Mexico & Florida: Growth rates of pelagic Sargassum species in the Mexican Caribbean

This study had two objectives: 1) to demonstrate the feasibility of both in-situ and ex-situ culture methods of sargasso, and 2) to examine the growth rates of the new morphotype S. natans VIII in comparison with S. fluitans III, a common species/morphotype in both the Sargasso Sea and the Great Atlantic Sargasso Belt (García-Sánchez et al., 2020, Schell et al., 2015).

2. Materials & methods
2.1. Collection and preparation of the algae
Prior to the experiments, drifting sargasso was collected in nearshore Puerto Morelos (20° 52 ´ N, 86° 52 ´ W) waters with dip net from a boat or kayak. The algae were placed in coolers with seawater and transported to the in-situ and ex-situ experiments within 20 min. Pelagic Sargassum species and morphotype (S. natans VIII and S. fluitans III) were sorted following the criteria of Parr (1939), Schell et al. (2015) and García-Sanchez et al. (2020). Sections of thalli were selected based on visible criteria such as light brown (golden) color, few epibionts, and lack of deterioration. Clippings were carefully brushed clean to remove debris from the thalli’s base for the experiments, and loosely attached epibionts were removed. The hydroids were left in place since they were firmly attached to the blades. The wet weight was determined in a balance (Ohaus Corporation, Pine Brook, New Jersey, USA) with a 0.01 g accuracy after a gentle dry to remove excess water.

2.2. In-situ growth experiment design
In-situ experiments were conducted approximately 40 m from shore at times when only small amounts of sargasso reached the coast. Two pairs of drifting parallel polypropylene ropes were anchored in a ∼2 m deep area where seagrass beds dominated the ocean floor. The distance between the ropes within each pair was ∼5 m and the rope pairs were ∼70 m apart from each other, separated by a physical barrier (a small pier). One pair of ropes served as control, while the other served as a nutrient treatment (Fig. 1). Each rope was ∼7 m long and anchored at one end to the bottom, with one buoy at the beginning and one at the end of the drifting section, allowing it to float freely with the dominant superficial currents and waves (Fig. 1). Four plastic mesh cylindrical cages (22 cm long and 15 cm in diameter) were fixed on each drifting section of the rope, so that they were floating at the surface (Fig. 1). Light attenuation by the plastic mesh was measured inside the cage and was not lower than 60% of natural irradiance (Io) as recommended Lapointe et al. (2014). Therefore, it was assumed not to have a limiting effect on photosynthesis, as the determined saturation irradiance (Isat) in natural conditions for sargasso was 220 − 470 µmol m−2 s−1 (Lapointe, 1995, Lapointe et al., 2014), which is well below 60% of the maximal Io (∼2000 µmol m−2 s−1, Enríquez et al., 2002) at noon in the study area.

Two 21-day experiments were conducted, one with a low nutrient treatment (February − March 2017) and one with a high nutrient treatment (April − May 2017). The experiments were carried out using a completely randomized 2 × 2 factorial design. The species (S. fluitans III and S. natans VIII) and nutrient level were the factors tested in the first and the second experiment. The nutrient treatment included addition of mesh bags containing 100 g of a slow-release fertilizer

, following previous experiments of nutrient addition to macroalgae (Chan et al., 2012, Sura et al., 2021). In the low nutrient experiment, one mesh bag was attached to each cage, whereas three bags were attached to each cage in the high nutrient experiment (Fig. 1). Mesh bags containing 100 g of pebbles were used for non-enriched/control treatments to ensure similar buoyancy. For each nutrient treatment and species combination, four replicate cylindrical cages were used with sections of thalli between 40 − 50 g wet weight. The growth rate was calculated using the initial and final wet weight and reported as specific growth rate in doubling day−1 (Hanisak and Samuel, 1987). At the end of each experiment, nutrient mesh bags were dried overnight in a 60 °C oven and remaining pellets were weighed to determine fertilizer release rates. Based on the mass lost from mesh bags, release rates were calculated per cage in g day−1. During the final week of each experiment, three 30 mL water samples were collected from both control and each treatment just below the cages to perform water nutrient analyses. Water samples were frozen at − 20 °C until they could be analyzed. The ropes and cages were cleaned every 3 − 6 days, with the frequency increasing as the experiments progressed and more algae began to grow on the mesh of the cylindrical cages. Water temperature was continuously measured using Onset HOBO® pendant loggers (UA-002–64, Onset Computer Corporation, Bourne, U.S.A., temperature accuracy: ± 0.53 °C, temperature resolution: 0.14 °C at 25 °C).

2.3. Ex-situ growth experiment design
Recirculating systems, consisting of a sump and a circular container, were made of plastic and commercially available PVC components. The sump was a 52 (high) × 35 (wide) × 25 (deep) cm tank (∼ 45 L) with a 100 W submersible heater and a 2000 L h−1 submersible pump (Fig. 1). The heaters were employed to keep the water at the appropriate temperature at night, when ambient and seawater temperatures commonly drop. Water was pumped from the sump to a cylindrical container (35 cm high, diameter 29 cm, ∼ 18 L) via a 90° elbow on the bottom connected to a cap with a 5 mm hole (Supplemental Video 1). When the water entered, the velocity increased, resulting in a circular water movement. A relief valve installed in the sump was used to change the flow velocity (Fig. 1). Once the water reached the drain, it returned to the sump by gravity. A plastic edge was added to the drain in the cylindrical tank to prevent the drifting thalli from becoming trapped and motionless (Fig. 1). All water used for the experiments was obtained from a reef lagoon (salinity 35 and pH 8.2), and previously filtered with a sand filter (Hayward, Model S360T2, Hayward Pool Products Inc., Elizabeth, NJ, USA).

The following is the Supplementary material related to this article Video S1.

Trial experiments were carried out at temperatures of ∼28 °C and ∼31 °C. Four recirculating systems were fitted in a water bath tank of 206 × 70 × 58 cm with a drain at a height of 20 cm (approximately 288 L). For treatments at 31 °C, the water bath tank had a continuous seawater flow at 28 – 29 °C and the recirculating system water temperature was 31.1 ± 0.1 °C. For 28 °C treatments, the water bath tank was kept at 25 °C using a Delta Star DS4 chiller (Aqualogic, San Diego, CA, USA), and the recirculation systems were 28.2 ± 0.1 °C. The water from the bath tanks was never mixed with water of the recirculating systems. The daily rate of water exchange, with water from the reef lagoon, in the recirculating systems was regulated manually at 10%, and no fertilizers were used in any experiment. Light was between 435 and 581 µmol m−2 s−1 (LI1500, LICOR Inc., USA). Small sections of either S. fluitans III or S. natans VIII thalli (∼ 12 wet g) were placed in the circular container, and their wet weight was determined every 5 days until day 20. Per water bath tank, two recirculating systems contained S. fluitans III, and the other two contained S. natans VIII. The experiment started on July of 2019 and lasted for 20 days. This was repeated in September of 2019 to achieve a total of n = 4 per species per temperature treatment. Growth rates were determined from the initial and final wet weight measurements, and the specific growth rate was calculated as doubling rate per day (Hanisak and Samuel, 1987). Water temperature (°C), salinity (g L−1) and pH were determined twice a day (09:00 and 17:00 h) in the recirculating systems with a portable multiparameter meter (Hanna HI98194, Hanna Instruments Inc., Woonsocket, RI, USA).

2.4. Analysis of algae for C, N and P content
Following the in-situ experiments, two tissue samples were taken from each cage in order to analyze total C, N and P tissue content (samples oven-dried at 60 °C). Before analysis, the tissues were dried and ground with a mortar and pestle. Total C and N were determined by combustion using a CNHS elemental analyzer (CE Flash 1112 Elemental Analyzer) and P content was determined by colorimetric analysis (Solórzano and Sharp, 1980) at J. Fourqueran laboratory, Florida International University, Miami, FL. The percent tissue content of C, N, and P were then used to calculate molar ratios.

2.5. Water analyses
For in-situ experiments, dissolved nutrient concentrations (μM) of ammonium (), nitrates + nitrites (, and phosphates () were determined colorimetrically using a SKALAR San Plus II segmented flow autoanalyzer with a precision of 0.01 μM (Centro Universitario de Investigaciones Oceanológicas de la Universidad de Colima). Ammonium was determined based on a reaction with sodium hypochlorite in the presence of phenol with a high pH (Solórzano, 1969). Nitrates and nitrites were determined based on the Griess reaction, where nitrates are reduced to nitrites and nitrites are converted into azo dyes (Strickland and Parsons, 1972). Phosphates were determined by a reaction of ammonium molybdate and potassium antimony tartrate in an acid medium with a dilute phosphate solution in the form of an antimony-phosphate-molybdenum complex (Grasshoff et al., 1983).

2.6. Statistical analysis
A factorial ANOVA followed by a Tukey’s test for unequal N post-hoc comparisons was applied to each of the in-situ trials (low and high nutrient), with specific growth rates and total C, N and P tissue content as variables and species (Species: S. fluitans III and S. natans VIII) and nutrient level, as fixed factors. The unequal N per treatment was due to a lost rope at the start of the second experiment (high nutrients), and placing of an additional rope to each treatment (with and without high nutrients). A Student´s T-test was used to compare the fertilizer release rate and the water nutrient concentration of the (low and high) nutrient treatments. All data were checked for normality and homogeny of variance.

For the ex-situ experiments, the growth rates were recorded every five days until day 20, to obtain average values ( ± standard error) by temperature and species. Because sargasso stopped growing after 10 days in our experimental setting (Fig. 4), the specific growth rate of day 10 was used for further analysis. A Student´s T-test was applied to discern possible significant differences in the specific growth rate per species and temperature level (28 °C and 31 °C). Differences were considered significant at p < 0.05.

2.5. Water analyses

2.6. Statistical analysis
A factorial ANOVA followed by a Tukey’s test for unequal N post-hoc comparisons was applied to each of the in-situ trials (low and high nutrient), with specific growth rates and total C, N and P tissue content as variables and species (Species: S. fluitans III and S. natans VIII) and nutrient level, as fixed factors. The unequal N per treatment was due to a lost rope at the start of the second experiment (high nutrients), and placing of an additional rope to each treatment (with and without high nutrients). A Student´s T-test was used to compare the fertilizer release rate and the water nutrient concentration of the (low and high) nutrient treatments. All data were checked for normality and homogeny of variance.

For the ex-situ experiments, the growth rates were recorded every five days until day 20, to obtain average values ( ± standard error) by temperature and species. Because sargasso stopped growing after 10 days in our experimental setting (Fig. 4), the specific growth rate of day 10 was used for further analysis. A Student´s T-test was applied to discern possible significant differences in the specific growth rate per species and temperature level (28 °C and 31 °C). Differences were considered significant at p < 0.05.

3. Results

The specific growth rates differed significantly between the species during both experiments, and S. natans VIII had consistently higher growth rates than S. fluitans III (Table 1). Although nutrient addition did not affect the specific growth rate during the low nutrient experiment (Table 1), it reduced the specific growth rates of S. natans VIII by 32.8%, and of S. fluitans III by 75.3% during the high nutrient experiment. No significant interactions were detected between species and nutrient level during both (low and high nutrient) experiments (Table 1).

In most cases N content in the tissues of both S. fluitans III and S. natans VIII were significantly higher in the nutrient addition treatments than the controls (Fig. 3, Supplementary Table 1). C:N tissue ratios were lower when nutrients were added, C:P ratios did not show consistent patterns between species and treatments, whereas N:P ratios were higher in the tissues of fertilized sargasso (Fig. 3).

3.2. Growth rates: ex-situ experiments
Regardless of temperature, both species gained weight until day 10; after that time, weight was maintained or slightly decreased over time (Fig. 4). Therefore, the specific growth rate for both species was calculated using the wet weight difference between day 1 and 10. For S. natans VIII, there was no significant effect of the temperature on its specific growth rate (Student´s T-test, p = 0.99; Fig. 5). S. fluitans III thalli cultured at 31 °C had a significantly higher specific growth rate than those at 28 °C (Student´s T-test, p = 0.03; Fig. 5).

4. Discussion
Setting up systems to study the growth rates of pelagic Sargassum species proved to be more difficult than initially thought. Several series of preliminary trials (2016–2017) in flow-through mesocosms with ambient temperatures and light regimes, resulted in fast decay and zero growth of sargasso, and we concluded that failure to maintain sargasso in a good condition may be related to its pelagic nature. Therefore, further experiments were conducted in specially designed systems, in which the thalli were kept in continuous movement at the water surface, either on the in-situ drifting ropes or in the ex-situ circular containers (Fig. 1, Supplementary video 2). The main aims of this study were to demonstrate the feasibility of both in-situ and ex-situ culture methods for sargasso, and to evaluate whether the application of nutrients (in-situ experiments) or manipulation of temperature (ex-situ experiments) would result in different responses (growth rates), which we achieved in some but not all experiments (Table 1; Fig. 4). Further evaluation of growth under a complete range of temperature or nutrient supply was beyond the scope of this work. The growth rates of S. fluitans III (there are no previous studies for S. natans VIII) for both in-situ and ex-situ experiments under the mesotrophic conditions in this study were on par with, or lower/higher than, those previously reported for neritic waters and higher than those reported for oceanic waters (Table 2; Lapointe, 1986; Lapointe et al., 2014).

Table 2. Growth rates expressed as doubling time (DT, number of days needed to duplicate biomass = 1/specific growth rate in doubling·day−1) of sargasso in different studies. NW: Neritic water. OW: Oceanic water. The morphotypes were nor established in earlier studies. In these studies, S. fluitans is also morphotype III, a morphotype of S. natans I was either deduced from illustrations or assumed, because S. natans VIII was (virtually) absent before 2011.

The following is the Supplementary material related to this article Video S2.

This study demonstrated that both the in-situ and ex-situ approach can be used to assess the growth of sargasso, and both approaches resulted in growth rates in comparable ranges (Table 2). The in-situ system has the advantage of being simple and inexpensive to implement, as well as exposing the sargasso to natural ambient conditions. It is, however, exposed to prevailing climate and physical and chemical conditions of the water, as well as biofouling, which depending on the local setting, may also pose logistical challenges, such as cleaning of the cages or difficulty in ongoing observation or handling of the algae. There may also be additional costs for transportation, cleaning, and cage setup. The ex-situ system allows for better physicochemical and epiphyte control, as well as easier algal handling and variable recording. Nonetheless, depending on the study site, special equipment to lower or increase the temperature of the water (i.e., heat pumps, chillers, or heaters) may be necessary, which may result in higher costs. Moreover, in the ex-situ trials of this study, the growth of both species ceased after ten days (Fig. 4). This cessation in growth could be attributed to various factors, including the nutritional history of sargasso at the start of the experiments (e.g. nitrogen, phosphorous and micronutrient tissue content; Harrison and Hurd, 2001). The daily rate of water exchange with water from the mesotrophic reef lagoon was 10%, and nutrient demand by the fast-growing algae may have outweighed the supply once the tissue reserves were finished. After 10–15d, some tissue decay of older parts of the thallus was observed, and recycling of nutrients by microorganisms and associated fauna (Lapointe et al., 2014) may be absent in the experimental settings. For future research, it is important to determine tissue nutrient contents before and after the experiments.

This is the first study to report growth rates of S. natans VIII, which used to be rare before the formation of the Great Atlantic Sargassum Belt (Schell et al., 2015). In-situ, at temperatures between 27 and 29 °C, S. natans VIII grew faster than S. fluitans III. However, the ex-situ growth rate of S. fluitans III increased with increasing temperature, whereas that of S. natans VIII decreased at ∼31 °C (Fig. 4), indicating that apart from morphological and genetic differences (Dibner et al., 2021), these species also have different physiology. The observation of increased growth above 30 °C for S. fluitans III differs from a previous report where this species, collected in the Gulf Stream, ceased growth above 30 °C (Hanisak and Samuel, 1987).

Although the nutrient addition trials were only preliminary, their results reveal interesting patterns. Increased nitrogen tissue content (Fig. 3), indicate that the algae absorbed (some of) the added nutrients. Deviations in C:N:P tissue composition in the nutrient treatments compared to the controls (Fig. 3), suggest that, when nutrients were added, nitrogen accumulated (indicated by decreasing C:N) and phosphorus could be the limiting nutrient, as indicated by increasing N:P ratio > 35 (Atkinson and Smith, 1983, Lapointe et al., 2014). The Liebig’s law of the minimum states that the nutrient in the smallest amount in relation to other nutrient requirements will limit the rate of growth (Harrison and Hurd, 2001), and as phosphorus was similar among treatments, this may explain why we did not find an increase in growth rate during the low nutrient addition treatment. During the initial experiment, the lack of growth response was misinterpreted as a treatment error (insufficient supply of nutrients). Thus, more nutrients were applied in the subsequent experiment (high nutrients), to which the algae unexpectedly responded with decreased growth (Table 1). The high nutrient enrichment treatment might have resulted in side effects such as increased shadow from the growth of epiphytes and algae on the cages, resulting in a reduction in photosynthesis, even if these were cleaned on a regular basis, or the formation of a distinct microbiome (Singh and Reddy, 2014) that inhibited optimal development of sargasso. It has recently been suggested that pelagic Sargassum species are currently benefiting from the global trend in nitrogen enrichment (Lapointe et al., 2021, Skliris et al., 2022); however, the results of this study reveal that the complicated relationship between nutrient enrichment and growth of these species needs to be explored further.

In conclusion, this study suggests that both in-situ and ex-situ culture systems are feasible to determine growth of sargasso. Which system can best be used will be determined by the study’s objectives, and local conditions and facilities. Maintaining sargasso in motion proved to be essential to keeping the algae in good condition. These results open the door to conducting a variety of studies related to the physiology of the pelagic Sargassum species in terms of environmental factors, nutrition, photosynthesis, mortality, and so on, which could aid in understanding the variables associated with the blooms of sargasso in the tropical Atlantic.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements
This research was supported by DGAPA, Project PAPIIT IN203417, and Caroline Graham received a Fulbright Garcia-Robles Student Research Scholarship. We thank Deborah Goodwin, Jeffrey Schell, Elisa Vera Vázquez, Fernando Negrete-Soto, Edgar Escalante-Mancera, Miguel A. Gómez, Gustavo Villareal-Brito, Guadalupe Barba Santos, Eduardo Ávila, Laura Celis-Gutierrez, for providing technical support or bibliographical resources. We thank Ligia Collado-Vides for her support in the tissue C, N and P analyses.

Source: Sciencedirect