Pyrodinium bahamense and Other Dinoflagellate Cysts in ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs2018/2ndQtr/pyrodi… · This important stage of the lifecycle of dinoflagellates

*Corresponding author: [email protected]

Key words: cyst, dinoflagellate, Gonyaulax, Gymnodinium, Lingulodinium, Protoceratium, Pyrodinium

Pyrodinium bahamense and Other Dinoflagellate Cysts in Surface Sediments of Cancabato Bay, Leyte, Philippines

Division of Natural Sciences and Mathematics, University of the Philippines Visayas Tacloban College,

Magsaysay Boulevard, Tacloban City, Leyte 6500 Philippines

Leni Yap-Dejeto*, Caryl Y. Durante, Irene L. Tan, and Coleen O. Alonzo

Cysts withstand hostile environmental conditions and are source of inoculum for recurrent blooms. In the Philippines, the first recorded bloom of the phytoplankton Pyrodinium bahamense was observed in Samar-Leyte areas, including Cancabato Bay in 1983. Since then, shellfish bans in these areas have been imposed periodically. Until the present however, there is no thorough cyst study done in this bay. This study has assessed the abundance and distribution of dinoflagellate cysts in the bay. Surface sediment samples collected and processed by palynological technique have revealed a total of 21 species of dinoflagellate cysts belonging to five groups: Gonyaulacoid, Protoperidinioid, Gymnodinioid, Calciodinellid, and Diplopsalid. Cysts have been detected in all stations, with cyst densities ranging from 1-80 cysts g-1 DW; and Operculodinium centrocarpum (Protoceratium reticulatum) dominated in four stations. Low levels of P. bahamense cysts, Polysphaeridium zoharyi, have been detected in 13 stations, the densest at 16 cysts g-1 DW. Concentrations of cysts that have been highest in the inner part of the bay could have been affected by several factors, including substrate type, bulk dry weight, and nitrogen content. This important stage of the lifecycle of dinoflagellates should be factored in future models to predict P. bahamense blooms in the bay.

Philippine Journal of Science147 (2): 209-220, June 2018ISSN 0031 - 7683Date Received: 10 Jul 2017

INTRODUCTIONThe major organisms responsible for harmful algal blooms (HABs) in the Philippines are dinoflagellates, mainly the toxic species of Pyrodinium bahamense (Siringan et al. 2008). In the Philippines, the first recorded bloom of P. bahamense was observed in Samar-Leyte areas, including San Pedro and Cancabato Bays during 1983. P. bahamense has been responsible for the toxic blooms in the country since then (Azanza 1997). Toxic red tide occurrences in Leyte were documented in 1983, 1988-1989, 1993, and 1994. A total of 322 cases of paralytic shellfish poisoning (PSP) was recorded, among which 18 people died (Furio & Gonzales 2002).

In recent years, P. bahamense blooms in San Pedro Bay were recorded during the years 2002, 2007, and 2009, and as of this writing in 2015, 2016, and 2017. At present, the Bureau of Fisheries and Aquatic Resources (BFAR) has been monitoring the bay twice a month if negative of HABs, and four times a month if reported positive (Rosalinda Cañas pers. com). However, only water samples and toxicity levels of shellfish in the area are monitored and no cyst studies are done. Cysts are important stages in the lifecycle of a dinoflagellate (Anderson 1989); thus, aside from monitoring of the abundance of vegetative cells, information of resting cysts of P. bahamense must be acquired.

A cyst is a non-motile cell with non-existent flagella and is incapable to swim. Two types of cysts can be found in the life cycle of dinoflagellates: the temporary cyst and

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Figure 1. Map of Cancabato bay showing 17 sampling stations. Fewer stations were plotted near the mouth of the bay because of sandy substrate and the selected stations were preferably muddy.

the resting cyst. The temporary cysts are formed asexually and observed only in laboratory cultures, while the resting cysts are formed sexually and occur naturally among dinoflagellates (Matsuoka et al. 1989; Matsuoka & Fukuyo 2000; Uchida 2001). P. bahamense cysts play an important role in bloom initiation, genetic recombination, species dispersal, environmental survival, and toxicity (Anderson 1989). PSP incidents caused by P. bahamense have been increasing while their geographical distribution are expanding i.e., these have been detected in areas previously P. bahamense free (Mizushima et al. 2007).

Cancabato Bay (11o00’N and 11o20’N;124o00’E and 125o14’E ) is a bay within San Pedro Bay, which is in between the islands of Leyte and Samar located at the eastern border of the Philippine archipelago. It has a total area of about 949 km2, which roughly contains 625 km3 marine water (Baleña 1995). The bay consists of primarily muddy or sandy substrate with reefs and seagrass beds found along most of the coast; an extensive assessment of the bay’s water quality and sediment nutrients was done by Baleña (1995). Moreover, Ingles (1997) underwent a year-wide data collection regarding the abundance, distribution, and occurrence of P. bahamense (cyst and motile cells) during its bloom in 1994. In Jul-Nov 1994, P. bahamense levels were so high that it caused the contamination of any filter feeding animal and posed hazard to life. A shellfish ban was declared that year (Ingles 1997).

In instances where HABs occur, the local fishing and shellfish industry are severely affected. More so, the community is at risk of poisoning if the blooms are not

detected early and shellfish ban bulletins are not properly disseminated.

There have been many cyst studies worldwide to explain distribution, expansion, and origin of harmful dinoflagellate blooms (Anderson 1989; Matsuoka & Fukuyo 2000; Devillers & Vernal 2000; Marret et al. 2001; Furio et al. 2012). However, aside from the survey done twenty years ago by Ingles (1997) on P. bahamense cysts alone, no study has been done to identify other dinoflagellate cysts present in Cancabato Bay. A recent study has mapped an atlas of dinoflagellate cysts involving 2,405 globally distributed data points (Zonneveld et al. 2013), but there is no data point for the Philippines, even on cysts of toxin producing P. bahamense. Thus, an updated list of the presence and abundance of dinocysts and an assessment of the surface sediments of San Pedro Bay is needed. The results presented in the paper could be useful for further assessment of the possibility of the recurrence of P. bahamense and understanding of its bloom dynamics. The data generated is hoped to contribute to the understanding of HAB ecology.

METHODS

Sampling StationsSurface sediment samples were collected in 17 sampling stations within the Cancabato Bay (Figure 1). Stations were predetermined by plotting points on the satellite map

Yap-Dejeto et al.: Dinoflagellate Cysts in Cancabato BayPhilippine Journal of ScienceVol. 147 No. 2, June 2018

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of the bay. In an S-fashion, each station was in estimated increments of 500 m within the horizontal plot, and 1,000 m increments within the vertical plot. However, not all tentative stations were included because of substratum considerations. Stations with sandy substrate were excluded because according to Pospelova and co-authors (2004), cysts were associated primarily in muddy sediments. The coordinates were plotted in QGIS v. 2.6.0, and the exact geographic locations were recorded on the field using a GPS receiver unit.

Sample CollectionGravity corer and grab sampler were used to collect surface sediments in Cancabato Bay. Samples were placed in individual plastic bags, kept in the dark, and stored in an ice box. Samples were stored in the refrigerator until they were processed. The upper 2 cm of the surface sediments were sub-sampled into three using a syringe with its lower tip cut-off.

Other physico-chemical parameters of the surface water such as temperature, salinity, pH, depth, turbidity, light intensity, and current velocity were also measured during the sample collection. Two sampling sessions were conducted, one in Nov 2014 across nine sites (C1-C9) and another in Mar 2015 across eight sites (C10-C17).

Physico-chemical Characterization of SedimentsCollected sediments were characterized based on their particle size, sediment moisture, organic content, and nutrient content (phosphorous and nitrogen). Particle size was determined by filtering them through a standard set of sieves (Fieldmaster Soil Sampling Sieve Set) measuring 4000 µm and 63 µm.

Classification of sediment particles were based on Wentworth grade scale with few modifications (Wentworth 1922). Three major categories were identified: gravel (>2 mm), sand (2-0.0625 mm), and mud (<0.0625 mm). Percentages of sand were plotted in a ternary diagram using a modified Folk’s classification scheme for texture classification (Folk et al. 1970). Sediments were classified as sand (≥90% sand), muddy sand (50-89% sand), sandy mud (10-49% sand), and mud (≤10% sand).

Sediment moisture was measured by heating the sediments to 110˚C for 24 h and calculating for the net weight loss (Lewis & McConchie 1994). Organic content was measured by heating the previously dried sediments to 600˚C for 15 min and calculating for the net weight loss (Heiri et al. 2001). For the concentration of phosphorous and nitrogen, samples were sent to Central Analytical Services Laboratory (CASL), Visayas State University for analysis.

Palynological TechniqueHandling of cysts were based on the methods of Matsuoka and Fukuyo (2000) with few modifications. First, 2 ml sediment was placed into a 50 ml polyethylene tube using a tip-cut syringe. Eight ml distilled water was added into the tube, which was mixed with the sediments. After centrifugation at 3200 RPM for 28 s, the supernatant water was decanted making sure light-weight cysts were not included. The washing procedure was repeated three times to remove salt. Ten ml of 10% HCl was added to remove calcium carbonate. Then, the sample was washed with distilled water to remove excess acid. To remove silicates, two ml of 38% HF was added to the sample. This was conducted under a fume hood and with proper protection because hydrofluoric acid is very reactive and highly toxic. Next, the sediments were heated in a water bath at approximately 70˚C for 2 h to neutralize the calcium carbonate. The sediments were again washed with distilled water. The samples were then placed in a water bath type Branson 3510 sonicator and were sonicated for 20 min at 40 kHz to break the cells of diatoms. To separate the sediments from the cysts, the sediments were then filtered through sieves of 125 µm and 25 µm mesh size. Particles collected in the 25 µm sieve were collected and stored in polyethylene tubes, covered in aluminium foil, and stored at 4˚C to avoid bacterial proliferation until microscopic analysis.

Cyst Identification and CountingAn aliquot of 1 ml for each sample, mounted on a Sedgewick-Rafter chamber grid slide was analyzed using a Carl Zeiss Axiovert 40 CFL light microscope at 100-400x magnification. Cysts were identified based on the characteristics described by Matsuoka and Fukuyo (2000) and other available sources. Preliminary identification was done with the help of Ms. Zoan Reotita and Ms. Mary Rose Ezperanza. These were verified and confirmed by Dr. Vera Pospelova, Dr. Fabienne Marret-Davies, Dr. Karin Zonneveld, Dr. Yasuwo Fukuyo, and Dr. Kazumi Matsuoka. At least 300 cysts were counted per sample and cyst concentration was determined using the formula:

Cyst concentration = number of cystsg of dry bulk (1)

RESULTS

Physico-chemical Character of SedimentsParticle size analysis revealed that all sites had zero to a few percentage of shell fragments and gravel. C1 and C11, which were found in areas near fish cages and wet markets, had the highest values for shell fragments and gravel with


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11.84% and 7.54%, respectively. Most stations located in or near the mouth of the bay were muddy except for C5, C7, C8, C9, C15, and C16. The average percentage of sand was 34.3% and therefore, in accordance with the textural classification of sediments modified after Folk and co-authors (1970), substrate of Cancabato Bay was generally sandy mud. Grain size analysis is shown in Table 1.

Dinoflagellate CystsDinoflagellate cysts were found in all 17 stations. A total of 21 species of dinoflagellate cysts were found in Cancabato Bay (Table 2). Cyst concentration ranged from 1 cyst per gram of dry weight (cysts g-1 DW) (C7) to 80 cysts g-1 DW (C11). Most of the cysts found were empty making up 95% of the total cysts, while the remaining 5% were living cysts. Autotrophs dominated the cyst population covering 65% of the total cysts, while heterotrophs occupied the remaining 35%. The highest cyst concentrations were observed in areas near the inner side of the bay (C11, C17, C13, and C14) (Figure 4).

Identified cysts belonged to five groups, namely Gonyaulacoid, Calciodinell id, Gymnodinioid, Protoperidinioid, and Diplopsalid. Gonyaulacoid occupied the largest percentage among all five groups, followed by Protoperidinioid, Gymnodinioid, Calciodinellid, and Diplopsalid (Table 2).

Table 1. Grain size analyses of sediments using Fieldmaster soil sampling sieve set (4000 µm and 63 µm).

Site Shell Fragments and Gravel (%)

Sand (%)

Mud (%)

TexturalClassification

C1 11.84 14.47 73.68 Sandy Mud

C2 0.0 12.50 87.50 Sandy Mud

C3 0.0 16.22 83.78 Sandy Mud

C4 0.23 45.20 54.57 Sandy Mud

C5 0.28 89.94 9.77 Muddy Sand

C6 0.00 14.75 85.25 Sandy Mud

C7 0.00 53.67 46.33 Muddy Sand

C8 1.12 61.45 37.43 Muddy Sand

C9 3.72 81.86 14.42 Muddy Sand

C10 0.00 4.35 95.65 Mud

C11 7.54 3.28 89.18 Sandy Mud

C12 0.00 21.36 78.64 Sandy Mud

C13 0.00 5.64 94.36 Mud

C14 0.00 7.73 92.27 Mud

C15 2.74 62.09 35.16 Muddy Sand

C16 1.92 85.34 12.74 Muddy Sand

C17 0.93 3.27 95.79 Mud

Moisture content for all stations ranged from 79% to 23%. Site C3 had the highest moisture content of 79%, while C5 had the least moisture content at 23%. Wet bulk density was highest for C16 with 1.83 g ml-1and lowest for C1 with 1.06 g ml-1. On the other hand, dry bulk density was recorded highest in C5 (1.73 g ml-1) and lowest for C11 (0.26 g ml-1).

For organic matter, C11 had the highest with 1.26% while C5 had the lowest organic matter content with 0.07%. Carbonate content analysis revealed that C12 had the highest carbonate percentage at 0.76%, while C5 had the lowest with only 0.1%. On the other hand, lithic was highest at 99.82% at C5 while C11 had the lowest lithic at 98.26%. Nitrogen and phosphorous content were high in areas near the inner east part of the bay (C1, C9, C13, and C14) (Table 1).

Figure 2 (Plate 1). Autotrophic dinoflagellate cysts in Cancabato Bay, Leyte, Philippines. (a) Spiniferites bulloideus;(b) Spiniferites mirabilis (c) Spiniferites ramosus (d) Spiniferites membranaceus; (e) Spiniferites sp.; (f) Gymnodinium catenatum; (g) Operculodinium centrocarpum; (h) Lingulodinium machaerophorum; (i) Polysphaeridium zoharyi (P. bahamense); (j) Pheopolykrikos hartmannii; (k) Scripsiella trochoidea. Scale bar is 20 µm.


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Table 2. List of dinoflagellate species found in surface sediments of Cancabato Bay, Leyte, Philippines and the stations these were found.

Cyst Species (Paleontological Name)

Motile Species (Biological Name)

Stations

AUTOTROPHIC

Gonyaulacoid

Lingulodinium machaerophorum Lingulodinium polyedruma C6, C12, C13, C15

Spiniferites bulloideus Gonyaulax scrippsae C5, C6, C12, C13, C14

Spiniferites ramosus Gonyaulax spinifera complexa C10, C12, C13, C16, C17

Spiniferites mirabilis Gonyaulax spinifera complexa C1, C2, C9, C15

Spiniferites membranaceus Gonyaulax membranaceus C17

Spiniferites sp. Gonyaulax sp. C2, C3, C4, C6, C13

Operculodinium centrocarpum Protoceratium reticulatuma C1, C2, C3, C6, C10,C13, C15

Polysphaeridium zoharyi Pyrodinium bahamensea C1, C3, C4, C8, C17

Calciodinellid

- Scripsiella trochoidea C6

Gymnodinioid

- Gymnodinium catenatuma C12

Pheopolykrikos hartmanii C10, C16

HETEROTROPHIC

Protoperidinioid

Brigantedinium cariacoense Protoperidinium avellana C12, C14, C15, C17

Brigantedinium majusculum Protoperidinium sp. C2, C14

Brigantedinium simplex Protoperidinium conicoides C1, C12, C15

- Protoperidinium conicum C5, C9, C11, C16, C17

Trinovantedinium applanatum Protoperidinium pentagonum C13

- Protoperidinium spp. C1, C2, C4, C5, C6, C12, C13, C14, C15

- Protoperidinium thorianum C1

- Protoperidinium subinerme C12

Diplopsalid

- Diplopelta sp. C2

- Dubridinium sp. C1, C2

- Oblea acanthocysta C5

Gymnodinioid

- Polykrikos kofoidii C10, C17aharmful species (Matsuoka & Fukuyo 2000)

Five of the identified cysts belonged to the Gonyaulacoids, namely Spiniferites sp. (Gonyaulax sp.), S. bulloideus (G. scrippsae), S. ramosus, S. mirabilis (G. spinifera complex), Operculodinium centrocarpum (Protoceratium reticulatum), and Polysphaeridium zoharyi (P. bahamense). Among which, G. spinifera (Matsuoka & Fukuyo 2000), P. reticulatum (Satake 1997), and P. bahamense (Siringan et al. 2008) were species that produce harmful toxin.

The Protoperidinioid group, characterized by round brown cysts, had five recorded species that included Brigantedinium majusculum (Protoperidinium sp.), B. simplex (P. conicoides), P. conicum, P. thorianum, and P. subinerme. For the Gymnodinioid group, both autotrophic (Gymnodinium catenatum and Pheopolykrikos hartmannii) cysts and cysts of heterotrophic (P. kofoidii) species were identified. Among these, G. catenatum can


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produce PSP toxin. The group of Calciodinellid had only a single species identified, namely cysts of Scripsiella sp. The heterotrophic Diplopsalid group had Diplopelta sp., Dubridinium sp., and Oblea acanthocysta.

Cyst DensityCysts were highly concentrated at C11 (80 cysts g-1 DW), which was only followed by C17 (39 cysts g-1 DW) and C12 (36 cysts g-1 DW) with cysts concentration lower than half of C11’s (Table 3). In contrast, the least dense cyst concentration was recorded for C7 having a density of only 1 cyst g-1 DW. Stations that were high in cyst concentration were located in areas near the innermost part of the bay (at the base), while stations with lower cyst concentrations were located near its mouth.

Species concentration varied widely among all 17 stations (Table 3). O. centrocarpum dominated in four stations (C2, C6, C10, and C12). For stations C7, C8, and C9, the dominant

Figure 3 (Plate 2). Heterotrophic dinoflagellate cysts in Cancabato, Bay, Leyte, Philippines. (a) Diplopelta sp.; (b) Polykrikos kofoidii; (c) Dubridinium sp.; (d) Brigantedinium sp. (Protoperidinium sp.); (e) Brigantedinium majusculum (Protoperidinium sp.); (f) Brigantedinium simplex; (g) Brigantedinium cariacoense; (h) Trinovantedinium applanatum; (i) Protoperidinium thorianum; (j) Oblea acanthocysta; (k) Round Brown Spiny Cyst. Scale bar is 20 µm.

cyst species was Scripsiella sp.; however, O. centrocarpum and Scripsiella sp. cyst density in these sites were not high compared to other dominant cysts in other sites. This is true for P. zoharyi (P. bahamense) and G. catenatum cysts. P. zoharyi (P. bahamense) dominated in station C17 with a cyst concentration twice denser than C14. Same is true for G. catenatum cysts, the dominant cyst in stations C11 that had cyst concentration three times more dense than C1. In stations C4 and C6, the dominant cyst was that from the species of Protoperidinium while for stations C5 and C15, P. conicum cyst was dominant. S. ramosus cysts of G. spinifera complex dominated station C16 while for C3, no dominant species were detected and equivalent cyst concentrations were recorded for each of the following: P. zoharyi (P. bahamense), O. centrocarpum (P. reticulatum), G. catenatum, and S. bulloideus (G. scrippsae).

Cysts of Pyrodinium bahamense, Polysphaeridium zoharyiP. zoharyi were detected in 13 stations; however, no cyst of this species was detected for the following stations: C2, C5, C6, and C7. The highest P. zoharyi cyst concentration was recorded for C17 with a density of 16 cysts g-1 DW, followed by C11 with 12 cysts g-1 DW and C14 with 9 cysts g-1 DW (Table 3). A map showing P. zoharyi cyst density is shown in Figure 5.

DISCUSSION

Physico-chemical Characters of Surface Sediments in the BayGrain size analysis has revealed that in areas near the fishing barangay and wet markets (C11 and C1), there is a high percentage of shell fragments and gravel. This may be due to the presence of shellfish farming and food waste coming from inhabitants near those sites. Surface sediments near the mouth of the bay (C5, C7, C8, C9, C15, and C16) have been found to be sandy and become muddy towards the inner part of the bay, which may be due to the U-shaped structure of the bay. Because the mouth part is exposed to strong currents from San Juanico Straight, sandy particles are carried along with it, producing a sandy surface (Ingles 1997). On the other hand, the inner parts of the bay (C10, C13, and C14) are isolated from strong currents that are also more stationary, allowing small muddy particles to settle (Pospelova et al. 2004).

Changes in microbial activity can be determined by changes in soil moisture content (Orchard & Cook 1983). Sediment moisture content has been highest in areas at the base of the bay (C1, C2, and C3), while stations on or near the mouth of the bay (C5, C16, and C9) have the


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Figure 5. Map of Polysphaeridium zoharyi (Pyrodinium bahamense) cysts in g-1 DW in Cancabato Bay, Leyte, Philippines.

Figure 4. Map of dinoflagellate cyst density (cysts g-1 DW) in Cancabato Bay, Leyte. Cyst density is more concentrated in the innermost part compared to the outer part of the bay.


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lowest moisture content. This may suggest that stations located at the northern part of the bay had higher soil respiration; therefore, it houses higher microbial activity than the southern part of the bay.

Sediment moisture is inversely related to organic carbon content (Avnimelech 2001). Calcareous nanoplankton, foraminiferous, and other remains of marine organisms that sink to the surface contribute to the carbonate content of the sediments (Matsuoka & Fukuyo 2000). The range of carbonate content has not varied (SD=0.18); however, C12 had a high carbonate content compared to other stations. This suggests that this site can be rich in remains of carbonate-forming organisms such as shells and skeletons. Reef rubble due to reoccurring storms may also have contributed to this increase in carbonate content.

Sediment organic matter is composed of carbon and nutrients in the form of carbohydrates, proteins, fats, and nucleic acids from plant and animal derivatives (Boyd 1995). A higher level of organic matter has the capacity to absorb organic components and pollutants (Avnimelech 2001). Stations that have the highest organic matter content were C11, C10, and C13 and are located near residential areas that may indicate higher levels of pollution in the water coming from sewage waste and effluent waste from fish farms, which were evident in these areas.

Lithic content is the amount of stones or rocks eroded down to sand size that are found in the sediments. Sites

with high lithic content are found in areas near the mouth of the bay (C5, C7, and C8), while stations with low lithic content are located in the inner part of the bay (C11, C10, and C12). Rocks and other stony particles are heavy and are not washed to shore easily. This may explain the high lithic content in areas near the bay’s mouth, while low lithic content is present in the inner part of the bay near the shore.

Carbonate, lithic, and organic matter content when summed up makes up 100% of the total dry bulk density. Bulk density can be divided into two types: wet and dry. Dry bulk density is the ratio of the mass of the solid phase of the soil to its total volume, whereas wet bulk is the ratio of the total mass of soil to its total volume (Buckman & Brady 1960). Stations near the north-western part of the bay (C16, C5, and C9) had high wet and dry bulk content. However, stations with low wet (C1, C6, and C3) and dry (C5, C9, and C10) bulk densities were not observed in one single, closely related area. Nitrogen and phosphorous content were high in areas in the inner east side of the bay. These stations (C1, C11, C10, C14, and C13) were found near coastal residential areas suggesting that high nitrogen and phosphorous content may be due to anthropogenic influences (Azanza et al. 2004).

Dinoflagellate Cysts DiversityThe number of dinoflagellate cysts species identified in Cancabato Bay (CB) was comparatively low compared

Table 3. Total cyst density, dominant cyst density and Pbc cyst density, of surface sediments in Cancabato Bay, Leyte, Philippines.

Station P. bahamense Cyst Density (cysts g-1 DW)

Total Cyst Density (cysts g-1 DW)

Dominant Species(Biological Name)

Density of Dominant Species (cysts g-1 DW)

C1 2 15 Gymnodinium catenatum 4

C2 - 19 Protoceratium reticulatum 7

C3 1 5 - 1

C4 2 14 Protoperidinium sp. 5

C5 - 3 Protoperidinium conicum 2

C6 - 22 Protoceratium reticulatum 8

C7 - 1 Scripsiella sp. 1

C8 2 14 Scripsiella sp. 5

C9 1 5 Scripsiella sp. 1

C10 3 14 Protoceratium reticulatum 6

C11 12 80 Gymnodinium catenatum 15

C12 6 36 Protoceratium reticulatum 9

C13 7 30 Protoperidinium sp. 7

C14 9 30 Pyrodinium bahamense 9

C15 1 8 Protoperidinium conicum 3

C16 2 7 Gonyulax spinifera 2

C17 16 39 Pyrodinium bahamense 16


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to other areas in the Philippines where cyst studies have been conducted (Table 4). The nearest bays – Maqueda and Villareal Bays in Samar, for example – reported higher number of cyst types (Furio et al. 2012). Previously recorded dinoflagellate cysts studies in CB only involved P. zoharyi (P. bahamense) (Ingles 1997) and no current study regarding cyst identification and diversity in this bay could be used to compare the number of species identified in this study. Nevertheless, the low number of species diversity may be due to several factors.

Cancabato Bay is located in the eastern region (Region VIII) of the Philippines wherein typhoons frequently occur. An annual average of 20 typhoons enter the Philippines and from 2007 to 2010, four typhoons were recorded by the Philippine Atmospheric Geophysical and Astronomical Services Administration (PAGASA) to enter the region (Israel 2012). This may contribute to disturbances in the surface sediments, which causes occasional mixing of the water substratum. Moreover, the U-shaped geography of the bay may have contributed to isolation of cyst species (Ingles 1997). Cyst-forming motile cells of other species may not be easily washed off to these areas, thus contributing to low species diversity. The low cyst count maybe further explained by low dinoflagellate cells in the water column, since the bay is diatom-dominated the whole year round (Yap-Dejeto & Batula 2016). Furthermore, sedimentation rate in the area is quite fast at 5 cm/yr (Baleña 1995), diluting cyst concentration.

Most dinoflagellate cysts found have been recorded to be present in their motile forms in the area in the survey of 2012 (Yap-Dejeto & Batula 2016) except for species under the genus Gonyaulax. Representative species of this genus, however, were detected the next year (2013) in the same area (Yap-Dejeto et al. 2016). These genera of dinoflagellates must have been regular residents in the bay for a long time, if not at least around those years when they were observed.

Table 4. Dinoflagellate cyst diversity studied in different areas in the Philippines.

Sites No. of Cystspecies References

Cancabato Bay, Leyte

21 This study

Maqueda & Villareal Bays, Samar

33 cyst-types Furio et al. (2012)

Bolinao, Pangasinan

34 Baula et al. (2011)

Manila Bay 23 Azanza et al. (2004)

Manila Bay 17 Siringan et al. (2008)

Cyst DensityOne of the major factors affecting cyst composition, abundance, and distribution is the physico-chemical characterization of the surface sediments and its overlying waters. Results show that total cyst density has significant correlation (Spearman Rank Correlation) with the following physico-chemical parameters: moisture content (p =0.0183), wet bulk density (p=0.0198), dry bulk density (p<0.001), organic matter content (p=0.0015), carbonate content (p=0.0028), lithics content (p=0.0034), and nitrogen content (p=0.0106). Further, total cyst has a moderate positive correlation with moisture content (rs=0.568), organic matter content (rs=0.708), carbonate content (rs=0.678), and nitrogen content (rs=0.602); a moderate negative correlation with wet bulk density (rs=-0.559) and lithics content (rs=0.-618); and a strong negative correlation with dry bulk density (rs=-0.868). These are almost the same correlations obtained by Baula and co-authors (2011) on the study of Alexandrium cysts in Bolinao, Pangasinan, except for nitrogen that showed negative/opposite correlation (Baula et al. 2011). There could be varied preference of N:P ratio for different dinoflagellate species. The dominant dinoflagellate species of the two bays are different. In Bolinao, Pangasinan, the dominant species are Xandarodinium xanthum, Diplopelta parva, cysts of P. kofoidii, Votadinium spinosum, Selenopemphix spp., and Quinquecuspis concreta (Baula et al. 2011); while most species here in Cancabato Bay, Leyte are: S. ramosus and S. mirabilis (G. spinifera complex); O. centrocarpum (P. reticulatum), P. zoharyi (P. bahamense), and Brigantedinium spp. (Protoperidinium spp.). It should be noted that S. bulloideus (G. scrippsiae) densities correlated with that of O. centrocarpum (P. reticulatum) (p=0.04754). Cyst densities of Protoperidinium sp. correlated with that of O. centrocarpum (P. reticulatum) (p=0.0034) and P. zoharyi (P. bahamense) (p=0.0071). This may hint at possible ecological relationship between these organisms. This still has to be verified by more field and laboratory studies.

The density of dinoflagellate cysts somehow is associated with the kind of surface sediments in the area. In this study, stations with sandy substrate contained very low cyst densities (1-14 cysts g-1 DW), while muddy sediment substrate contained some of the highest levels of cyst densities. These observations agree with Pospelova and co-authors (2004) and Nehring (1993). The cysts here, which were 65% empty, were associated primarily with muddy sediments. These, being almost similar to the sediments comprising the mud – that being of light and fine particles – will be carried similarly by water currents to settle in the same location. Water circulation patterns also determine the spatial distribution of cysts in the bay (Pospelova et al. 2004).

Baula and co-authors (2011) observed that the amount of carbonates in the sediment is correlated to dinoflagellate


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cyst species richness in Bolinao, Pangasinan, Northern Philippines. In this study, the highest cell density was in C11, with high carbonate content. However, there was no significant difference of the carbonate content within stations.

Depth is also associated to cyst density. Lower depth reveals a direct proportion to cyst concentration. In stations wherein the depth is less than 2 m, the level of cysts density was high (C11, C17, and C15), while in areas with depths 4 m and above, cyst densities were low. As depth increases, it will take a longer time for cysts to settle which permits other factors such as current to act upon the settling cysts. However, this is contradictory to the results achieved by Pospelova and co-authors (2004) in shallow lagoons of southern New England (USA), wherein the deepest stations sheltered the highest cyst concentration. This difference may be due to differences in sedimentation rates and other geohydrobiological specific factors.

Nutrient concentration and dinoflagellate cyst distribution are closely related (Devillers & Vernal 2000). Species richness and diversity are negatively associated to total nitrogen, therefore high total nitrogen contained low cyst concentration (Azanza et al. 2004; Baula et al. 2011). However, according to Zonneveld and co-authors (2013), cyst production may be increased by anthropogenic pollution, especially nitrate. In this study, nitrogen and phosphorous contents were high in areas with high cyst densities. High concentration of nitrogen and phosphorous may be due to anthropogenic influences (Azanza et al. 2004), since these stations were located near coastal inhabitants.

Aside from surface sediment characteristics, water temperature and current speed may also affect cyst density. Baula and co-authors (2011) observed that levels of dinoflagellate cysts densities in temperate countries were higher compared to tropical regions. The low cyst density in Cancabato Bay is in accordance to this observation. In addition, water current speed also affects the cyst density. Fast moving waters are associated with fine sandy particles, which indicate a lesser possibility for cysts to settle (Baula et al. 2011). In a concurrent study of dinoflagellate cyst along the coasts of Tacloban City where current speeds are faster, average cyst density was <1 cyst g-1DW (Yap-Dejeto et al. 2018). Low cyst density was probably due to strong water current speed and anthropogenic disturbances.

Cyst of Pyrodinium bahamense and implications for PSP mitigation

Ingles (1997) studied P. bahamense during its bloom in CB in 1994. It showed that the density of abundance of P. bahamense cyst (P. zoharyi) in the sediments ranged 29-770 cysts/cm3. High levels of P. zoharyi (≥200 cysts/cm3) were observed during Jul-Nov 1994 throughout the

bloom, while a decrease in cyst density (≤80 cyst/cm3) was recorded during Jan-May 1995 after the bloom. However, in this study, the highest level of P. zoharyi was only 16 cysts g-1 DW. In addition, only C17 and C14 had P. zoharyi as the dominant cyst species. The low cyst densities are probably due to the fact that the sampling events were not during or right after a bloom. Sampling events were within the northeast monsoon, while most P. bahamese blooms are during the southwest monsoon (Azanza 2013).

Since the first red tide occurrence in the Leyte-Samar areas in 1983, the BFAR has been monitoring the bay for HABs, twice a month if reports come as negative, and four times a month if positive. A shellfish ban was announced during the years 2002, 2007, and 2009. However, no red tide bulletin was declared since 2009 (Rosalinda Cañas pers. com) until the sampling of this study from 2014 to Mar 2015. This could be due to lack of detectable bloom as shown in the low Pyrodinium cyst concentration in 2014-2015 with the cysts mostly from autotrophic dinoflagellates (71%), or this may also imply that there are other sources of the 2015, 2016, and 2017 Pyrodinium blooms.

Physico-chemical characterization of surface sediments revealed that the substrate of Cancabato Bay is predominantly sandy mud. Notably, P. bahamense cysts were found in mostly muddy stations (C10, C13, C14, and C17). Due to the U-shaped geography and strong water currents affecting the northern part of the bay, stations near its mouth were found to be sandy and become muddy towards the inside. Other dinoflagellate cysts do not follow this pattern.

High level of P. bahamense was expected to settle in Cancabato Bay substratum; however, low P. zoharyi (P. bahamense) levels were detected. P. bahamense cysts were also dominated by other species such as O. centrocarpum (P. reticulatum), Protoperidinium sp., and P. conicum in other sites within the bay. It should be remembered that Cancabato Bay and Manila Bay have recurrent PSP cases, with P. bahamense as the causative organism with recorded human deaths. In fact, at the time of this writing (2017), red tide ban is implemented in Cancabato Bay and its nearby bays (BFAR 2015-2017). Bolinao, Pangasinan, on the other hand, has no recorded PSP case involving P. bahamense. PSP cases in Bolinao happened only recently, caused by Alexandrium species, not P. bahamense and must have been caused by artificial increase of organic nutrients in the water (Azanza & Benico 2013). Given the fact that the first record of P. bahamense bloom and PSP case was in these parts, it can be speculated that the P. bahamense blooms in this bay are more of a natural occurrence, which may be exacerbated by anthropogenic interference. However, more data that spans to decades of years are needed to prove this claim. Further studies need to be done because this might represent changing cyst densities depending on the dinoflagellates' life cycle


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strategies (Azanza et al. 2017). Based from shellfish bans, there seems to be no pattern as to when the bloom comes. It is also disconcerting to note that the highest P. bahamense cyst density is found in the middle of the bay; that makes it imperative to closely monitor P. bahamense in this bay. Research should be more sophisticated and rigorous including cyst, molecular analyses, among others to infer origin and create models for bloom prediction.

ACKNOWLEDGMENTSThe authors thank the following: Dr. Fernando Siringan of UP-MSI for the use of his laboratory and his RA’s, Ms. Zoan Reotita and Ms. Mary Rose Ezperanza; the scientists who verified the cysts identification, Dr. Vera Pospelova, Dr. Fabienne Marret-Davies, Dr. Karin Zonneveld, Dr. Yasuwo Fukuyo, and Dr. Kazumi Matsuoka; Mr. Jay-ar Ragub and Ms. Brenda Gajelan, for the digital maps; Dr. Nancy Dayap of BFAR, for the field instruments, Ms. Rosalinda Cañas for the P. bahamense bloom data; Ms. Imelda Sievert of LMBTC for information on red tide occurrences; laboratories of RTRMF and DOST RO VIII, with staff, Aries Mazo and Ingrid Peliño Cormero respectively; Rosabella B. Montes for the statistics. This work is funded by the University of the Philippines Visayas, OVCRE.

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Pyrodinium bahamense and Other Dinoflagellate Cysts in ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs2018/2ndQtr/pyrodi… · This important stage of the lifecycle of dinoflagellates