Grazer-induced defence mechanisms in Skeletonema marinoi

Transkript

1 Grazer-induced defence mechanisms in Skeletonema marinoi Andreas Persson Uppsats för avläggande av naturvetenskaplig kandidatexamen i Biologi 15 hp Institutionen för Marin Ekologi Göteborgs universitet Handledare: Lars Gamfeldt och Erik Selander Examinator: Susanne Baden

2 Content Abstract 3 Introduction 4 Material and methods 5 Results 7 Discussion 10 References 10 2

3 Sammanfattning Skeletonema marinoi är en kedjebildande kiselalg som i stor utsträckning betas av olika typer av zooplankton, framförallt olika arter av hoppkräftor. Kedjebildande kiselalger justerar längden på kedjorna för att hålla sig utanför det storleksspann som är mest utsatt för betning av zooplankton. Målet med den här studien var att undersöka huruvida kloner av S. marinoi som inte utsatts för betning tidigare fortfarande uppvisar betarinducerad kedjelängdsplasticitet. Kloner från tre olika populationer på den svenska västkusten (Vinga) samt den danska västkusten (Mariagerfjord) användes i studien. Kiselalgerna från Mariagerfjord bestod av både nutida kloner samt 90 år gamla vilosporer som återaktiverades för experimentet. S. marinoi-populationen från nutida Mariagerfjord hade i stort sett ingen tidigare erfarenhet av betning. Den danska fjorden har varit kraftigt övergödd sedan tidigt 1980-tal och hoppkräftor har i princip varit helt frånvarande sedan dess. Klonerna från vilosporerna, däremot, levde med största sannolikhet i en miljö med hoppkräftor innan de övergick till sitt vilostadie. Hypotesen var att klonerna från nutida Mariagerfjord, utan tidigare erfarenhet av betning, inte längre skulle ha förmågan att reagera på kemiska signaler från hoppkräftor genom att justera sin kedjelängd. Experimenten genomfördes genom en rad försök där de olika S. marinoipopulationerna utsattes för betning av Acartia-hoppkräftor. Medellängd på cellkedjorna dokumenterades före och efter betning. Resultaten visade en klar skillnad i kedjelängd mellan populationerna i frånvaro av betare. Efter betning, däremot, fanns ingen tydlig skillnad i kejdelängd. Hypotesen stöds i den bemärkelsen att klonerna från de mest betade populationerna (Vinga) uppvisade ett starkare svar på betning genom att förkorta kedjelängden. Dock så uppvisade klonerna från Mariagerfjord betydligt kortare kedjelängder från början, innan de utsattes för betning. På grund av detta så kan hypotesen varken godtas eller förkastas. Förutom betningsexperimentet så beräknades tillväxthastigheter för de olika populationerna med hjälp av data från en partikelräknare. Resultaten visade ingen signifikant skillnad i tillväxthastighet mellan de olika populationerna. Skillnad i tillväxthastighet kan således inte förklara skillnaden i kedjelängd mellan populationerna. Abstract Skeletonema marinoi is a chain forming diatom that is readily grazed by different types of zooplankton, primarily different types of copepods. Chain forming diatoms adjust the length of their chains in order to stay out of the critical size range that make them a potential prey. The aim of this study was to determine whether strains of the marine diatom S. marinoi that have not previously been exposed to grazing also exhibit grazer-induced chain length plasticity. Strains of the diatom from three different populations on the Swedish west coast (Vinga) and Danish east coast (Mariagerfjord) were used in the study. The diatoms from the Mariagerfjord were collected as recent isolates and in the form of 90 years old resting stages that were subsequently hatched for the experiment. The S. marinoi population from present day Mariagerfjord had experienced virtually no previous copepod grazing. The fjord has been highly eutrophic since the early 1980 s and copepods have been more or less absent since. The population hatched from resting stages, however, most likely coexisted with copepods before laying to rest on the bottom. The hypothesis was that the strains derived from Mariagerfjord with little previous exposure to copepod grazing would no longer have the ability to respond to chemical cues from copepods by shortening the length of their chains. Experiments was carried out through a series of 3

4 trials were the different S. marinoi populations were exposed to grazing by Acartia copepods. Average chain length was recorded before and after grazer exposure. The results showed a clear difference in chain length between the populations when grazers were not present. After grazer exposure, however, there was no clear difference in chain length. The hypothesis is supported in the sense that strains derived from the populations most exposed to copepod grazing (Vinga) displayed stronger response to grazing by shortening of the chain length. However, the clones from Mariagerfjord displayed considerably shorter chain lengths to begin with, before exposure to grazing. Because of this it is not possible to falsify or retain the hypothesis. In addition to the grazing experiment, growth rates for the different populations were calculated and analyzed statistically using data obtained from a particle counter. The results showed no significant differences in growth rate between the different strains of S. marinoi. This means that difference in growth rate does not explain the difference in chain length between the populations. Introduction Skeletonema marinoi is a chain forming marine diatom. It is very common on the Swedish west coast and it is particularly prevalent during spring blooms (Tiselius och Kuylenstierna 1996). During most of the year (apart from the spring blooms) S. marinoi experiences a high grazing pressure. One important group of grazers is different species of copepods (Mauchline, o.a. 1998). It has been shown that several species of diatoms adapt their size to stay out of the critical size range that makes them a potential prey. (Bergkvist, et al. 2012). It is usually the grazer s ability to detect, capture and handle the prey that dictates the lower limit of this size range. (Hansen, Bjornsen and Hansen 1994). This chain length plasticity is not fully understood, but a study performed by Bergkvist and colleagues (2102) shows that chemical cues from grazing copepods triggers a suppression of the chain lengths. Subsequently, the shorter chains are detected and grazed to a considerably lower extent than the longer ones. This chain length plasticity is documented in two strains of S. marinoi from populations that are readily exposed to copepod grazing (Bergkvist, et al. 2012). However, much less is known about populations that are not exposed to copepod grazing. In order to explore the generality of grazer induced chain length plasticity in relation to different evolutionary histories of grazing exposure, S. marinoi from three different populations were used in the experiment. The three populations came from Vinga (in Skagerak, by the inlet of Göta Älv just outside of Gothenburg), present day Mariagerfjord (at the east coast of the Danish mainland), and sediment sources from Mariagerfjord. The strains from sediment sources in Mariagerfjord were hatched from 90 years old resting stages. Today, Mariagerfjord is a highly eutrophic location (Härnström, o.a. 2011), but this was not the case 90 years ago. The current hypertrophic state of the Mariagerfjord has resulted in a very scarce presence of grazing zooplankton, in particular copepods (Fallesen, Andersen och Larsen 1999). The anthropogenic loadings of nitrogen and phosphorus in Mariagerfjord has increased since at least the 1950s (Härnström, o.a. 2011). However, close to a century ago, when the sediment with the clones used in this study was deposited, it is reasonable to assume that nutrient levels and copepod abundance were approximately the same as in the adjacent sea. The hypothesis was that strains of S. marinoi from present day mariagerfjord would have lost the ability to respond to copepod cues. Accordingly, S. marinoi strains from Vinga and the strains from the sediment cores in Mariagerfjord should both display a stronger response in chain length plasticity when exposed to copepods. 4

5 Figure 1 Map with Skeletonema marinoi strain origins Materials and Method Algal cultures In this study, 9 clones from GUMACC (Gothenburg University Marine Algal Culture Collection) were used. Three of the clones were from Vinga in the Gothenburg archipelago, three from Mariagerfjord in northern Denmark, and three were hatched from 90 years old sediment resting stages from Mariagerfjord (Figure 1). Grazing experiment A growth experiment was performed with a control sample and a treatment from each of the three strains of Skeletonema marinoi. Before the experiment started, cell concentrations were estimated using a Sedgewick rafter counting chamber. Acartia tonsa copepods were used as grazers. The Copepods were collected at the Sven Lovén Center for Marine Sciences at Tjärnö, Sweden. Three copepods were placed in each experimental bottle. Throughout the experiment, dead copepods were replaced daily to maintain an even grazing pressure. The samples were contained in 250 ml glass bottles. The bottles were filled to the brim and plastic wrap was used to make sure that the bottles were free of air bubbles. The seawater used for the growth experiment was collected at the Sven Lovén Center for Marine Sciences at Kristineberg, Sweden. The seawater was gently suction filtered (Whatman GF/F) before use and the salinity was 33 psu. The experimental bottles were incubated on an illuminated, rotating plankton wheel in an ambient temperature of 17 C for the duration of the experiment. The light intensity was kept at approximately 40. F-medium was added to the filtrated water in order to maintain high nutrient levels throughout the experiment. The 5

6 experiment was terminated after four days. The reason that the plankton growth was not allowed to continue for more than four days is that the growth rate needs to be kept in within the exponential phase. If S. marinoi reaches the upper limit density that the sample volume can sustain, the cell chains will start to break up due nutrient limitation and grazer-induced chain length plasticity will not be observable. After the growth experiment was terminated, average chain lengths were recorded for all replicates. 250 µl was extracted from each replicate and deposited into separate wells on a counting chamber. One drop of lugol s iodine was deployed in each well. Numbers of cells for all chains were then recorded by counting under a microscope. Growth rate In order to determine if there was a difference in growth rate between the different populations, samples from all treatments were analyzed in a Beckman & Coulter Multisizer III particle counter that measures the volume of all particles in the water. After the grazing experiment was completed, 50 ml from each treatment was transferred into plastic vials and preserved with lugol s iodine. The data obtained from this measurement was then used to estimate the growth rate using the following equation: =(ln ln )/ Where = growth rate, = concentration ( ) at the end of the grazing experiment, = start concentration and = time in days. Statistical analysis IBM SPSS was used for the statistical analyses. A spread vs. level plot was performed in order to determine if there was heterogeneous variance between the different variables (population, grazing, and strain). The plot showed heterogeneous variance between the variables. To minimize the effect of this uneven variance, chain length data was log transformed. A 3-way ANOVA with the strain factor nested under population and grazing was performed. The interaction between population and grazers relates to the hypothesis testing. The strain factor was nested under population since the strains were chosen at random to give a good representation of each population. A post hoc test was performed on the grazing/population interaction in the computer program SUPERANOVA. In addition, a 2-factor ANOVA was run in SPSS on the data obtained from the growth rate calculations in order to verify if there was any significant differences between the populations. Lastly, a regression analysis was performed on the data from the particle counter. 6

7 Figure 2 Rotating plankton wheel with experimental bottles attached Results The results show that Skeletonema marinoi from Mariagerfjord was overall shorter to begin with, which gives less room for variation. The statistical analysis showed significant difference in chain length by both population and treatment (Table 1). Moreover, the difference in chain length is considerably more apparent between the control replicates from the different locations than between the grazed replicates (Figure 3). Chain length (number of cells) A A A A B A Control Grazed Figure 3 Average chain length for grazed and ungrazed treatments by location Mariagerfjord (O): clones from sediment cores Mariagerfjord (N): clones from present day Mariagerfjord 0 Mariagerfjord (O) Mariagerfjord (N) Vinga 7

8 Table 1 3-factor ANOVA from grazing experiment Source df Sum of squares Mean square F-value P-value Population 2 92,108 46,054 7,017 0,0269 Grazers 1 33,829 33,829 15,777 0,0073 Strain(Population) 6 39,378 6,563 25,005 0,0001 Population*Grazers 2 13,002 6,501 3,032 0,1230 Grazers*Strain(Population) 6 12,865 2,144 8,169 0,0001 The 3-factor ANOVA shows that there is a difference in grazing response between different strains (Table 1). However, there is also a clear difference in chain length between the strains when grazers are not present. After grazing the chain lengths appear to be similar for all strains (Figure 3). Apparent growth rate Control Grazed Figure 4 Results of the growth rate calculations Mariagerfjord (N) Mariagerfjord (O) Vinga 8

9 Source Corrected Model Type III Sum of Squares,063 a Intercept 34,609 1 Population,040 2 Grazing Population * Grazing Error,005,018, Total 34, Corrected Total,285 a. R Squared =,222 (Adjusted R Squared = -,103 df 5 Mean Square F Sig.,013,683,645 34, ,801,000,020 1,071,373,005,280,606,009,497,018,620 Table 2 2-factor ANOVA on growth rate data The 2-factor ANOVA on the growth rate data showed no significant differences between populations (Table 2). Difference in growth rate between control and grazed treatment (µ-g) y = x R² = Average chain length ( number of cells) Figure 5 Regression curve for grazing, presented as the difference between growth rate in control and grazed treatments versus average chain length over the experiment P=0.006 Table 3 Regression analysis on the apparent growth rate versus average chain length The regression analysis revealed a significant relationship between average chain length over the experiment and grazing rate expressed as the difference between growth rate in the controls and the grazed cultures. Shorter cell chains are less susceptible to grazing than longer ones. 9

10 Discussion The initial hypothesis of this study was that strains of Skeletonema marinoi that have not been exposed to grazing in their native habitat have lost their grazer-induced chain length plasticity. The results from the grazing experiment seem to support this hypothesis since the Mariagerfjord strains showed no significant response to copepod grazing. However, this result is misleading because the results clearly show that cell chains are shorter in Mariagerfjord (both present day and sediment resting stages) than at Vinga regardless of grazer presence. This indicates inherently shorter chain lengths in Mariagerfjord than at Vinga, and it is the main explanation to why the differences in chain length between grazed and control treatments were smaller for the Mariagerfjord populations than for the Vinga populations. The difference between control samples and grazed samples will be smaller at locations with inherently shorter cell chains since there is a lower chain length limit (a S. marinoi-unit cannot be shorter than one cell), but theoretically no upper chain length limit. Furthermore, the growth experiment lasted for four days and the assumption was that the growth rate would still be in the exponential phase when the experiment was terminated. If the growth was different in different strains, it is possible that it had already peaked after four days in some treatments. This would obscure the results since cell chains start to break up when it becomes too crowded. Since there was no significant differences in the growth rates between the populations, however, it is reasonable to assume that they did not affect the results. In addition, there was a clear positive relationship between chain length and susceptibility to grazing (Figure 5). Shorter cell chains appear less likely to be consumed by grazing zooplankton as the encounter rate between prey and grazer is lower. Longer chains, on the other hand, are more likely to be eaten since they are more easily detected. This is confirmed by Bergkvist et al. (2012). In conclusion, although there was a clear difference in grazer-induced chain length plasticity between the strains from Vinga and from Mariagerfjord, there was almost no difference between the two different strains from Mariagerfjord that were separated in time. From this observation, it would appear as though there is an intrinsic chain length in strains from different populations. However, there is no strong support that history of coexistence with grazers has any effect on either chain length or grazer-induced chain length plasticity from this study. Bergkvist, Johanna, Peter Thor, Hans Henrik Jakobsen, Sten-Åke Wängberg, and Erik Selander. "Grazer-induced chain length plasticity reduces grazing risk in a marine diatom." Limnology and Oceanography, 2012: Fallesen, G., F. Andersen, och B. Larsen. Life, deat and revival of the hypertrophic Mariager Fjord, Denmark. Journal of Marine Systems, 1999: Hansen, Benni, Peter Koefoed Bjornsen, and Per Juel Hansen. "The Size Ratio Between Planktonic Predators and Their Prey." Limnology and Oceanography, March 1994: Härnström, Karolina, Marianne Ellegaard, Thorbjorn J. Andersen, och Anna Godhe. Hundred years of genetic structure in a sediment reviveddiatom population. Proceedings of the National Academy of Sciences of the United States of America, 2011:

11 Mauchline, J, JHS Blaxter, AJ Southward, och PA Tyler. The biology of calanoid copepods. San Diego: Academic Press, Tiselius, Peter, och Mats Kuylenstierna. Growth and decline of a diatom spring bloom: phytoplankton species composition, formation of marine snow and the role of heterotrophic dinoflagellates. Journal of Plankton Research, 1996: