Arginine kinase activity in

Littorina fabalis

Felix Englund Örn

Degree project for Bachelor of Science in

Biology

BIO602 Biology: Degree project 15 hec

Spring 2015

Department of Biological and Environmental Sciences

University of Gothenburg

Examiner: Susanne Eriksson

Department of Biological and Environmental Sciences

University of Gothenburg

Supervisor: Kerstin Johannesson

Department of Biological and Environmental Sciences - Tjärnö University of Gothenburg

(Leeuwen, 2012)

Table of Contents

Abstract ...................................................................................................................................... 4

1.Introduction ............................................................................................................................. 6

1.1.Arginine kinase ................................................................................................................. 7

2.Methods and materials ........................................................................................................... 8

2.1.Data sampling ................................................................................................................... 8

2.2.Enzymatic Activity Procedure ........................................................................................... 8

2.3.Calculations ...................................................................................................................... 9

2.4.Statistics.......................................................................................................................... 10

3.Results ................................................................................................................................... 10

3.1.Two-Way ANOVA ............................................................................................................ 11

4.Discussion .............................................................................................................................. 13

7. Acknowledgments .......................................................................................................... 14

8. References ...................................................................................................................... 15

Abstract

Littorina fabalis, a snail living in the intertidal zone of much of northeastern European shores, has been found to differ by several non-synonymous mutations in the locus coding for the phosphagen - arginine kinase (Ak). Which type of allele, Ak100 or Ak120, which is present in the genome of an individual of this species has been linked to what environment the snail lives in. The environment of these snails is either sheltered or exposed in relation to waves. Ak120 being almost exclusively tied to individuals from sheltered shores whilst Ak100 is linked to the exposed environment. The alleles are present in the form of homozygotes of Ak100 or Ak120 in each respective environment (with less frequency of heterozygotes than expected by chance in most sites). This study sets out to test if there is a difference in the enzymatic activity in the proteins (alloenzymes) coded for by the different genotypes dominant in each environment. Different enzyme activities would support a hypothesis that there is a functional difference between snails that have different Ak genotypes. This will support that it is in fact the Ak locus that is under selection and not a locus linked to Ak. By sampling snails from the hybrid zones of these two environments I collected all three possible genotypes (homozygotes for both Ak100 and Ak120 and heterozygotes). In a later study all snails will be genotyped, but here I tested for differences between sex (male and female) and location (two sites sampled) in two different types of snail tissue (foot and hepatopancreas) in order to assess other factors that may affect enzyme activity. Activity was measured by spectrometry. The results showed a significant difference between locations in the foot tissue but not in the hepatopancreas. Sex had no significant effect on activity in either tissue.

Sammanfattning

Littorina fabalis, en snäcka som lever i tidvattenzonen över stora delar av nordvästra Europas kuster har upptäckts visa stora skillnader med flera icke-synonyma mutationer i genen kodande för fosfagenatet, arginine kinase (Ak). Vilken typ av allel, Ak100 eller Ak120, som finns i en individs genom av denna art har kopplats till vilken typ av miljö snäckan lever i. Snäckornas livsmiljö är antingen skyddad eller exponerad i relation till vågor. Ak120 är nästan exklusivt kopplat till individer från skyddade kuster och Ak100 till den exponerade miljön. Allelerna är närvarande som homozygoter för både Ak100 och Ak120 i respektive miljö (med en lägre frekvens av heterozygoter än förväntat av slumpen i de flesta lokaler). Denna studie företar att test om det finns en skillnad i den enzymatiska aktiviteten i proteinerna (alloenzymer) de olika genotyperna, som är dominanta i respektive miljö, kodar för. Olika enzymaktiviteter skulle ge stöd åt hypotesen om det finns en funktionell skillnad mellan snäckor som har olika Ak genotyper. Detta ger stöd åt att det faktiskt är Ak lokuset som är under selektion och inte ett lokus länkat till Ak. Genom att plocka snäckor från hybrid zonen av dessa två miljöer samlar jag in alla tre möjliga genotyper (homozygoter av både Ak100 och Ak120 och heterozygoter). I en senare studie kommer all snäckors genotyp bestämmas, här testar jag för skillnader mellan kön (hane och hona) och lokal (två utvalda platser) i två olika typer av vävnader (fot och hepatopancreas) för att undersöka andra faktorer som kan påverka enzymaktiviteten. Aktivitet mäts genom spektrometri. Resultaten visade en signifikant skillnad mellan lokaler hos fotvävnaden men ej hos hepatopancreasvävnaden. Kön hade ingen signifikant effekt på respektive vävnad.

Introduction

The arginine kinase locus of Littorina fabalis has been under the gaze of investigation for just over two decades now. The reason is that the snail species has been found to differ genetically in, arginine kinase (Ak), while invariant in a number of other loci over small environmental transects (Tatarenkov & Johannesson 1994). What alleles of arginine kinase are present in Swedish shores are linked to what microhabitat the individual of snail originated from, that is microhabitats that are either exposed or sheltered to wave exposure, with Ak120 most common in the former type of habitat and Ak100 dominant in the latter (Tatarenkov & Johannesson 1998). Later studies found both a genetic difference and a size related difference (Tatarenkov & Johannesson 1998). Kemppainen et al. (2005) emphasized this point and referred to the two morphs of L. fabalis as “Large-moderately exposed” (LM) and “Small-Sheltered” (SS). Recent studies (Duvetorp et al. in prep) have shown that the two alleles differ by several non-synonymous mutations (natural selection being present). Additionally, in intermediate microenvironments where the two ecotypes overlap in distribution, homozygotes of Ak120

alleles are almost exclusively present in the SS ecotype while homozygotes of Ak100 are predominate in the LM ecotype, and heterozygotes of the two alleles are relatively rare in numbers (Tatarenkov & Johannesson 1994). The same genetic difference in the Ak locus over microhabitats has also been found in other parts of NW Europe (France and Wales) (Tatarenkov & Johannesson 1999).

These local adaptions and forming of ecotypes are not in themselves surprising to find within L. fabalis. The species does not have pelagic larvae, a somewhat common facet in the Littorina genus but by no means the standard. Consequently, spread of offspring will be very local which in turn promotes local adaptation (Johannesson 2003). Living in a heterogeneous habitat, the intertidal zone, with exposed and sheltered habitats just meters apart, the snail has a potential for local adaptation due to sharp environmental differences. However, forces working against forming of genetically distinct populations are also present. Gene flow acts to homogenize the population (Futuyma 2013), but the differentiation in the Ak locus has nevertheless occurred. All of this suggests that strong directional selection present in both environments is the underlying cause of the difference in Ak allele frequencies (Duvetorp et al. in prep). Under a hypothesis of differential selection we expect the enzyme products (alloenzymes) of the different Ak alleles to show different activities. By analyzing the actual activity of the different Ak alloenzymes, through a kinetic assay, and comparing their activity it is possible to test the hypothesis of different selection acting on the different genotypes. An alternative hypothesis that may explain strong differentiation in the Ak locus is that the Ak locus is linked to another locus under strong differential selection. Activity differences between the different Ak genotypes would support that it is specifically the Ak locus that selection is working on, and not a locus linked to Ak.

To eliminate the risk of enzyme activities induced by different environments, samples from the hybrid zones (where both homozygotes live side-by-side) of two localities were collected. Genotyping the snails will occur in a later study where Ak activity will be analyzed looking at differences between genotype. In the present study, alloenzyme activity was compared over two locations (Långholmen and Lökholmen), and two sexes (male and female). This is an

important first step to analyze and expel factors other than genotype which might influence Ak activity.

Arginine kinase

Arginine kinase is a part of the enzymatic group of phosphagen kinases whose role often means to replenish ATP levels in cells that are demanded to maintain, or produce, in bursts, high levels of energy (Kotlyar et al., 2000, Newsholme et al., 1978). The energy metabolism present in the foot of both ecotypes is likely to require Ak, suggesting the foot a suitable tissue to use in activity measurements. The foot is essentially a large muscle and is used by the snails to move and hold on to substrata. Besides the foot, the hepatopancreas (liver) will also be homogenized and tested for Ak activity as expect to find Ak in this tissue as well for its role in synthesis of amino acids and protein synthesis where the enzyme might play an important role (K. Sundell 2015, pers. comm., 01-06). Arginine kinase has also been found in unusually high levels in the hepatopancreas of the blue crab Callinectes sapidus (Chen & Lehninger 1973) which further suggests that activity measurements should target this tissue.

The activity of arginine kinase is measured indirectly by measuring the disappearance of NADH at the wavelength of 340 nm. The following reaction described by Blethen (1970) is what occurs when all the reagents for the assay have been mixed together:

Figure1 Explanation of reaction. Abbreviations used: ATP = Adenosine 5’-Triphosphate, AK = Arginine Kinase, ADP = Adenosine 5’-Diphosphate, PEP = Phospho(enol)pyruvate, PK = Pyruvate Kinase + β- Nicotinamide Adenine Dinucleotide, Reduced Form, LDH = L-Lactic Dehydrogenase, β- Nicotinamide Adenine Dinucleotide, Oxidized Form CO.

Arginine kinase is necessary for the first part of the reaction and this reaction is coupled with the conversion of phosphoenolpuryvate (PEP) to pyruvate by pyruvate kinase (PK). This is in turn coupled to the conversion of pyruvate to lactate by lactate dehydrogenase (LDH). It is this last reaction that NADH is oxidized to NAD+ (McCormick 1993). The disappearance of NADH can be followed by monitoring the absorbance at the wavelength of 340 nm with a spectrometer. This wavelength can be measured by kinetic analysis which allows for continuous data points to be taken for the entire run and measured by the program SoftMaxPro 6.0. Using these data point an estimation of the Ak activity in the tissues can be measured. The samples have all been standardized by volume of tissue to weight. Derived from the kinetic analysis the NADH decrease per 10μg tissue per hour (NADH/10μg/h) can be calculated as a final measurement of enzymatic activity and used for statistical comparisons.

Methods and materials

Data sampling

Collection of the snail Littorina fabalis used for the enzymatic assay was done by Kerstin Johannesson. Snails were sampled from Lökholmen (N 58° 53' 22", E 11° 6' 34") and Långholmen (N 58° 53' 5", E 11° 7' 3") May 2015. A total of 50 snails were collected and 20 were used for this study.

The sample snails were brought to Göteborg and kept in saltwater aquaria with algae (Fucus vesiculosus) in the laboratory.

Enzymatic Activity Procedure

I used an enzymatic assay of EC 2.7.3.3 by Blethen (1970) with some adjustments to adapt to measurements in 96-well plates. Additionally, a second glycine buffer but with added mercaptoethanol (Reagent B) was not mixed or replaced as our spectrometry measure was “Kinetic” and not an “End Point” assay. Reagent J, an arginine kinase enzyme solution, was replaced by a homogenized, diluted sample acting as the source of Ak in the reaction. Finally, the reaction cocktail was not adjusted to 30°C and 8.6 pH, but were kept at room temperature until use.

All reagents except for the PK/LDH solution were mixed before homogenization of the snail samples and stored at room temperature. The PK/LDH solution was prepared after all tissue samples had been diluted to preserve its enzyme activity. It was kept on ice until loading it into the microplate.

Chemicals were bought from Sigma Aldrich unless otherwise stated.

The snails were brought from the aquarium to the lab. Shells were crushed and the pieces removed from the body. The body was placed on a glass dish placed on ice. The foot and hepatopancreas were separated and saved for weighing. The head was put in a RNA free eppendorf tube containing 5ml of RNAlater and stored in a fridge for 24 h before put into a freezer and stored for genotyping at a later stage. After weighing the hepatopancreas it was placed in a 1.5 ml eppendorf tube and the foot in a 2 ml micro tube (PP). A Precellys 24 Lysis & Homogenizer machine was used to homogenize the foot tissue after adding three ceramic beads form a soft tissue homogenizing CK14 tube into the micro tube containing the foot tissue. The hepatopancreas was homogenized with a Pellet Pestle Motor manufactured by KONTES. Before homogenization of either hepatocpancreas or the foot, a 250 mM Glycine buffer solution mixed at pH 8.6 at 30°C was added. Volume of buffer, measured in µl, was added in a 1:1 ratio (v/w) in order to support homogenization and make it a liquid homogenate.

Homogenized samples were diluted (1:15) with deionized water into another eppendorf tube.

To fill a well the following reagents were pipetted into the microplate in the following order and volume:

1. 10µl L-arginine (Reagent G, ARG)

2. 10µl PK/LDH (Reagent I)

3. 10µl Diluted sample

4. 270µl Reaction Mix

Thereafter the microplate was put into a Spectramax i3 machine (Molecular Devices). In the program SoftMaxPro 6 a kinetic assay was performed with a run time of 6 minutes at room temperature and with a medium shake before start.

Calculations

In order to calculate each individual activity as a measure of decrease NADH/10µg/h the mean rate of absorbance was calculated from absorbance per second into absorbance per hour by multiplying with 60 twice. That value was divided by the extinction coefficient of β-NADH at 340 nm and the standardized amount of tissue per sample to get the decrease of NADH in mM per 10µg of tissue. The calculation is the following

Mean rate of Absorbance x 60 x 60 6, 22 mM extinction coefficient of β-NADH at 340 nm x 0, 67 µg

Statistics

Analysis in SPSS were carried out to test if there was a significant difference between sexes, or between the two different hybrid zones. This was done through a Two-Way ANOVA in SPSS.

Results

Figure 1. Mean rate of absorbance (measured every 15 seconds for 6 minutes) for hepatopancreas samples from L. fabalis. Legend shows location and sex of each individual line. n = 20.

Figure 2. Mean rate of absorbance (measured every 15 seconds for 6 minutes) for foot samples from L. fabalis. Legend shows location and sex of each individual line. n = 20.

The compiled mean absorbance for each individual show some clear differences between tissues. Most notably is the higher degree of variance between individuals in the foot samples (Fig. 2) compared to hepatopancreas (Fig. 1). The foot samples host both the highest and lowest mean absorbance as well as a more curved decline of absorbance whereas the heptopancreas are more uniform in mean absorbance and linear in their decrease. Certain

groupings appear distinguishable as specific lines lie on top or close to each other for the full 6 minutes in both the hepatopancreas and, even more clearly, in the foot samples. It is hard to distinguish if these are by location but the individuals from Lökolmen (red lines) generally appear together. Groupings appear not to be by sex as the lines of the different sexes of jumble together. Whether these grouping are by genotype would be interesting to investigate further.

Both Fig. 1 and Fig. 2 were used to analyze which data points to use for statistical analysis for each tissue. This was done in order to determine the data points in which the slope was at its highest gradient reflecting therefore the highest rate of reaction, where the reaction is not limited by any enzyme or substrate. As many data points as possible were included to increase the accuracy of the rate but at the same time I wanted to avoid points where the gradient began to decrease. This led to a difference in data points included in the statistics between the two tissues. The more linear hepatopancreas samples allowed for 3 more data points to be included (time frame of 15-200 seconds) than the foot (15-150 seconds). The first data point at 0 seconds for either tissue was not included due to general errors that can occur in the machines’ first measurement and are exclude by that principle. The number of males in the foot samples (Fig.2) is only 8 as two individuals (one from each location) produced results that had no apparent activity; a user error must have occurred when measuring these and have therefore been discarded from the analysis.

Two-Way ANOVA

Table.1. Two-Way ANOVA results summarized for both tissues. Hepatopancreas n = 20, Foot n = 18.

Tissue Factor F values Significance

Hepatopancreas Sex F1 = 0.016 0.144

Location F2 = 2.362 0.902

Interaction F3 = 0.656 0.430

Foot Sex F4 = 0.000 0.985

Location F5 = 24.238 0.000

Interaction F6 = 0.183 0.675

Figure 3 The Ak activity in the hepatopancreas of L. fabalis in males and females from two localities, Lökholmen and Långholmen. Bars show means ± standard error. For all samples n = 10

Figure 4 The Ak activity in the foot of L. fabalis in males and females from two localities, Lökholmen and Långholmen. Bars show means ± standard error. Males from Lökholmen and Långholmen n = 8, females from both locales n = 10

The bar plots from the Two Way-ANOVA in SPSS show similar results in regards to sex in both tissues, however there is a clear difference between locations in the foot samples (Fig.4) which is not present in the hepatopancreas samples (Fig.3). In general, the foot tissue showed a higher activity and the error bars for females from Långholmen suggested data from those individuals were more variable in both samples

The Two Way-ANOVA also tested for an interaction between the two factors (not illustrated by the bar plots, see Table.1). In the hepatopancreas samples, neither sex (F1 = 0,016) nor location (F2 = 2,362) affected Ak activity significantly, and there was no significant interaction (F3 = 0,656); they did not potentiate each other effects (Table.1). Sex did not either significantly affect the foot samples (F4 = 0,000) but location (F5 = 24.238) does (Table.1).

Their interaction did not affect Ak activity significantly (F6 = 0,183); they did not potentiate each other effects (Table.1).

Discussion

The results indicate no difference in activity due to sex, suggesting the Ak activity is not skewed towards any particular sex. Between locations, however, there is a significant difference in activity in the foot tissue between Lökholmen and Långholmen, where individuals from Långholmen show a higher level of activity. Yet sampling has been done on hybrid zones to try and ascertain a level of similarity in environment between sample locations. Notably, this significance does not show up in the hepatoapancreas tissue despite both tissues were from the same individuals with the same genotypes. A hypothesis for the how this can be could come from the environmental difference existing in the hybrid zone. Snails from the exposed transect would have to use their foot to hold on tightly to a substrate more often and more intensely than those from the sheltered part. Therefore snails in this environment (homozygotes of Ak100) would require more muscular work from the foot which is the tissue that holds on to the substratum.

Indeed, it was found in a proteomic study in Littorina saxatillis by Martínez-Fernández et al. (2008) that snails from a wave exposed environment had higher activity than those from sheltered shores. Since it is the foot which resulted in the significance we might assume it requires higher levels of activity to replenish ATP levels from holding on more often/intensely and the hepatopancreas need lower levels of activity to endure the wave stress. This assumes individuals from Långholmen in general have a higher number of individuals from a wave exposed environment for this hypothesis to hold. This might prove to be an inaccurate hypothesis since the genotype data might group individuals differently, creating new groups of snails which would be compared to each other leading to new statistical results.

Another factor that might influence the results of this study is the fact that the Ak activity that is due to the levels of arginine kinase present has not been analyzed. The NADH to NAD+ reaction, which is what is being measured, is not limited to the reaction described in the method as it could be driven by other enzymes or substrates in the tissue. This background activity could skew the results to be higher than they actually are. But if we could use an inhibitor to block the activity caused by arginine kinase we would get a measurement of the background activity that then could be taken into account to calculate the actual Ak activity. A study by Arockiarja et al. (2011) on the giant freshwater prawn Macrobrachium rosenbergii points towards α-ketoglutarate being an effective inhibitor of Ak activity. The same study also suggests ATP and Glucose serving as inhibitors. Two studies in insects (Brown & Grossman 2004, Wang et al. 2011) conclude that L-Arginine the flavonoids quercetin and luteolin are as well inhibitors of Ak activity. It might also be worthwhile to test ouabain which has been used to block Na+, K+ ATPase activity in fish gills (McCormick 1993) and could be used to eliminate the Na+, K+ ATPase activity present in the samples. Whether any of these will work in L. fabalis remains to be tested. Finding and testing an efficient inhibitor of Ak in L. fabalis would give more accurate results in future studies.

In addition, all activities were measured using double replicates and the data used for the statistical evaluation is the mean of these two replicates. All the replicates showed, to a varying degree, a difference between them, some more than others, for example, the higher

error bars of the females from Långholmen (Fig. 3 and Fig. 4) are caused by higher levels of variance in this group. Measuring activities using three or four replicates is time consuming, but would probably reduce variation and increase precision of the measurements.

Acknowledgments

I would like to thank Kerstin Johannesson, Kristina Sundell, Henrik Sundh, Marina Panova, Linda Frank, Kerstin Ebefors, and Oliver Englund Örn for all their help with theory, equipment and other instructions during the bachelor's thesis project.

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