Co-occurrence of the dinoflagellates Margalefidinium polykrikoides, a known fish killer, and the neurotoxic species Pyrodinium bahamense is commonly observed in the coastal waters of Sabah, Malaysia. During most of these events, M. polykrikoides dominated the bloom, apparently suppressing the growth of P. bahamense. To increase our understanding on the nutrient conditions of this phenomenon, a study was conducted to explore the interaction between these species. The specific aim was to document the allelopathic effects, if any, of M. polykrikoides o n P. bahamense when varying ratios of the two species were co-cultured under different nitrogen (N) and phosphorus (P) concentrations. The bioassay experiments started with three cell abundance proportions, which were 5:5 (500 cells mL-1 of each species, M. polykrikoides, and P. bahamense); 1:5 (100 cells mL-1 of M. polykrikoides and 500 cells mL-1 of P. bahamense); and 5:1 (500 cells mL-1 of M. polykrikoides and 100 cells mL-1 of P. bahamense). Additionally, culture filtrates (10, 20 and 50 mL) from the late exponential phase of M. polykrikoides were added to 150 mL of P. bahamense to determine if cell filtrates were allelopathic. Results indicate that M. polykrikoides was allelopathic to P. bahamense when nutrients were abundant, but not when nutrients were limiting or N was limiting relative P. The production of allelopathic compounds was supported by abnormal morphological changes in P. bahamense when co-cultured with M. polykrikoides. This capacity to suppress P. bahamense growth, combined with the inherently faster growth rate of M. polykrikoides relative to P. bahamense can account for why M. polykrikoides forms nearly monospecific blooms when nutrients are high. The filtration studies indicated the allelopathic capacity of M. polykrikoides required direct cell contact or that the allelopathic compounds degraded rapidly and were inactive when added to P. bahamense cultures. These results are important in understanding the bloom mechanisms of these two harmful algal blooms (HABs) species

Adam, Aimimuliani

Al-Has, Asilah

Ayub, Mohd Nor Azman

Darnis, Deny Susanti

M. Shaleh, Sitti Raehanah

Mohammad Noor, Normawaty

Mustakim, Ghaffur Rahim

Mustapha, Shuhadah

Co-occurrence of the dinoflagellates Margalefidinium polykrikoides, a known fish killer, and
the neurotoxic species Pyrodinium bahamense is commonly observed in the coastal waters of
Sabah, Malaysia. During most of these events, M. polykrikoides dominated the bloom, apparently
suppressing the growth of P. bahamense. To increase our understanding on the nutrient conditions
of this phenomenon, a study was conducted to explore the interaction between these species.
The specific aim was to document the allelopathic effects, if any, of M. polykrikoides on P.
bahamense when varying ratios of the two species were co-cultured under different nitrogen (N)
and phosphorus (P) concentrations. The bioassay experiments started with three cell abundance
proportions, which were 5:5 (500 cells mL-1 of each species, M. polykrikoides, and P. bahamense);
1:5 (100 cells mL-1 of M. polykrikoides and 500 cells mL-1 of P. bahamense); and 5:1 (500 cells
mL-1 of M. polykrikoides and 100 cells mL-1 of P. bahamense). Additionally, culture filtrates (10,
20 and 50 mL) from the late exponential phase of M. polykrikoides were added to 150 mL of P.
bahamense to determine if cell filtrates were allelopathic. Results indicate that M. polykrikoides
was allelopathic to P. bahamense when nutrients were abundant, but not when nutrients were
limiting or N was limiting relative P. The production of allelopathic compounds was supported
by abnormal morphological changes in P. bahamense when co-cultured with M. polykrikoides.
This capacity to suppress P. bahamense growth, combined with the inherently faster growth rate
of M. polykrikoides relative to P. bahamense can account for why M. polykrikoides forms nearly
monospecific blooms when nutrients are high. The filtration studies indicated the allelopathic
capacity of M. polykrikoides required direct cell contact or that the allelopathic compounds
degraded rapidly and were inactive when added to P. bahamense cultures. These results are
important in understanding the bloom mechanisms of these two harmful algal blooms (HABs)
species

Al-Has,Asilah

Mohammad-Noor,Normawaty

Shaleh,Sitti Raehanah M.

Mustapha,Shuhadah

Darnis,Deny Susanti

Ayub,Mohd Nor Azman

Adam,Aimimuliani

Mustakim,Ghaffur Rahim

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

Allelopathic effects of Margalefidinium polykrikoides on the growth of Pyrodinium bahamense in different nutrient concentrations

The International Islamic University Malaysia Repository

058HA ECOLOGYAllelopathic effects of Margalefidinium polykrikoides on the growth of Pyrodinium bahamense in different nutrient concentrations Asilah Al-Has1, Normawaty Mohammad-Noor1*, Sitti Raehanah M. Shaleh2, Shuhadah Mustapha3, Deny Susanti Darnis1, Mohd Nor Azman Ayub4, Aimimuliani Adam1, Ghaffur Rahim Mustakim21 International Islamic University Malaysia, 25200, Kuantan, Pahang, Malaysia; 2 Universiti Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia; 3 Department of Fisheries Sabah, 89400, Kota Kinabalu, Sabah, Malaysia; 4 Fisheries Research Institute, 11960, Batu Maung, Pulau Pinang, Malaysia.*corresponding author’s email: normahwaty@iium.edu.myAbstractCo-occurrence of the dinoflagellates Margalefidinium polykrikoides, a known fish killer, and the neurotoxic species Pyrodinium bahamense is commonly observed in the coastal waters of Sabah, Malaysia. During most of these events, M. polykrikoides dominated the bloom, apparently suppressing the growth of P. bahamense. To increase our understanding on the nutrient conditions of this phenomenon, a study was conducted to explore the interaction between these species. The specific a im was to document the a llelopathic effects, i f any, o f M. polykrikoides o n P. bahamense when varying ratios of the two species were co-cultured under different nitrogen (N) and phosphorus (P) concentrations. The bioassay experiments started with three cell abundance proportions, which were 5:5 (500 cells mL-1 of each species, M. polykrikoides, and P. bahamense); 1:5 (100 cells mL-1 of M. polykrikoides and 500 cells mL-1 of P. bahamense); and 5:1 (500 cells mL-1 of M. polykrikoides and 100 cells mL-1 of P. bahamense). Additionally, culture filtrates (10, 20 and 50 mL) from the late exponential phase of M. polykrikoides  were added to 150 mL of P. bahamense to determine if cell filtrates were allelopathic. Results indicate that M. polykrikoides was allelopathic to P. bahamense when nutrients were abundant, but not when nutrients were limiting or N was limiting relative P. The production of allelopathic compounds was supported by abnormal morphological changes in P. bahamense when co-cultured with M. polykrikoides. This capacity to suppress P. bahamense growth, combined with the inherently faster growth rate of M. polykrikoides relative to P. bahamense can account for why M. polykrikoides  forms nearly monospecific blooms when nutrients are high. The filtration studies indicated the allelopathic capacity of M. polykrikoides required direct cell contact or that the allelopathic compounds degraded rapidly and were inactive when added to P. bahamense cultures. These results are important in understanding the bloom mechanisms of these two harmful algal blooms (HABs) species.Keywords: Allelopathy, bi-algal cultures, Margalefidinium polykrikoides, nutrients, Pyrodinium bahamensehttps://doi.org/10.5281/zenodo.7034906059HA ECOLOGYIntroductionHarmful algal blooms (HABs) are natural events where increased phytoplankton biomass can result in adverse impacts on both public and ecosystem health. In recent years, Margalefidinium (Cochlodinium) polykrikoides blooms have gained attention because of their increasing geographical distribution worldwide (Kudela and Gobler, 2012). This species has been reported to produce cysts, is a fast swimmer, mixotrophic, and has Allelopathic properties (Kudela and Gobler, 2012). Allelopathy is a phenomenon where a species produces chemicals that inhibit the growth of, or kill, other competing species (Hattenrath-Lehmann and Gobler, 2011). Species which have been adversely effected by M. polykrikoides include Gymnodinium catenatum (Band-Schmidt et al., 2020), Chattonella subsalsa, Isochrysis galbana, Heterocapsa rotundata, Thalassiosira weissflogii (Tang and Gobler, 2010) and Akashiwo sanguinea (Yamasaki et al., 2007). These studies indicate the allelopathic effects of M. polykrikoides are not species-specific. The impacts of other allelopathic species has been shown to be influenced by nutrient limitation, i.e. Alexandrium tamarense (Zhu and Tillmann, 2012) and Prymnesium parvum (Uronen et al., 2005). In contrast, the effects of nutrient availability on the allelopathic impacts of M. polykrikoides are less studied.Another HAB species of concern, which often co-occurs with M. polykrikoides is Pyrodinium bahamense. This species is a saxitoxin producer reported from the Indo-Pacific and Caribbean Sea (Usup et al., 2012). Although the distribution of P. bahamense is more limited compared to M. polykrikoides, the species can have devastating impacts where it blooms due to its ability to produce higher intracellular saxitoxin concen-trations relative to other saxitoxin producers which cause paralytic shellfish poisoning (PSP) (Usup et al., 2012). The co-occurrence of M. polykrikoides and P. bahamense has been monitored by the Sabah Fisheries Department of Malaysia since 1976 (Jipanin et al., 2019). Since 2005, blooms of M. polykrikoides have also been observed in the coastal waters adjacent to Kota Kinabalu, the  capital city of Sabah (Anton et al., 2008). These later blooms sometimes co-occur with P. bahamense (Adam et al., 2011; Chong et al., 2020). Shaleh et al. (2010) found that in the laboratory, salinity and pH did not significantly affect interactions between these species. However, field reports indicate elevated nutrient concentrations (N and P) can trigger blooms dominated by M. polykrikoides blooms and containing only a small subpopulation of P. baha-mense (Mohammad-Noor et al., 2014). The present study used bi-species cultures to simulate bloom conditions where either M. polykrikoides or P. bahamense dominated, or co-dominated the bloom population. These different simulated populations were further allowed to develop under different nutrient conditions. The goal was to quantify how the relative cell densities of M. polykrikoides versus P. bahamense in conjunction with different nutrient levels might affect the allelopathic interactions between these two species. Also, the effects of cell free filtrates from M. polykrikoides on the growth of P. bahamense were investigated to evaluate if 060HA ECOLOGYdirect cell contact was required to achieve any observed allelopathic effects. Materials and MethodStrains of M. polykrikoides and P. bahamense were obtained from the Borneo Marine Research Institute (BMRI), University Malaysia Sabah, Malaysia. Strains of M. polykrikoides and P. bahamense were isolated during blooms in Sepanggar Bay in 2019 and 2013, respectively, and established into unialgal non-axenic cultures in f/2 media. The experiments were conducted by inoculating M. polykrikoides into f/2 media containing three nutrient concentrations: (a) f/2 medium, 882 µM NO3-N and 32 µM PO4-P, N:P = 27.6 (b) nutrient enriched media (NE), 30 µM NO3-N and 5 µM PO4-P, N:P = 6 and (c) cells were added from the late log stock cultures without nutrient addition, low concentration (LC). Pyrodinium bahamense was grown in f/2 medium. Trace metals and vitamins were the same as listed in Guillard and Ryther (1962). All media were prepared using autoclaved filtered seawater. The cultures were maintained at 25-26 oC with a 12:12 light–dark cycle illuminated by LED light with an intensity of 100 µmol quanta m-2 s-1. Cell contact experiments Bi-algal culture experiments were conducted in 250 mL flasks containing 150 mL of f/2 medium which augmented with nutrients as described above. Both species were harvested during their late exponential phase from the unialgal cultures and mixed at three cell abundance ratios, which were 1:5 (100 cells 100 mL-1 of M. polykrikoides and 500 cells mL-1 of P. bahamense); 5:1 (500 cells mL-1 of M. polykrikoides and 100 cells mL-1 of P. bahamense); and 5:5 (500 cells mL-1 of each species). Treatments were run in triplicate.Culture filtrate experiments Cell-free filtrates of M. polykrikoides cultured in different nutrient concentrations (f/2, EN and LC treatments) were harvested at the late exponential phase and filtered through 47 mm GF/F filter (Whatman). Three volumes (10, 20 and 50 mL) of the culture filtrates of M. polykrikoides were added to 250 mL Erlenmeyer flasks containing 150 mL of P. bahamense containing an initial density of P. bahamense was 500 cells mL-1. A three mL aliquot was collected from each experiment daily during seven days for cell enumeration and morphological observations. One milliliter of each sample was counted twice using a Sedgewick Rafter cell under a light microscope (Zeiss, Axiostar). Growth rate (µ) was determined based on the following formula (Guillard and Ryther, 1962). Results and DiscussionIn the f/2 nutrient treatment, P. bahamense was able to initially compete well with M. polykrikoides when it started with a 5:1 concentration advantage. However, by day four of the experiment, P. bahamense began declining relative to the control as M. polykrikoides cell concentration began slowly increasing. In the corresponding treatment where M. polykrikoides had a 5:1 starting advantage, M. polykrikoides  increased slowly relative to the control whereas  P. bahamense failed to increase in the presence of M. polykrikoides. Interpretation of these experimental results is complicated because the control culture of P. bahamense did not grow well. The treatment starting with the 5:5 (equal concentration of both 061HA ECOLOGYspecies) showed that M. polykrikoides cell concentrations steadily increased while the P. bahamense cell population steadily declined, with both controls showing steady growth. Cumulatively, these results are consistent with M. polykrikoides inhibiting the growth of P. bahamense either through allelopathy or an ability to outcompete P. bahamense for nutrients. Filtrates from M. polykrikoides cultures did not effect the cell morphology or the growth of P. bahamense.Evidence for the production of allelopathic com-pounds is supported by abnormal changes in the morphology of P. bahamense after two more days with direct cell? contact with M. polykrikoides. The main changes observed were loss of the thecate plate and cell lysis (Fig. 2). These observations were consistent with allelochemicals increasing the cell membrane penetrability thereby suppressing cell growth (Xue et al., 2018). Future studies where ambient nutrient concentrations are monitored closely are needed to further separate the extent to which the inhibition is related to nutrient competition versus production of allelo-chemical(s). Interestingly all three experiments showed that M. polykrikoides growth in the bi-species cultures was slower than that in the control. This reduced growth rate indicates a cost for competing with P. bahamense, but that cost is not likely due to the production of allelochemicals (Zhu and Tillman 2012). Another observation from the control cultures is that M. polykrikoides has an inherently faster growth rate than P. bahamense. This would give M. polykrikoides a growth advantage when nutrient levels are elevated.The high nutrient treatment corresponds to field conditions where nutrient concentrations are elevated, but there is an excess of P relative to N (N:P = 6). In the treatment where there were five P. bahamense relative to one M. polykrikoides, the population of P. bahamense steadily declined while the M. polykrikoides cell counts gradually increased. The 5:1 M. polykrikoides: P. baha-mense experiment failed after three days and provided no results. In contrast, the 5:5 experiment showed that under the more N-limited conditions, growth of both species in the bi-cultures was very similar to the 1:5 treatment. The only difference from that initial 1:5 treatment is that the control population rapidly increased indicating the media is capable of supporting rapid growth of M. polykrikoides. Though these nutrient conditions were not optimal for P. bahamense growth, at high enough cell concentration this species inhibited the growth of M. polykrikoides. Because P. bahamense growth was exceeding slow this inhibition is unlikely due to direct nutrient competition indicating under certain nutrient regimes P. bahamense may inhibit M. polykrikoides, instead of the reverse situation.The low nutrient condition, where cells grown in f/2 media, with an N:P ratio closer to the Redfield ratio, and then allowed to become more nutrient limited represents what might happen more toward the end of a bloom. In the 1:5 M. polykrikoides: P. bahamense treatment the controls grew while the populations in the bi-species cultures steadily declined. In the 5:1 treatment the growth of both species in the controls and the cells in the bi-species cultures were essentially the same (Fig. 1) as was also true for the 5:5 treatment. These results are consistent with cells growth largely 062HA ECOLOGYdependent on internally stored nutrient with no indication of any allelopathic interactions. Cumulatively these results suggest elevated nutrient concentrations favor M. polykrikoides over P. bahamense because of its inherently higher growth rate.  The data are further consistent with high densities of nutrient replete M. polykrikoides having the ability to produce allelopathic compounds that impeded the growth of P. bahamense. This allelopathic inhibition of P. bahamense, however, did not occur at higher nutrient concentrations where the N:P ratio was skewed toward an excess of phosphate or when the cells were nutrient limited (Fig. 1). This may indicate N is a structural component of the allelo-pathic compound itself. Similarly observed high allelopathic activity of A. sanguinea when nutrient concentrations were high. Tang and Gobler (2010) also demonstrated the allelopathic effect of M. polykrikoides was dependent on the initial cell abundance and Band-Schmidt et al., (2020) showed the duration of the inhibition was proportional to cell density. In summary, M. polykrikoides likely dominates over P. bahamense when nutrients are high due to its inherrently higher growth rate (Kudela and Gobler, 2012). Evidence from this and other studies futher support allelopathic supression of P. bahamense when M. polykrikoides cell concentration are high. This would account, at least in part, for why there are relatively few P. bahamense present when M. polykrikoides cell densities are high and nutrients abundant. Fig. 1. Cell abundance proportions of M. polykrikoides (mean ± standard deviation) and P. bahamense in co-cultured cell contact experiments: (i) 1:5, (ii) 5:1, (iii) 5:5 at different treatments (A) f/2 medium (f/2); (B) nutrient enriched (EN); and (C) low concentration (LC). Closed circles: M. polykrikoides in bi-algal culture; open circles: M. polykrikoides control; closed triangles: P. bahamense in bi-algal culture; open triangles: P. bahamense control. 063HA ECOLOGYFig. 2.  Detachment of the thecal plate of P. bahamense when exposed to M. polykrikoides. ReferencesAdam, A., Mohammad-Noor, N., Anton, A., Saleh, E., et al., (2011). Harmful Algae 10, 495-502.Anton, A., Teoh, P.L., Muhamad Shaleh, S.R., Mohammad-Noor, N. (2008). Harmful Algae 7, 331-336. Band-Schmidt, C.J., Zumaya-Higuera, M.G., López-Cortés, D.J., Leyva-Valencia, I., et al., (2020). Harmful Algae 96, 101846. Chong, B.W.K., Leong, S.C.Y.L., Kuwahara, V.S., Yoshida, T. (2020). Reg. Stud. Mar. Sci. 37, 101326. Guillard, R. 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Allelopathic effects of Margalefidinium polykrikoides on the growth of Pyrodinium bahamense in different nutrient concentrations

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NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

The International Islamic University Malaysia Repository

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY