Introduction

For generations, humans have established their cities near coastal areas due to the benefits offered by these ecosystems.¹ Fishing is one of the most important economic activities developed in these areas.

According to Food and Agriculture Organization (FAO)² data, fish supply has increased at an average annual rate of 3.2% in the last 50 years, outpacing world population growth (1.6%). This production is supported by aquatic ecosystems in which photosynthetic organisms (eg macrophytes, benthic and planktonic microalgae, and cyanobacteria) conform the base for the food web.³ However, the abundance of some taxa can reach harmful levels to humans and other organisms. These proliferations are known as Harmful Algal Blooms (HABs) and are formed by a variety of microalgae species including dinoflagellates (eg Gambierdiscus, Prorocentrum, Ostreopsis, Coolia, etc) and diatoms (eg Coscinodiscus).

Harmful Algal Blooms are a natural phenomenon and occur in all aquatic environments (eg freshwater, brackish, and marine) and at all latitudes. However, since the 1960s, impacts on the economy and public health seem to have increased in intensity, frequency, and geographical distribution.⁴

About 300 harmful microalgae species have been described. Of those, more than 100 are able to produce natural toxins harmful or even lethal to humans and animals, due to their strength and persistency.^3,5,6

There are several classes of marine biotoxins, such as saxitoxin (STX), domoic acid (DA), ciguatoxin (CTX), brevetoxin (BTX), tetrodotoxin (TTX), okadaic acid (OA), azaspiracid (AZA), and palytoxin (PLTX).⁷ Many of these toxins represent a threat to human health not only in the form of food-borne illness but also by skin contact with contaminated water or by inhalation of toxic aerosols.⁷

Growth, distribution, and abundance of some HAB species are largely temperature driven and expected to shift in response to climate-induced changes as ocean temperatures rise.^8,9 Nevertheless, conditions such as salinity, irradiance, turbulence, and substrate availability also should be considered.¹⁰

The Greater Caribbean Region (GCR) has been an area with high incidence of Ciguatera Fish Poisoning (CFP) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa.¹¹ Ciguatera Fish Poisoning occurs upon consumption of fish containing relatively high concentration of CTX. This toxin tends to be highest in the Greater Caribbean and Lesser Antilles, Bahamas, and Southern Florida, with occasional outbreaks in the Gulf of Mexico, the Yucatan Peninsula, and Central America.¹¹ Another dinoflagellate found in the Caribbean is the genus Ostreopsis. Toxins produced by Ostreopsis have been suspected to play a role in CFP by some researchers.¹² Other toxins associated with food-borne poisoning and respiratory and cutaneous irritation in Mediterranean beaches are PLTX, Ostreocin, and Ovatoxins.¹³

In an effort to establish an observation network for ocean acidification and its impact on HABs, the International Atomic Energy Agency (IAEA) carried out the Regional Project RLA/7/020 along with 10 countries in the Caribbean area, including Panama to evaluate using nuclear and isotopic techniques for this objective. The work presented in this article was developed within the scope of this IAEA project. Therefore, this research is aimed to evaluate the influence of different substrata (macroalgae and screens) and environmental factors on growth and distribution of potential HAB organisms. We expect that the data collected will contribute to increase information for the Great Caribbean Region, which does not include much information of the Southern part of the Caribbean.

Figure 1.

Aerial view of the Punta Galeta location in the Province of Colon in Panama. The red star indicates the sampling area.¹⁴

Material and Methods

Processing of HAB samples

Approximately 20% of the seawater from each plastic bag was poured through a 300-µm pore metal sieve into a 1 L graduated cylinder with a variation of ±5 mL. The bag was closed and vigorously shaken between 5 and 10 seconds to dislodge the microalgae bound to the artificial or natural substrate. The remaining homogenate was poured through the sieve into a test tube, and the total volume contained in each bag was recorded. The specimens of macrophytes were reserved to determine the weight with a scale; the screens used in the sampling were discarded and new ones were used for each experiment.¹⁸

The sieved seawater sample was re-homogenized and the rest of the seawater was filtered by gravity through a piece of 25 mm diameter nylon with 20 µm pore size to collect the microalgae cells. Any particulate material left in the specimen was rinsed on the screen with filtered seawater. Then, the 20 µm mesh was transferred to a conical 15 mL screw cap centrifuge tube containing 10 mL of filtered seawater in the 300 µm mesh and the sample was preserved with 2 drops of a neutral iodine lugol solution.¹⁹

Cellular abundances of potential HAB (Ostreopsis, Prorocentrum, Coscinodiscus, and others) in each sample in screens or macrophytes were determined using microscopy. A semi motorized vertical fluorescence microscope was used, observing the samples in the objectives with magnifications of 10× and 40×. A 1.3 megapixel Moticam 1000 camera was also used, which allows the display of the lens samples on the computer screen. These 2 devices, together with the Motic Image Plus software, allowed the images to be projected on a computer and made captures of images and videos in microscopy.

Samples were shaken to suspend the cells of the microalgae and screens, then an aliquot of 0.1 mL was transferred to a slide and each cell found was counted and photographed.²⁰ For the screen samples, microalgae concentrations were expressed as cells per 100 cm², calculated using equation (1), surface area of each screen (166 cm⁻²).

For macrophyte samples, microalgae concentrations were determined using a similar method, except that the concentrations were normalized to wet weight of the macrophyte sample and expressed as cells g⁻¹ of algae or seagrass.¹⁸

Results

Table 1 shows the descriptive statistics for physicochemical parameters measured for water in the field (pH, salinity, dissolved oxygen, and temperature). Monthly variations in these parameters are shown in Figures 2 to 5. Salinity data were obtained from transforming conductivity results. All parameters seem to behave without much apparent variation.

Table 1.

Descriptive statistics for pH, salinity (ppt), dissolved oxygen (mg L⁻¹), and temperature (°C) monthly values measured in situ at the Punta Galeta site from June 2016 to November 2017.

Figure 2.

Field pH values corresponding to sampling points at Punta Galeta from June 2016 to November 2017.

Figure 3.

Field salinity (ppt) results corresponding to the sampling points at Punta Galeta from June 2016 to November 2017.

Figure 4.

Field dissolved oxygen (mg L⁻¹) results corresponding to sampling points in Punta Galeta from June 2016 to November 2017.

Figure 5.

Field temperature (°C) results corresponding to sampling points in Punta Galeta from June 2016 to November 2017.

For artificial substrates, organisms from genera Prorocentrum, Ostreopsis, and other microalgae were found, with the genus Coscinodiscus (other microalgae) being the more abundant (Table 2). Abundance of Prorocentrum ranged from 0 to −271.1 cells 100 cm⁻², while Ostreopsis ranged from 0 to 813.3 cells 100 cm⁻². Counts for other microalgae, including Coscinodiscus, ranged from 3524 to 9217 cells cm⁻² (Figure 6A). For samples taken from macrophytes, Prorocentrum counts ranged from 0 to 818 cells g⁻¹, while Ostreopsis and other microalgae were observed at 0 to 25 912 cells g⁻¹ and 0 to 6314 cells g⁻¹, respectively (Figure 6B).

Table 2.

Microalgae genera (P: Prorocentrum, O: Ostreopsis, OM: other microalgae) found on screens during the period of study at each sampling point.

Figure 6.

(A) Artificial substrates. (B) Microalgae collected on natural substrates (macrophytes) from August 2016 to November 2017. Green boxes represent the genus Prorocentrum, red triangles the genus Ostreopsis, and inverted orange triangles other microalgae.

One-way ANOVA results using monthly values as a factor showed a significant difference between the microalgae groups for each month, F(13) = 2.172, P value = .012. Prorocentrum, Ostreopsis, and other microalgae were also found in samples from the macrophytes employed (see Table 3). Here, one-way ANOVA analysis was also applied, to count for differences among macrophytes in the sampling points. Basically, no significant differences between the macrophytes specimens in the 5 sampling points were found, except for February 2017, F(4) = 4, P value = .0343.

Table 3.

Microalgae genera (P: Prorocentrum, O: Ostreopsis, OM: other microalgae) found on macrophytes during the sampling period at Punta Galeta.

Regarding the host preference of the microalgae groups in the macrophytes, the total values of the cells counted for each group (Prorocentrum, Ostreopsis, and other microalgae) were used in all the macrophytes, emphasizing the genera that showed the most cells of each group. In this way, the cells of the genus Prorocentrum showed a preference of 81.6% in H tuna, while the genus Ostreopsis had greater affinity for Dictyota sp. with 85.3%. Other microalgae maintained an affinity of 42.7% with T testudinum (Figure 7). For the host preference of microalgae in macrophytes, there were no significant differences between the groups (F = 1.408, degree of freedom [DF] = 2), as the data in A spicifera were not taken into account due to the absence of organisms in the samples.

Figure 7.

Preference of microalgae in macrophytes. Cells were found in 4 macrophytes (Thalassia testudinum, Halimeda monile, Halimeda tuna, and Dictyota sp.). The genus Prorocentrum and the genus Ostreopsis are shown in blue and red, respectively. Other microalgae are shown in yellow.

Normalized data from natural and artificial substrates based on results shown in Figure 6A and B were compared to determine possible differences among microalgae host preferences, abundances, and substrates. From this analysis, clear differences between artificial and natural substrates, especially for Coscinodiscus, were obtained for the months of November 2016 and June 2017. Also, a 2-way ANOVA was applied, using as factors the type of microalgae and substrates. From this exercise, significant differences were found between the microalgae, F(2) = 5.351, P value = .0113, and type substrates, F(1) = 8.106, P value = .0085, for November 2016. Significant differences were also found between the microalgae, F(2) = 9.951, P value = .0006, and type substrates, F(1) = 5.444, P value = .028, for June 2017.

Discussion

This study is the first to detect the presence of potentially harmful dinoflagellates from the genera Prorocentrum and Ostreopsis and a diatom from the genus Coscinodiscus in Punta Galeta, Panama. These findings are important as the OA and derivatives produced by Prorocentrum lima can cause Diarrhetic Shellfish Poisoning (DSP). Also, this genus is present worldwide, affecting seafood and fisheries activities.^25262728-29 For the case of the Coscinodiscus, this genus was the most abundant found in Punta Galeta throughout the present study. According to toxicology, benthic organisms can be damaged by mucilage produced by Coscinodiscus, which can aggregate, sink, and cover the sea bed. Bloom decay is also likely to cause anoxic conditions, whereas fisheries and aquaculture plants can be impacted by clogging of fishing nets and cages.⁴

The greatest abundance for the genus Prorocentrum in the Caribbean Sea was observed in the dry season at 27.4°C and salinity at 35 ppt³⁰ in a study conducted in 2 coastal sites in Guadalupe Island to evaluate the abundance of dinoflagellates on an invasive macrophyte Halophila stipulacea. Other researchers indicate that the abundance of Prorocentrum is very low in temperate cold-water environments; although a direct comparison with warmer waters cannot be made as there have not been many samplings in cold waters.^313233-34 The results in Punta Galeta contrast with the findings of Boisnoir et al,³⁰ as we found that this genus had greater abundance in the rainy season, at an average temperature of 30°C and a variable salinity in each month of sampling.

Muciño-Márquez et al,³⁵ in a case study in Mexico, indicated that the genus Prorocentrum is abundant in high salinities finding maximum abundance of cells at 30 ppt and presenting a decrease when the salinity reached a value of 20 ppt. It is also known that these species are sensitive to sudden changes in salinity.³⁶ Different species of phytoplankton with specific requirements respond differently to changing environmental conditions.^373839-40 The constant changes in the environment (natural and anthropic) can condition environments that favor the development of certain species. In Punta Galeta, the cell density in the genus Prorocentrum was correlated with high salinity (around 32.83 ppt), in accordance with the studies carried out in Mexico and Guadalupe mentioned above.

Marasigan et al⁴¹ point out that for the genus Prorocentrum and the dinoflagellates in general, a preference and better adaptation to seagrasses can be observed. Cells of this genus also have been found in detritus of floating mangroves.^42,43 Contrary to previous investigations, our results show a preference for H tuna (81.58%) by this genus, followed by T testudinum (9.85%) and finally Dyctiota sp. (8.57%).

The genus Ostreopsis is usually found on hard substrates, such as rocks and mussel shells and plankton.⁴⁴ Most of the organisms of this genus (9 species) have been observed on macro algae samples, except for Ostreopsis belizeanus and Ostreopsis carribeanus, which have been observed in plankton. Aligizaki and Nikolaidis⁴⁵ identified this genus in macro algae, sediments, and water column. In New Zealand, Ostreopsis has been observed in plankton and in drifting macroalgae.⁴⁶ In the Virgin Islands, Ostreopsis has been reported in samples of Dictyota, which grows in dead coral.⁴⁷ Monti et al⁴⁸ showed that the genus Ostreopsis prefers to stay in brown and red algae in the northern Adriatic.

In Tasmania, Ostreopsis has been described as a common group in the seagrass.⁴⁹ The 9 species of Ostreopsis presented differences in preferences. For example, in Belize, Ostreopsis lenticularis was abundant in A spicifera and was less prevalent in T testudinum, Dictyota dichotoma, and Halimeda opuntia. Ostreopsis hetagona was abundant in T testudinum, but also was present in A spicifera, H opuntia, and D Dichotoma.⁵⁰ In Hawaii, Ostreopsis was abundant in Martensia fragilis and A spicifera.⁵¹ The results presented in other investigations are different from those found in Punta Galeta, with the exception of the one by Kohler and Kohler,⁴⁷ as they had a greater preference for Dictyota sp. (85.30%), followed by T testudinum (12.13%), H tuna (2.33%), and finally H monile (0.24%).

Boisnoir et al³⁰ indicate that for Gosier in Guadalupe Island, the highest abundances of Ostreopsis spp. occur during the rainy season with a temperature between 31.3°C and 31.4°C, but in Rivière Sens the highest abundance is given at 26.8°C. It also reached great abundance during the dry season with a salinity of 36 ppt. Similarly, our results showed greater abundance during the rainy season at an approximate temperature of 30°C. The changes in salinity at Punta Galeta were not associated with changes in abundance during the whole sampling period. Another important characteristic found in Punta Galeta is the high Dissolved Oxygen (9.07-13.71 mg L⁻¹), this appears to be related to the proliferation of Prorocentrum, which is probably in direct relation with the rapid growth of many macrophyte.

Some factors that condition the occurrence and intensity of dinoflagellate blooms are the specific nutritional needs of each species, water temperature, solar radiation, meteorological phenomena that lead to movements of water masses, mixing by the action of rising currents, tides, and development of a thermoclimate.^52,53

The genus Coscinodiscus has been reported as a marine planktonic group distributed in all waters of the world. Von Stosch⁵⁴ indicates that the species related to warm waters of the tropics are between 18°C and 30°C and that they die below 15°C and above 33°C. Our results agree as this diatom type was found within these temperature ranges. Santiago et al,⁵⁵ in a study conducted in the port of Recife (Brazil), indicate that for sites associated with estuarine waters there is a dominance of diatomaceous groups as they are favored by their Euryhaline characteristics. This is consistent with our results as the sampling sites were close to an estuary and the Coscinodiscus genus remained the dominant group during all cell counts. Coscinodiscus is considered an invasive species probably introduced with ballast water discharge⁴ from the ships at the entrance of the Panama Canal. This fact could be one of the reasons why this genus is found year round in the waters of Punta Galeta. Another hypothesis to explain the abundance of this genus could be that environmental conditions such as temperature, salinity, dissolved oxygen, and nutrients present in Punta Galeta favor its growth, as also the gender competition.

It is important to notice that Gambierdiscus was not observed in this study. Nowadays, the rise of ocean temperature has lead to increases in Ciguatera Fish Poisoning (CFP) at higher latitudes due to a broadening of the distribution of Gambierdiscus and Fukuyoa species poleward.¹⁰ However, these CFP-associated genera still present high cell densities in warm, shallow bays and where temperatures are high and relatively stable throughout the year.⁵⁶ Although the sea surface temperature is higher and more stable in the Eastern Caribbean Sea (approximately 24°C-29°C), the temperature-CFP relationship weakens at temperatures >30°C, suggesting that an upper thermal limit may restrict Gambierdiscus occurrence.⁵⁶ An upper threshold is supported by experimental data showing a precipitous decline in Gambierdiscus and Fukuyoa growth rates at temperatures approaching ~31°C,⁹ but more substantial warming may cause cell mortality. The GCR shows optimal growth conditions for Gambierdiscus and Fukuyoa¹⁰ supporting the hypothesis that CFP occurrence is associated with these conditions; however, the environmental conditions presented in Punta Galeta (Caribbean Sea) do not favor the growth of these genera due to its high temperature (around 32°C).

Conversely, the climate at both sites of the Isthmus of Panama presents marked seasonal variations, related to the Intertropical Convergence Zone. Winds from the north move the intertropical convergence zone away from the isthmus, while the winds from the south generally push it toward the isthmus.⁵⁷ The effect of the winds is more intense at the Caribbean Sea (in Panama), resulting in bigger waves than in the Pacific Ocean. Gambierdiscus inhabits certain substrates (macroalgae, sea grass, sand, and death corals) and requires special conditions for its survival, such as zones without waves and away from contributions of continental waters.^18,5859-60 As we mentioned before, the Panamanian Caribbean coast presents large waves with irregular tides (1 m amplitude) influenced by prevailing weather conditions. As for the waters of Panama, the superficial marine currents at the Caribbean have a predominant direction from coast to coast to the East and 500 km offshore to the West shaping a counter clockwise ring of circulation during all seasons of the year. This constant dynamic does not favor the presence of this genus.

Another hypothesis could be that the allelopathic effect produced by Prorocentrum and Ostreopsis may lead to a competition among them and the production of chemical compounds inhibiting the proliferation of Gambierdiscus or another microalgae.⁶¹

Conclusions

In Punta Galeta, dinoflagellates of the genera Prorocentrum and Ostreopsis were found. Both are considered potential bloom-forming genera worldwide. In addition, the taxonomic genus Coscinodiscus which is known to cause damage to marine life was also found, showing the highest cellular abundance among the microalgae studied. The field sample collections indicate that Punta Galeta microalgae differ in their preference to adhere to natural substrates (macroalgal) or artificial substrates (screens). However, the physicochemical characteristics of the waters in Punta Galeta present favorable conditions to sustain the life of the place. However, some parameters as water temperature, salinity, marine currents, the effect of the wind, and the contribution of continental waters neither favor the growth of certain genera vectors of marine toxins nor the blooms of these genera at Punta Galeta.