Individual specialization
in a marine gastropod
Littorina littorea
Kristie Rigby
Degree project for Master of Science (Two Years) in
Biology
Degree course in Marine Ecology 45 hec
Autumn 2014 and Spring 2015
Department of Biological and Environmental Sciences
University of Gothenburg
Examiner: Kerstin Johannesson
Department of Biological and Environmental Sciences
University of Gothenburg
Supervisor: Gunilla Toth
Department of Biological and Environmental Sciences
University of Gothenburg
Front page image by Kristie Rigby Table of Contents Abstract ................................................................................................................. 4 Introduction ........................................................................................................... 5 Materials and Methods .......................................................................................... 8 Field observations ............................................................................................................................................. 8 Individual specialization experiment ....................................................................................................... 9 Morphology and chemical defence experiments ............................................................................... 10 Bioassay-­‐guided fractionation ................................................................................................................... 11 Results ................................................................................................................. 12 Field observations ........................................................................................................................................... 12 Individual specialization pilot experiment ........................................................................................... 14 Main individual specialization experiment .......................................................................................... 14 Morphological and chemical defence experiments ........................................................................... 16 Discussion ............................................................................................................ 18 Acknowledgements ............................................................................................. 21 References ........................................................................................................... 22 Abstract The common European periwinkle (Littorina littorea) is considered a generalist herbivore. To date there has been no previous research investigating individual specialization with L. littorea. Individual specialization can be affected by inter-­‐ and intraspecific competition and ecological opportunity. It is hypothesized that hard bottom communities in comparison with soft bottom communities should feature higher ecological opportunity, higher interspecific competition and higher intraspecific competition. In accordance I hypothesize that individuals of L. littorea from hard bottom substrates express a higher degree of individual specialization than soft bottom individuals. Field observations confirmed the hypothesis of hard bottom populations having higher ecological opportunity. However, there was no evidence to support the presence of inter-­‐ or intraspecific competition. Multiple-­‐choice assays were performed with Fucus vesiculosus, Chondrus crispus, Furcellaria lumbricalis and Fucus serratus and 10 individuals from 2 hard bottom sites and 2 soft bottom sites. Results after 6 trials indicated there was no individual specialization, however a pilot study indicated a significant preference for Ulva. lactuca. F. vesiculosus and F. lumbricalis were the least preferred from the multiple-­‐choice assays and were subjected to morphological and chemical defence assays with artificial agar food. Morphological trials indicated a significant preference for U. lactuca when paired with F. lumbricalis. Chemical defence trials indicated no sign of a chemical deterrent suggesting it is a nutritional aspect deterring L. littorea. F. vesiculosus crude extract showed a significant preference for artificial agar food without F. vesiculosus compounds, suggesting a chemical defence. Separation of F. vesiculosus compounds by polarity indicated no significant results, proposing there is a multi-­‐compound defence system. The results of this investigation can only confirm that there is no individual specialization with the species of assayed algae. As a species, L. littorea are specialized on U. lactuca, however, individual specialization may be present in another season and with other algal choices. 4 Introduction Individual specialization is a phenomenon where individuals use a small subset of the available resource base within their population (Araujo et al. 2011). Araujo et al. (2011) reviewed 189 species across 9 major taxonomic groups and found only 12 species that reported low or absent individual specialization, indicating that individual specialization is common in natural populations. However, not all organisms exhibit individual specialization and non-­‐
significant outcomes of individual specialization experiments are likely to be under-­‐published. As a result of this, the average degree of individual specialization may be overestimated. Furthermore, many taxonomic groups exhibit individual specialization to varying degrees. For example less than 10 species in the gastropod class showed individual specialization (Araujo et al. 2011). One of the underlying theoretical frameworks for understanding the evolution of individual specialization (i.e. if the theory is true, then individual specialization is a logical consequence) is the optimal foraging theory. The optimal foraging theory is a strategy, which individuals may develop by restricting their diet from an array of suitable profitable resources. Svanbäck and Bolnick (2005) suggest that optimal diets vary between individuals due to different factors such as search, handling and/or digestive abilities causing individual specialization to arise. Individuals can optimize their diets by ranking their resources according to energetic value per unit handling time, it reduces their energy expenditure and maximizes their fitness (Svanbäck & Bolnick 2005). Optimal foraging theory suggests that the lower ranked resources will be ignored as more time can be spent searching and handling for a more valuable resource. Different ecological mechanisms can influence the degree of individual specialization. For example competition between individuals (intraspecific competition) is hypothesized to have varying effects on individual specialization. Intraspecific competition can arise when the same species compete for limited resources. At higher consumer densities the preferred resource may become limited due to higher intraspecific competition. The consumers may then add an alternative resource to their diet, increasing individual specialization. However, if individuals use different resources, higher densities may cause them to converge to the same alternative resource. That is, if their initial preferred resources become limited the variation in diet will decrease, thereby reducing individual specialization. (Araujo et al. 2011). However, empirical evidence shows that intraspecific competition increases individual specialization. Several studies have 5 reported a positive correlation between population density (an indication of intraspecific competition) and individual specialization (Svanbäck & Persson 2004). Other studies showed that a decrease in resource abundance corresponded to a behavioural diversification in microhabitat use (Kobler et al. 2009) or to the inclusion of new resources through increased inter-­‐individual variation (Tinker et al. 2008; Svanbäck & Persson 2004). Interspecific competition results from a variety of species competing for the same resources. With competitors present it may constrain the population niche to one resource, limiting the level of individual specialization. With competitors absent individuals exploit different resources and individual specialization should be high. The effect that interspecific competition has on the direction of individual specialization can vary as it depends on the rank-­‐preference variation by the focal species as well as the nature of the diet overlap with the competing species (Araujo et al. 2011). Empirical evidence shows conflicting results (Araujo et al. 2011), however it has been supported that interspecific competition reduces individual specialization. It has been assumed that interspecific competition reduces ecological opportunity. While these 2 concepts are related they are still distinct from each other. Opportunity also depends on factors such as patch size, environmental stability, microhabitat diversity and resource diversity (Masello et al. 2013). Higher ecological opportunity should increase individual specialization, as habitat fragmentation and low resource diversity has been shown to decrease individual specialization (Layman et al. 2007; Darimont et al. 2009). Seasonal availability of resources has also been shown to influence the degree of individual specialization. Herrera et al. (2008) reported fruit bats (Rousettus aegypiacus) have a higher degree of individual specialization in the spring when plants began to display more fruits. An additional factor that can affect the food preference of herbivores is morphological and/or chemical defences in plants and seaweed.. Some species of seaweeds use chemical defences as a method of protection from herbivores, these are commonly in the form of secondary metabolites. These secondary metabolites can be induced as a response to grazing and have previously shown to deter or reduce herbivores (Hay & Fenical 1992). Numerous studies have reported conflicting results between herbivore feeding behaviour and chemical defence(Molis et al. 2008; Rohde et al. 2004; Geiselman & McConnell 1981). It has also been reported that the presence of herbivores can induce production of chemicals in plants, e.g. phlorotannins, in seaweeds (Deal et 6 al. 2003). The effect of phlorotannins on herbivores varies between species and under certain conditions (Pavia et al. 2012). These fluctuations of chemical defence levels in seaweeds could be a factor in herbivore feeding preference, thus influencing individual specialization. Littorina littorea is natively a European periwinkle that is widely distributed along rocky coasts in northern Europe. L. littorea have also been introduced to the Atlantic coast of North America more than 100 years ago (Chapman et al. 2007). They are commonly found on the upper shore into the sublittoral zone (Newell 1958). L. littorea reproduces by releasing pelagic larvae, which can stay in the plankton for over a month. This gives considerable prospect for larvae to transport to considerably different habitats from the parents (Currey & Hughes 1982). On the population level, L. littorea is considered a generalist herbivore, which grazes on a variety of algal species, however, previous experiments denote that L. littorea have strong food preferences (Lubchenco 1978; Watson & Norton 1985). Chlorophyceae (greens) algal species are highly preferred on the population level over Phaeophyceae (browns) and Rhodophyceae (reds). Previous preference experiments with L. littorea have been done on the population level, using individual snails as replicate units. In order to investigate individual specialization, the preference of each individual should be replicated (i.e. several measurements with the same individual). No previous studies have examined L. littorea to determine if there is underlying ecological influences impacting individual specialization. On the Swedish west coast, populations of L. littorea are found in reasonably sheltered habitats with different substrate characteristics (hard and soft bottom sites). The overall aim of this study is to determine if individuals of L. littorea collected from different substrate characteristics (hard-­‐and-­‐
soft bottom) are specialized to feed on different seaweed species (i.e express individual specialization within populations and between habitats). Additionally, the abundance of L. littorea and other Littorina sp. individuals (i.e. inter-­‐ and intraspecific competition) as well as the algal species richness (ecological opportunity) between different habitats were assessed. We hypothesized that hard-­‐bottom communities would have higher abundance of Littorina sp. individuals (i.e. higher intra-­‐ and interspecific competition) and higher algal species richness, (higher ecological opportunity) compared to soft-­‐bottom habitats. In line with these hypotheses, we hypothesized that L. littorea individuals from hard-­‐bottom communities would be more specialized compared to individuals from soft-­‐bottom communities. 7 Figure 1. Locations of sampling sites, Site1-­‐Eskilholmen N58.892063 E11.118593, Site 2-­‐ Liggstill N58.898038 E11.133205, Site 3-­‐Sven Lovén Centre N58.876765 E11.145345, Site 4-­‐Saltö Canal N58.874223 E11.146139
Materials and Methods Field observations Field observations were conducted in September 2014 outside the Sven Lovén Centre for Marine Sciences-­‐
Tjärnö (SLCT) in order to quantify algal species richness and the abundance of different Littorina species (L. obtusata, L. littorea, and L. fabalis) at 4 different sites. The sites represented two different habitats and were either characterised by slightly sloping rocky cliffs (hard bottom sites: Eskilholmen N58.892063 E11.118593 and Liggstill N58.898038 E11.133205) or by sandy/muddy substrate (soft bottom sites: Sven Lovén Centre N58.876765 E11.145345 and Saltö Canal N58.874223 E11.146139, Figure 1). Data on algal species richness and snail abundance were collected by noting the algal species and counting the number of different Littorina species within a haphazardly placed metal frame (0.0625 m2, n = 10) at 0 -­‐ 0.5 m depth. Data on the species richness of algae was statistically analysed with a two-­‐sided t-­‐test. Data on the abundance of Littorina species were statistically analysed using a nested analysis of variance (ANOVA) with Habitat (2 levels) as a fixed factor and Site (2 levels) as a random factor nested within Habitat (Table 1). This study did not comprise of any endangered or protected species therefore no special permits or approvals were required. 8 Individual specialization experiment In order to investigate if individual L. littorea are specialised to feed on different seaweed species, several multiple-­‐choice feeding preference experiments were conducted during October to November 2014. Ten L. littorea individuals were collected at < 1m depth from each of the 4 different sites outside the SLCT in late September 2014 (a total of 40 snails). The L. littorea were individually marked with plastic tags designed for honey bees, glued to their shells and maintained in an indoor glass aquarium with running filtered surface seawater. 5 macroalgae species were chosen for the multiple choice feeding experiment based on the results from the field observation. Ulva lactuca, Fucus vesiculosus, Chondrus crispus, Fucus serratus and Furcellaria lumbricalis were collected from 5 haphazardly chosen sites outside the SLCT (Figure 1). Specimens were brought back to the laboratory, cleaned from macroscopic epiphytes, and stored indoors under running filtered surface seawater for a maximum of 42 days, before used in experiments. Macroalgal thalli were cut to approximately 0.5 -­‐2.0 g blotted wet weight and randomly cable tied to pieces of plastic mesh, which were placed in plastic containers (13.0 x 9.5 x 8 cm, 750 ml) that were filled to 7.5 cm with filtered surface seawater (≈ 15 °C). A L. littorea individual was placed in the centre of the container and left for 48 hours, after which the blotted wet weight of each seaweed was determined. An equal number of control aquaria (i.e. a total of 40) without snails was prepared to control for autogenic seaweed weight changes and used to calculate consumption (see below). The first trial consisted of 4 macroalgae species (U. lactuca, F. vesiculosus, C. crispus, F. serratus). U. lactuca was replaced with F. lumbricalis in later trials as U. lactuca was highly preferred by all snail individuals (see results). Trials were repeated 6 times and separated in time so that each trial was considered independent from the previous trial (i.e. snails were allowed to recover between each trial). Consumption (mg) by each individual L. littorea was calculated using the following equation: C= (Stop wwG-­‐ Start wwG)-­‐(Stop wwC-­‐Start wwC) wwG, blotted wet weight of the grazed algal pieces, wwC was the control alga pieces. Data on the consumption of different seaweeds by individual L. littorea were statistically analysed using univariate analysis of variance with repeated measures (ANOVAR) with Food (4 levels) as a within subject factor and Habitat (2 levels) as a fixed between subject factor, Population (2 levels) as a random between subject factor nested within Habitat and Individual (10 levels) as 9 a random between subject factor nested within Population (Table 2). ANOVAR was used because consumption data on the different seaweed species within an aquarium were not independent. Morphology and chemical defence experiments F. lumbricalis and F. vesiculosus were the least preferred seaweed species in the individual specialization experiment (see Results), and therefore a series of two-­‐choice preference experiments were performed in order to investigate if these species contain defences (morphological or chemical) against L. littorea grazing. Because U. lactuca was highly preferred in the previous experiment, this species was used as a control food. Tests for morphological defences were carried out by freeze drying U. lactuca, F. lumbricalis and F. vesiculosus, and incorporating the freeze-­‐dried, homogenized algal material into artificial agar food. Dry weight: wet weight percentage was calculated for each species and used to calculate the amount of dry seaweed material added to each type of artificial food. Artificial food was prepared by heating agar powder (1.5 % wet weight) with seawater to boiling point, adding the dried seaweed powder (10, 8.4, and 8.65 % wet weight for U. lactuca, F. lumbricalis, and F. vesiculosus respectively) after the agar solution had cooled to ≈ 50 ° C, and decanting the solution into a petri dish. The resulting solidified agar plate was divided into 20 square pieces and the blotted wet weight of each piece was recorded. Each replicate featured one piece containing U. lactuca and one piece containing either F. lumbricalis or F. vesiculosus together with 3 L. littorea collected from a soft bottom site (n = 10). An equal number of controls without snails were prepared to control for autogenic agar wet weight changes. The L. littorea individuals were starved 3 days prior to the experiments. After the feeding period of 2 days the blotted wet weights were recorded and consumption was calculated as described above. Tests for chemical defences were carried out by extracting secondary metabolites from F. lumbricalis and F. vesiculosus and incorporating the extracts to artificial agar food containing extracted F. lumbricalis or F. vesiculosus material. Crude extracts were obtained by extracting 20g of F. lumbricalis or F. vesiculosus 3 times with 200ml dicholoromethane (DCM) and 3 times with 200ml methanol (MeOH) for 1 hour each on a shaking table (132 rpm). The supernatant was filtered through a glass fibre filter and the solvent evaporated using a rotary evaporator. The crude extract was then redissolved with 16ml DCM and 16ml MeOH. For the bioassay, the crude extract was added in natural concentration to the dried algal powder and the solvent was evaporated. Controls 10 where only solvent was added to algal powder and evaporated were also prepared. Artificial agar food was prepared as described above (1.5 and 10 % wet weight agar and algal material respectively. Figure 2: Modified Kupchan method for F. vesiculosus artificial food bioassay guided fractionation with 3x L. littorea, n=10. Freeze dried F. vesiculosus material (60g) homogenized Bioassay-­‐guided fractionation For the artificial food bioassays, grazing preferences were statistically analysed using 2-­‐tailed, paired t-­‐tests. Crude extract (4x 300ml DCM 4x 300mlMeOH) Hexane + Me0H:H20 9:1 Hexane (A) Me0H:H20 9:1 Chloroform + Me0H: H20 6:4 Chloroform (B) Evaporate Me0H Because L. littorea preferred the control food containing extracted F. vesiculosus (see Results), further separations were performed on the crude F. vesiculosus extract. An adapted Küpchan fractionation method was performed to separate high polarity compounds from low polarity compounds ( Küpchan 1971, see Figure 2). Each fraction (A-­‐E) was redissolved in 16ml of the appropriate solvent (A = hexane, B = chloroform, C = ethyl acetate, D = water, and E = methanol), and artificial agar food and solvent controls was prepared as described above. Ethyl acetate + H20 Ethyl acetate (C) H20 + Me0H Filtered Water (D) Methanol (E) 11 Field observations There was a statistically significant difference in algal species richness between habitats with different substrate characteristics (t-­‐test, t1, 9 = 24.2, p = 0.04). The mean algal species richness was 7.0 ± 1.0 and 1.5 ± 0.5 (Mean ± SE, Table 1) at sites with hard-­‐ and soft-­‐bottom respectively. These results indicate that there is a higher ecological opportunity (i.e. more variety of algal species to select from) for the L. littorea in hard-­‐ than in soft-­‐
bottom habitats. There were no statistically significant differences in Littorina abundance between hard and soft bottom habitats for any of the species (Table 2). The number of L. littorea was similar between sites and habitats (Table 2, Figure 3), indicating no difference in intraspecific competition between different habitats. However, except for a few L. obtusata individuals at site 4, only L. littorea was found in the soft-­‐bottom habitats. These results indicate that there could be a difference in interspecific competition between habitats, although the power of the statistical test was too low to detect it (i.e. only 2 sites were studied for each habitat). Hard site Soft site 1 2 3 Ascophyllum nodosum x x Ceramium virgatum x x Chrondrus crispus x Cladophora x Pylaiella littoralis x Fucus serratus x Species Fucus spiralis Fucus vesiculosus x Ulva lactuca x x x x 4 x x x Ulva intestinalis x x 4.0
3.5
Abundance (no)
Results Table 1. Biodiversity of macroalgae recorded from in situ field observations. Site 1 and 2 from hard bottom substrate. Site 3 and 4 from soft bottom substrate. (t-­‐test, t1, 9 = 24.2, p=0.04. Hard =>x=7 SE=1.0 Soft => x=1.5 SE=0.5). Littorina littorea
Littorina obtusata
Littorina fabalis
3.0
2.5
2.0
1.5
1.0
0.5
0
P1
P2
Hard-bottom
P3
P4
Soft-bottom
Figure 3. Nested analysis of variance (ANOVA) represents the species and mean abundance of Littorina found at the 4 sampling sites. Error bars show ± SEM (n= 10). 12 Table 2. Nested ANOVA results from the abundance of Littorea observed at 4 sampling sites (n=10) L. littorea Source of variation df SS MS F-­‐Value P-­‐Value Error Term 3 Habitat 1 21.025 21.025 0.215 Site Site 2 13.05 6.525 Residual 36 145.7 4.047 L. obtusata Source of variation df SS MS F-­‐Value P-­‐Value Error Term 1.612 Habitat 1 2.5 2.5 10 Site 2 0.5 0.25 0.366 Residual 36 24.6 L. fabalis Source of variation df SS 0.214 Residual 0.087 Site 0.696 Residual 0.683 MS F-­‐Value P-­‐Value Error Term Habitat 1 12.1 12.1 2.469 Site 2 9.8 4.9 3.528 36 50 1.389 Residual L. obtusata & L. fabalis Source of variation df 0.257 Site 0.04 Residual SS MS F-­‐Value P-­‐Value Error Term Habitat 1 24.025 24.025 4.688 0.163 Site Site 2 10.25 5.125 1.836 0.174 Residual 36 100.5 2.792 Residual 13 Individual specialization pilot experiment After 6 trials, with 40 individuals there was no significant difference in consumption by individual L. littorea on F. lumbricalis, C. crispus, F. vesiculosus and F. serratus (as indicated by the non-­‐significant interaction between the factors Food and Individual, Table 3, Figure 5a). Therefore, the hypothesis that there is individual specialization in L. littorea was discarded (Figure 5a). There was a significant difference in overall consumption between snails originating from hard and soft bottom populations (as indicated by the significant factor Habitat, Table 3, Figure 5b). However, although individual L. littorea are not specialized on different food types, there was a clear preference from C. crispus and F. serratus by all individuals, as shown by the significance factor Food (Table 3, Figure 5a). Consumption (mg)
250
200
150
100
50
0
-50
Fucus
vesiculosus
Chondrus
crispus
Ulva
lactuca
Fucus
serratus
Figure 4. Individual specialization pilot multiple-­‐choice experiment performed with U. lactuca, F. vesiculosus, C. crispus and F. serratus. Consumption measured in milligrams. Statistical measures ANOVAR (Error bars represent ± SEM n=20, F3, 16 = 8.43, p= 0.001). A 60
50
Consumption (mg)
Main individual specialization experiment 300
40
30
20
10
0
Fucus
vesiculosus
B Consumption (mg)
There was a statistically significant difference in consumption by L. littorea on U. lactuca, C. crispus, F. vesiculosus and F. serratus. (Figure 4, ANOVAR, F3, 16 = 8.43, p= 0.001). These results indicate that there was a significant preference for U. lactuca by all L. littorea individuals included in the experiment and therefore, U. lactuca was replaced by F. lumbricalis in the main individual specialization experiment. 350
Chondrus
crispus
P1
P2
Furcellaria
lumbricalis
P3
Fucus
serratus
P4
40
30
20
10
0
Hard-bottom
Soft-bottom
Figure 5. Mean consumption (mg wet weight) of A) 4 different seaweed species (F. vesiculosus, C. crispus, F. lumbricalis and F. serratus) by L. littorea snails from B) hard bottom and soft bottom populations. Statistical measures ANOVAR (Error bars show ± SEM (n = 6 trials)
14 Table 3. ANOVAR results, trials with F. vesiculosus, C. crispus, F. lumbricalis and F. serratus with 40 L. littorea individuals (n= 6 trials). Source of variation df SS MS F-­‐
Value P-­‐
Value Habitat 1 .027 .027 5.545 .0195 Population (Habitat) 2 .001 4.948E-­‐4 .101 .9041 Individual (Habitat, Population) 36 .224 .006 1.267 .1573 Subject (Group) 200 .982 .005 Seaweed 3 .162 .054 8.284 .0001 Seaweed * Habitat 3 .012 .004 .598 .6167 Seaweed * Population (Habitat) 6 .026 .004 .667 .6761 Seaweed * Individual (Habitat, Population) 108 .787 .007 1.116 .2159 Seaweed * Subject (Group) 600 3.918 .007 Fucus vesiculosus
350
Chondrus crispus
A
Furcellaria lumbricalis
Fucus serratus
B
300
250
200
150
100
50
Consumption (mg)
0
-50
-100
I1
350
I2
I3
I4
I5
I6
I7
I8
I9
I10
C
I11
I12
I13
I14
I15
I16
I17
I18
I19
I20
I32
I33
I34
I35
I36
I37
I38
I39
I40
D
300
250
200
150
100
50
0
-50
-100
I21
I22
I23
I24
I25
I26
I27
I28
I29
I30
I31
Figure 6. Consumption (mg wet weight) of 4 different seaweed species (F. vesiculosus, C. crispus, F. lumbricalis and F. serratus) by individual L. littorea snails from A) and B) hard bottom and C) and D) soft bottom populations. Statistical measures ANOVAR (Error bars show ± SEM (n = 6 trials). 15 Morphological and chemical defence experiments Morphological bioassays show that there was a statistically significant preference by L. littorea for U. lactuca over F. lumbricalis (Table 4, Figure 7a). No significant difference in consumption was found between U. lactuca and F. vesiculosus (Table 4, Figure 7b). Bioassays using crude extracts indicated a statistically significant Consumption (g) Consumption (g) (7A) 0.2 preference for the artificial control food over the food with F. vesiculosus extract (Table 4, Figure 8b). However, no statistically significant deterrence was found for artificial food with F. lumbricalis extract (Table 4, Figure 8a). Results from statistical analyses of data from bioassays including fractionated extracts of F. vesiculosus showed no significant preferences (Figure 9, Table 5). (8A) 0.3 0.15 0.1 0.05 0.25 0.2 0.15 0.1 0.05 0 Furcellaria with Extract 0 Ulva Furcellaria (8B) (7B) 0.3 Consumption (g) 0.2 Consumption (g) Furcellaria Control 0.15 0.1 0.05 0.25 0.2 0.15 0.1 0.05 0 0 Ulva Fucus Figure 7. Mean consumption (g wet weight) morphological bioassays A) 2 choice assay with U. lactuca and F. lumricallis B) 2 choice assay with U. lactuca and F. vesiculosus 2-­‐
tailed t-­‐test results. Error bars show ± SEM (n = 10). Fucus with Fucus Control Extract Figure 8. Mean consumption (g wet weight) crude extract bioassays A) 2 choice assay with F. lumbricalis extract and F. lumbricalis control . B) 2 choice assay with F. vesiculosus extract and F. vesiculosus control 2-­‐tailed t-­‐
test results. Error bars show ± SEM (n = 10). 16 Table 4. Two tailed paired t test results from morphological and crude extract bioassays (n=10) T-­‐value P-­‐Value (t1,18) Mean Morphological U. lactuca & F. lumbricalis -­‐0.09 -­‐2.401 0.04 U. lactuca & F. vesiculosus -­‐0.03 -­‐1.062 Extract Mean 0.316 T-­‐value (t1,18) P-­‐
Value A 0.08 1.995 0.077 B -­‐0.04 -­‐0.863 0.411 C 0.1 1.832 0.1 D 0.02 0.665 0.522 E 0.06 1.568 0.151 Crude extracts F. lumbricalis extract & control 0 -­‐0.048 0.962 0.2 4.725 0.001 F. vesiculosus extract & control Consumption (g) (9A) 0.3 0.25 0.2 Consumption (g) Extract A Control Control 0.2 0.15 0.1 0.05 Extract B 0.25 0 Extract D Control Extract E Control (9E) 0.3 Consumption (g) 0.2 0.15 0.1 0.05 0 (9C) 0.3 0.25 0.2 0.15 0.1 (9D) 0.3 Consumption (g) 0.15 0.1 0.05 0 (9B) 0.3 0.25 Consumption (g) Test Table 5. F. vesiculosus fractionation p values for bioassays from a modified Küpchan method (A-­‐Hexane, B-­‐Chloroform, C-­‐Ethyl Acetate, D-­‐Water, E-­‐Methanol). 2-­‐
tailed paired t-­‐test indicated no significant values p= <0.05 0.25 0.2 0.15 0.1 0.05 0 Figure 9. F. vesiculosus control and F. vesiculosus extracts artificial agar bioassays. 2-­‐tailed paired t-­‐test analysis of fractions A-­‐
E from a modified Küpchan method. (A-­‐
Hexane, B-­‐Chloroform, C-­‐Ethyl Acetate, D-­‐
H20, E-­‐Methanol) (Error bars show ± SEM, n=10) 0.05 0 Extract C Control 17 Discussion No significant difference in the abundance of Littorina sp. was found between hard-­‐ and soft bottom habitats from the data collected in the present study, the data indicates that there were no differences in inter-­‐ or intraspecific competition between snails in the different habitats (Figure 3, Table 2). This insignificant result maybe due to the low power of the test, therefore I cannot fully accept the null hypothesis of no competition. However, the hard bottom habitats had significantly higher species richness of macroalgae (i.e. higher ecological opportunity) compared to soft bottom habitats (Table 1). A higher ecological opportunity should in theory promote a higher degree of individual specialization (Araujo et al. 2011). However, my results did not confirm the hypothesis that L. littorea individuals express individual specialization both within populations and from different habitats. Instead the first pilot experiment using periwinkles from hard bottom sites indicated that all tested L. littorea individuals prefer U. lactuca (Figure 4). The main experiment where U. lactuca was changed to F. lumbricalis showed that all individuals preferred C. crispus and F. serratus to F. vesiculosus and F. lumbricalis (Figure 5a, Table 3). Furthermore, there were significant differences in the consumption between L. littorea individuals from hard bottom and soft bottom communities (Figure 5b, Table 3). Soft bottom individuals consumed more algal biomass in comparison to individuals from hard bottom sites. The higher consumption by soft bottom snails may be explained by exploitation. In their natural habitat L. littorea from soft bottom populations are exposed to less diverse resources (Table 1). L. littorea can survive for several months on a unialgal diet but thrive best if offered a mixed diet (Norton et al. 1990). In the experiment soft bottom individuals were exposed to more resources in comparison to their natural habitat. Therefore it is possible these individuals seized the opportunity of more diverse resources to increase their fitness. An additional explanation for the higher food intake observed in the soft bottom populations could be the sporadic distribution of resources in soft bottom habitats and the risk of predation and desiccation when foraging. Foraging excursions by L. littorea are typically no longer than 1.5-­‐2m from their original location, and as more often than not no resources are found when foraging, they tend to return to their initial location (Norton et al. 1990). L. littorea from soft bottom habitats may therefore select a less preferred food source in closer proximity over the chances of finding a better resource when foraging (Norton et al. 1990). The difference in food intake between soft and hard bottom individuals could also be explained 18 by a lower intake in the hard bottom individuals as a result of parasitic infection (Clausen et al. 2008). Individuals from hard bottom communities are exposed to higher ecological opportunity, and thereby also a wider range of potential intermediate hosts (Bolnick et al. 2003). In northern European waters, L. littorea is regularly a host for at least 6 trematode species (Cercaria lebouri, Cryptocotyle lingua, Himasthla elongata, Podocotyle atomon, Renicola roscovita and Microphallus pygmaeus)(Clausen et al. 2008). These parasites commonly infect the gonad-­‐digestive gland system and have been reported to reduce food consumption. L. littorea parasitic load increases with age which is correlated with size (Byers et al. 2008) Clausen et al. (2008) report that uninfected individuals of L. littorea consumed up to 65% more macroalgae biomass than infected snails. Although evidence suggests the presence of parasites decreases consumption rates, there has been no empirical evidence considering parasites affecting the magnitude of individual specialization (Araujo et al. 2011). The pilot multiple-­‐choice experiment revealed a significant preference for U. lactuca (Figure 4). U. Lactuca is an opportunistic seaweed, with fast, sporadic and patchy growth, making it hard for large populations of specialize on. Furthermore, because L. littorea also is an opportunistic species they can utilize the periodical availability of such a valuable food source. The pilot was run with 20 individuals from the 2 hard bottom habitats. The observed preference of U. lactuca may be explained by the optimal foraging theory. L. littorea have sharp points on their radular teeth which can tear the relatively thin U. lactuca foliose thallus efficiently gaining more food to be ingested per unit time (Norton et al. 1990). The rate of replacement of the radula is 5-­‐6 rows per day at 20°C (Reid & Mak 1999). As the replacement of the radular teeth maybe energy demanding, is it likely that L. littorea prefer algae species that are thinner and easier to graze, such as U. lactuca. In the main individual specialization experiment L. littorea expressed no significant individual preference (Figure 5a, Table 3). However, as a species there was a statistically significant preference towards C. crispus and F. serratus over F. vesiculosus and F. lumbricalis. Preference ranking in L. littorea has been reported from most preferred seaweed to least; ephemeral greens, browns and reds (Norton et al. 1990). In previous studies there has been conflicting evidence concerning consumption preference of C. crispus. Vives I Batlle et al. (2004), report that they used C. crispus as a food source, as L. littorea have a preference to graze it. In a conflicting study by Norton et al. (1990) C. crispus has also been shown to be a less preferred food source in comparison to fucoids 19 (brown algae). It was believed that adult C. crispus was not consumed, as a result of its morphological structure being too tough. Reproductive plants of C. crispus were observed to have increased palatability after they had shed their spores (Unpublished observation D.P Cheney). In my experiments C. crispus and F. serratus were significantly preferred over F. lumbricalis and F. vesiculosus, suggesting a more complex preferential ranking than previously denoted. After the individual specialization experiment, possible reasons for differences in palatability were investigated. In the morphological trials U. lactuca was significantly preferred when paired with F. lumbricalis (Figure 7a). There was also a trend preferring the U. lactuca in the F. vesiculosus trial, although not significant perhaps due to too few snails (n=10) and the low power of the test (Figure 7b). The preference to U. lactuca even after morphological differences are removed suggests that it has a higher nutritional value or that F. lumbricalis and F. vesiculosus feature a chemical defence. Fleurence (1999), reports that the protein content in U. lactuca is between 10-­‐
21% dry weight and in the brown seaweeds such as F. vesiculosus less than 15%. F. lumbricalis has a reported protein content 13-­‐25% dry weight (Bird et al. 1991). This suggests that if nutritional value plays a role in preference of U. lactuca, it is not due to protein content but perhaps other nutritional aspects. However, the observed preference of U. lactuca could also be an effect of a chemical defence in F. lumbricalis and F. vesiculosus. L. littorea has been seen to reject algae without physically sampling them (Newell 1958). It is unlikely due to visual cues since they lack sophisticated vision (Newell 1958). Perception of food is from the olfactory and gustatory stimuli, therefore responding to the exudates from algae (Norton et al. 1990). In the crude extract chemical defence assays no preference was found in the F. lumbricalis assay (Figure 8a), therefore it seems the un-­‐
palatability of F. lumbricalis may be due to nutritional value. However, there was a significant preference for the control F. vesiculosus, suggesting the presence of chemical defence in F. vesiculosus (Figure 8b). Chemical defences (in the form of secondary metabolites) such as phlorotannins can serve as a defence against herbivores. Phlorotannins are water-­‐soluble secondary metabolites, commonly found as a chemical defence in F. vesiculosus (Toth & Pavia 2000). When investigating the chemical defence of F. vesiculosus further in a bioassay guided fractionation, L. littorea showed no significant preference for any of the fractions (Figure 9). If phlorotannins were the only chemical deterrent we would have expected L. littorea to prefer the 20 control in Fraction D (Figure 9d). However, in F. vesiculosus galactolipids have been found to deter herbivores rather than phlorotannins (Deal et al. 2003). Galactolipids have been reported in the ethyl acetate fraction (Deal et al. 2003) and therefore could be a plausible explanation for the trend preferring the control in this experiment (Figure 9c). Previous studies have found that the production of defence compounds sometimes can be induced by grazers (Toth & Pavia 2000). In this study no grazers were present before fractionation, which could explain why no chemical defence could be observed in the bioassay guided fractionation. Although, since no significant preferences were detected in any fraction but a significant preference was found in the crude extract, this would suggest it is a multi-­‐compound defence that is acting as a deterrent for L. littorea. It is often unknown if individual specialization is maintained over time (Masello et al. 2013). Algae choices can vary throughout the year and between seasons. Therefore individual specialization may seem absent in varying seasons and in fluctuations of resources. L. littorea are the most active between late spring and early autumn. Observations of L. littorea gathered in winter months and acclimatized to summer temperatures, have shown to consume less algae than L. littorea gathered in the summer (Bertness et al. 1983). In the experiments we manipulated the water temperature and air temperature to warmer summer like conditions to make the L. littorea more active, which may have affected the results. If these experiments were performed in another season the results may have been different. The results of this investigation can only confirm that there is no individual specialization with the species of assayed algae. As a species, L. littorea are specialized on U. lactuca, however, individual specialization may be present in another season and with other algal choices. Acknowledgements First and foremost I am forever grateful to my supervisor Gunilla Toth for excellent guidance, encouragement and insightful discussions. 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