Research Article |
Corresponding author: Carlotta Meriggi ( meriggi.c@gmail.com ) Corresponding author: Stina Drakare ( stina.drakare@slu.se ) Academic editor: Kęstutis Arbačiauskas
© 2024 Carlotta Meriggi, Richard K. Johnson, Ane T. Laugen, Stina Drakare.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Meriggi C, Johnson RK, Laugen AT, Drakare S (2024) Effects of temperature and N:P ratio on the invasion success of the cyanobacterium Raphidiopsis raciborskii. Aquatic Invasions 19(3): 275-286. https://doi.org/10.3391/ai.2024.19.3.134464
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The tropical invasive freshwater cyanobacterium Raphidiopsis raciborskii, first recorded in Central Europe about two decades ago, is now a relatively widespread species, expanding its geographic range. Currently, however, there are no records of this species in Sweden. As a bloom-forming and toxin-producing species, future population growths of R. raciborskii could negatively affect local biodiversity and ecosystem services. Hence, there is an urgent need to understand the factors controlling its capability of establishment in Northern European lakes. We performed a laboratory experiment to study the competitive success of R. raciborskii when interacting with other phytoplankton from major taxonomic groups typically found in Scandinavian lakes (diatoms, green algae, and cyanobacteria). The experimental settings included three temperature conditions (17; 22; 26 °C) and three different nutrient conditions (N:P ratios 8:1; 16:1; 32:1). The experiment was performed in a semi-continuous culture setup to test the invasion success of R. raciborskii. Raphidiopsis raciborskii did not become the dominant species in any of the tested conditions; however, it was able to grow and maintain its biomass in all treatments, also in relatively low temperature (17 °C). Temperature played an important role in the phytoplankton community composition, especially for the cyanobacterial group. Raphidiopsis raciborskii was more successful than Planktothrix agardhii, but less dominant than Microcystis aeruginosa. Temperature is thus important in determining the potential survival and settlement of the invasive R. raciborskii in lakes.
invasive species, competition, semi-continuous culture, Scenedesmus, Planktothrix, Microcystis, Chlamydomonas
Climate change and eutrophication are the two main causes of increased cyanobacterial blooms (
One species of concern is the invasive freshwater cyanobacterium Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii). Raphidiopsis raciborskii is a nitrogen-fixing, toxin-producing and bloom-forming filamentous cyanobacterium, known to impact local biodiversity and ecosystem services worldwide (
Human activities have greatly increased the bioavailability of nitrogen (N) and phosphorus (P) in freshwaters and, combined with climate change, have increased the number of areas suitable for invasive cyanobacteria (
A laboratory competition experiment was performed with R. raciborskii (NIVA-CYA 399) and five phytoplankton species from three major functional groups: chlorophytes (Chlamydomonas reinhardtii, NIVA-CHL 152 and Scenedesmus sp., NIVA-CHL 114); cyanobacteria (Planktothrix agardhii, NIVA-CYA 429 and Microcystis aeruginosa, NIVA-CYA 123/1) and diatoms (Asterionella formosa, NIVA-BAC 3). These five phytoplankton species are native to many Scandinavian lakes and therefore likely to encounter R. raciborskii if/when it invades. All isolates were purchased from the Norwegian Culture Collection of Algae (NORCCA) and were not axenic. The phytoplankton were originally isolated from Scandinavian lakes except for R. raciborskii (Hungary) and A. formosa (UK) (Suppl. material
The choice of experimental temperatures was based on a pilot study to identify the minimum, optimal and maximum growth temperature of R. raciborskii (NIVA-CYA 399) when exposed to a gradient of temperatures ranging from 3.5 °C to 35 °C. Biomass was measured daily as fluorescence in a TD-700 Fluorometer (Turner Designs, Sunnyvale, CA) with a 436 nm excitation filter and a 680 nm emission filter for 1 to 3 weeks depending on the growth rate at each temperature (Suppl. material
We performed a 3×3 factorial experiment with three replicates for each treatment combination. The experimental settings included three temperature conditions (17 °C; 22 °C; 26 °C) and three N:P ratios to achieve N-limiting, optimal and P-limiting growth conditions (8:1; 16:1; 32:1 mole ratio of N and P, respectively). The individual nutrient ratios were achieved by modifying the N source from the Z8 growth medium and by keeping the P source constant (Suppl. material
Experimental setup performed for a 3×3 factorial experiment with three replicates for each treatment combination. The cultures were exposed to several experimental conditions, including three temperature ranges (17 °C; 22 °C; 26 °C) and three different N:P mole ratio concentrations (8:1; 16:1; 32:1). The experiment was performed using an irradiance level between 70 and 90 µE m-2 s -1 and a 16:8 light/dark cycle.
The samples were analysed by a FlowCam (VS1, Fluid Imaging Technologies Inc., Scarborough., ME, USA), which is a flow imaging microscope that combines imaging and laser light to detect particles from a fluid sample (
Phosphorous concentrations were measured as orthophosphate (PO4-P) and analyzed photometrically according to Murphey et al. (1962), with discrete or continuous flow analysis depending on concentration (ISO 15923-1:2013, SS-EN ISO 15681-2:2018). Nitrogen concentration, as the sum of nitrate and nitrite measurements (NO2 + NO3-N), was analyzed with continuous flow analysis according to SS-EN ISO 13395 after reducing nitrate to nitrite according to
Repeated-measures ANOVA was performed to test the effects of temperature, nutrient ratios and their combined effects on each individual species’ biomass over the course of the experimental period. The statistical results presented used data from day 1, 10, 16, and 25, which give a good overview of the phytoplankton development over time. The last time point (day 31) was not taken into account as the FlowCam analysis was affected by bacterial growth. Asterionella formosa was present in low numbers at the beginning of the experiment but not recorded in any of the experimental treatments in later measurements, therefore it was excluded from the statistical analysis. The primary measurements prior to analysis were not transformed and statistical analyses was performed using the R version 4.2.1 (R Core Team 2022).
The evident increase in phytoplankton cell densities in all the treatments was reflected in declines in nutrient concentrations throughout the experiment (Figs
Nitrogen levels throughout the experiment (sampled approximately every 10th day, starting from day 2). Nitrogen-limitation was most severe in the N-limited treatment (below detection limit of 0.003 mg L-1 at day 11). In the optimal treatment, N levels were low at and after day 20. In P-limited treatments, nitrogen was available in high levels throughout the experiment. The error bars represent SE.
Chlamydomonas reinhardtii and M. aeruginosa became the dominant species in all treatments (Fig.
Species’ growth (log-transformed biovolume in µm3mL-1) over time (days 1, 10, 16, 25) for each experimental treatment. The last time point (day 31) was removed as the FlowCam analysis was affected by bacterial growth. The most dominant species reached saturation from day 10. The error bars represent SE.
Chlamydomonas reinhardtii was significantly and positively affected by nutrients (F(2, 18) = 9.41, p < 0.05), temperature (F(2, 18) = 5.01, p < 0.05), interactions between nutrients-time (F(6, 54) = 4.68, p < 0.05) and temperature-time (F(6, 54) = 4.30, p < 0.05). The post-hoc analysis showed that on day 25, C. reinhardtii’s biomass was significantly higher in the N-limiting treatment (8:1) (p < 0.005) compared to the optimal (16:1) and P-limiting ones (32:1). Additionally, on day 25, C. reinhardtii biomass was significantly higher at 17° (p < 0.05) compared to 22 °C. Microcystis aeruginosa increased significantly by the interaction between temperature-time (F (6, 54) = 8.600, p < 0.001). The post-hoc analysis showed that on day 10, 16, 25 M. aeruginosa’s biomass was significantly lower at 17 °C compared to 22 °C and significantly lower at 17 °C compared to 26 °C (p < 0,05). Raphidiopsis raciborskii was predominantly influenced by temperature (F (2, 18) = 4.52, p < 0.05) and by the interaction between temperature-time (F (6, 54) = 8.600, p < 0.001). Post-hoc analysis showed that on day 10, 16, 25, R. raciborskii’s biomass was significantly lower at 17 °C compared to 22 °C and on day 16 it was significantly lower at 17 °C compared to 26 °C (p < 0.05). For Scenedesmus sp., temperature (F (2, 18) = 13.377, p < 0.05) and several other interactions between variables significantly affected its biomass, specifically, the interaction between temperature-time (F (6, 54) = 6.443, p < 0.001), nutrients-time (F (6, 54) = 3.216, p < 0.05), and the combination of temperature-nutrients-time (F (12, 54) = 3.063, p < 0.05). The post-hoc analysis showed that on day 16 and 25 Scenedesmus sp. biomass was significantly higher at 17 °C compared to 26 °C (p < 0.05) and on day 25 its growth was significantly higher at 22 °C compared to 26 °C. On day 25 at 26 °C Scenedesmus sp. biomass was significantly higher in the N-limiting (8:1) treatment compared to the optimal one (16:1) (p < 0.05) and its growth was significantly lower at the optimal (16:1) treatment compared to the P-limiting one (32:1). The biomass of P. agardhii was significantly affected by temperature (F (2, 17) = 3.59, p < 0.005) and the post-hoc analysis showed that on day 16 its growth was significantly lower at 17 °C compared to 26 °C and significantly lower at 22 °C compared to 26 °C (p < 0.05).
This experiment showed an increase in species’ biomass with an increase in temperature in all treatments, except for Scenedesmus sp., which showed the opposite trend. In our study, by using a gradient of temperatures similar to those found in Scandinavian lakes during summer, we were able to show that none of the three cyanobacteria grew well at the lowest temperature tested (17 °C), especially P. agardhii. Raphidiopsis raciborskii was able to maintain a relatively stable biomass throughout the experiment even at the lowest temperature (17 °C). These results imply that if R. raciborskii establish viable populations in Swedish lakes, it might be able to outcompete a common filamentous cyanobacteria, like P. agardhii, by growing earlier in the season when lake temperatures are colder.
Chlamydomonas reinhardtii and Scenedesmus sp. were the only two species affected by nutrients. Chlamydomonas reinhardtii had a positive response to the different nutrient ratios, being mostly successful in the N-limiting treatment (8:1). Scenedesmus sp., towards the end of the experiment, was also positively affected by N-limiting conditions (8:1). We could speculate that C. reinhardtii, due to its high biomass, controlled the nutrient availability, creating a severely nutrient limited environment for all other species influencing their growth, despite the daily exchange of media.
Filamentous cyanobacteria like P. agardhii, R. raciborskii, and the colonial M. aeruginosa are usually the most successful bloom-forming cyanobacteria in temperate shallow lakes (
Raphidiopsis raciborskii and P. agardhii have similar phenotypical traits and morphology, indicating that they might be functionally equivalent and occupy a similar ecological niche (
Raphidiopsis raciborskii did not dominate in any of the treatments; however, it was able to maintain a stable biomass throughout the experimental period. This suggests a potential high capacity of R. raciborskii for coexistence with other species, supporting the hypothesis of its plasticity (
Differences between experimental conditions and natural ecosystems are unavoidable. Species-traits, environmental conditions and presence of grazers affect competition and invasion outcomes within aquatic systems. Our experimental study showed that the combination of temperature and nutrients ratio controlled the growth of each species within the experiment, providing an overview of species’ niches in a controlled environment. The dominant species did not inhibit the growth of R. raciborskii and its presence was consistent in all treatments. The results show that R. raciborskii might also be able to survive in relatively low temperatures and be able to grow despite competition from native phytoplankton. This means that, based on our experimental conditions, R. raciborskii might be able to establish and survive at relatively high latitudes, like in Scandinavian lakes.
C.M., R.K.J., A.T.L. and S.D. designed the study. C.M. ran the experiment, performed FlowCam analysis and measurements. C.M. performed data analyses and interpreted the results. C.M. wrote the manuscript. All authors read and approved the manuscript.
This work was supported by Oscar and Lili Lamm’s Memorial Foundation (DO2017–0053) and Carl Trygger Foundation (CTS 16:267).
We thank the reviewers for their helpful comments and advice that improved the manuscript.
Species’ details purchased from the Norwegian Culture Collection of Algae (NORCCA)
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R. raciborskii growth rate and temperature choice for the experiment
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Z8 media stock solutions
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Flowcam analysis
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