: 19344.38 µg. VH: 12671. Fish

length: 36.5 mm. Fish weight: 0.517 g.

Upon commencement of de-staining this gel, it was dropped from the staining dish onto the

bench surface. In the process of transferring it back to the dish, it tore in multiple places.

However, the areas possessing the spots used in this study remained in-tact, as well as the

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ladders remaining whole. Aside from the tearing of the gel, the only criticism with it is the high

degree of streaking across the gel, in places it made it particularly difficult to distinguish

between protein spots.

(d) G. aculeatus Liver sample 11. Quantity of liver protein: 14850.00 µg. VH: 12671. Fish

length: 37 mm. Fish weight: 0.552 g.

As can be immediately seen with this gel, it was significantly under-run, with a distinct band

across the lower half of the gel, where the dye front resides, which resulted in variable

expression patterns in comparison to the other samples, as well as missing Spot 1. Besides

this, the image came out relatively well, with some of the more distinct expression patterns

due to relatively small spot sizes, and less streaking.

(ii) Single Infection

(e) G. aculeatus Liver sample 2. Quantity of liver protein: 28164.38 µg. VH: 10721. Fish

length: 32 mm. Fish weight; 0.300 g. Parasite weight: 0.071 g.

As with Liver sample 4, there was slight clustering towards the base of the gel, which may

have been more successfully resolved if a longer run was performed. On this gel, there was a

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significant degree of streaking in the low molecular weight ladder, but aside from these there

was no significant streaking on the gel.

(f) G. aculeatus liver sample 3. Quantity of liver protein: 9000.00 µg. VH: 12541. Fish

length; 30 mm. Fish weight: 0.209 g. Parasite weight: 0.067 g.

Of the single infection group, this sample possessed the lowest quantity of protein, which was

surprising considering the dimensions of the fish. This low quantity of protein produced a faint

gel, but the majority of the spots referenced in the main text were not entirely indistinguishable.

Upon imaging there were a few noticeable stains towards the center-right of the gel, and an

odd expression pattern in the area circled in red.

(g) G. aculeatus liver sample 5. Quantity of liver protein: 17251.88 µg. VH: 12671. Fish

length: 29 mm. Fish weight: 0.245 g. Parasite weight: 0.068 g.

This sample was chosen as a representative gel as it appeared as one of the most clearly

resolved gels, with relatively discrete patterns of expression observable. As well as best

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37

encompassing the majority of variation seen on the other three samples. There was relatively

little streaking observed on the gel in comparison to the other samples, allowing to distinguish

spots with relative ease. As well as this, the low molecular weight ladder appeared most easily

distinguishable on this gel. Although there was a tear in the gel, it did not directly affect the

analysis.

(h) G. aculeatus liver sample 6. Quantity of liver protein: 30532.50 µg. VH: 12541. Fish

length: 31 mm. Fish weight: 0.296 g. Parasite weight: 0.076 g.

Of the ‘Single Infection’ gels, this possessed some of the highest levels of vertical streaking,

particularly along the right hand side, which may have affected the fidelity of the image it

produced. However, this did not present an issue in assessing the expression patterns of the

identified spots within the representative gels.

(iii) Multiple Infection

(i) G. aculeatus liver sample 1. Quantity of protein; 23827.5 µg. VH: 12541. Fish length: 27

mm. Fish weight: 0.181 g. Total weight of parasites: 0.090 g.

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Sample 1 proved one of the most challenging gels to analyze due to high degrees of vertical,

and horizontal streaking, causing some run-in between spots, especially when identifying the

relative spots on the left hand side of the image. Aside from the streaking, there were expression

patterns observable in the image, just not to as high of a fidelity as other samples.

(j) G. aculeatus liver sample 7. Quantity of protein: 34171.88 µg. VH: 12541. Fish length:

31 mm. Fish weight: 0.312 g. Total weight of parasites: 0.086 g.

As with sample 11, it can be immediately seen that this gel was significantly under-run, with

a distinct band across the lower half of the gel, where the dye front resides. This resulted in

variable expression patterns in comparison to the other samples, as well as missing Spot 1.

There was slight horizontal streaking in this gel, with slightly blurred expression patterns.

(k) G. aculeatus liver sample 15. Quantity of liver protein: 12746.25 µg. VH: 12671. Fish

length: 25 mm. Fish weight: 0.180 g. Total weight of parasites: 0.060 g.

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(l) G. aculeatus liver sample 27. Quantity of liver protein: 13055.63 µg. VH: 12671. Fish

length: 22 mm. Fish weight: 0.107 g. Total weight of parasites: 0.012 g.

Samples 15, and 27 both showed relatively similar patterns of expression, with well-defined

protein spots, and relatively little streaking. Both were contenders for the representative gel, as

they were both relatively consistent representations of the expression patterns observed in the

‘Multiple Infection’ sample group. However, of the two, sample 27 proved a higher fidelity

image, with lesser degrees of streaking, both in the horizontal and vertical axes.

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