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Boletín de Investigaciones Marinas y Costeras - INVEMAR

Print version ISSN 0122-9761

Bol. Invest. Mar. Cost. vol.46 no.2 Santa Marta July/Dec. 2017

https://doi.org/10.25268/bimc.invemar.2017.46.2.730 

Research Articles

Production of (+)-discodermolide by the sponge Discodermia dissoluta under fixed and suspended culture systems

Javier Gomez-León 1  
http://orcid.org/0000-0001-5015-7071

Johann Lopez-Navarro 1  

Alicia Millanguir 2  

Jesus David Castaño 1  

Sven Zea 3  

1 Instituto de Investigaciones Marinas y Costeras - Invemar [Institute of Marine and Coastal Research]. Calle 25 # 2-55 El Rodadero, Santa Marta, Colombia. javier.gomez@invemar.org.co; johann.lopez@invemar.org.co; jesus.castano@invemar.org.co

2 Universidad Católica de Temuco, Campus Temuco, Chile. amilla2007@alu.uct.cl

3 Universidad Nacional de Colombia - Sede Caribe - Instituto de Estudios en Ciencias del Mar-Cecimar, Atte. Invemar, Calle 25 # 2-55, El Rodadero, Santa Marta, Colombia. sezeas@unal.edu.co


ABSTRACT

The macrolide (+)-discodermolide produced by the marine sponge Discodermia dissoluta shows promising antitumor, antimitotic, and immunosuppressive activity. However, the sustainable supply of any molecule requires much in situ and in vitro research to optimize and later obtain the molecule of interest. In this study, two culture systems-fixed and suspended-were evaluated at 15-m depth in two sites, Punta de Betín and Nenguange, in Santa Marta (Colombian Caribbean). Survival, growth, and production of (+)-discodermolide were recorded, with the suspended system resulting in better growth and survival, depending on the culture site. The influence of the different environmental factors on survival was also studied, and a negative correlation with temperature was observed. Salinity, solar radiation, organic matter, and water flow were not correlated. Finally, no significant differences in the production of (+)-discodermolide existed between the culture systems and study sites. Production ranged from 20 to 270 μg per gram of dry sponge.

Keywords: Discodermia dissoluta; (+)-discodermolide; Sponge fragments; Sponge culture; Aquaculture

RESUMEN

El macrólido antitumoral (+)-discodermólido, producido por la esponja marina Discodermia dissoluta, tiene promisorios resultados como antitumoral, antimitótico e inmunosupresor. Sin embargo, el suministro sostenible de cualquier molécula necesita mucha investigación de trabajo in situ e in vitro para lograr la optimización y posterior obtención de la molécula de interés. En este estudio se evaluaron dos sistemas de cultivo fijo y suspendido a 15 m de profundidad, en las localidades de Punta de Betín y Nenguange en Santa Marta (Caribe colombiano). En cada caso se registró la supervivencia, el crecimiento y la producción de (+)-discodermólido, encontrando mejores resultados para el crecimiento en el sistema suspendido y una dependencia de la supervivencia según la localidad del cultivo. También se estudió la influencia de diferentes factores ambientales sobre la supervivencia, observando una correlación negativa con la temperatura. La salinidad, radiación solar, materia orgánica y flujo del agua no presentaron variaciones importantes, impidiendo establecer una correlación. En cuanto a la producción de (+) -discodermólido, no se encontraron diferencias significativas entre los sistemas y localidades, registrando una producción entre 20-270 pg por gramo de esponja seca.

Palabras clave: Discodermia dissoluta; (+)-discodermólido; Fragmentos de esponja; Cultivo de esponjas; Acuicultura

INTRODUCTION

The marine environment accounts for more than half of the global diversity and is estimated to contain up to 500 million different species, offering an immense source of different compounds with potentially useful biological activity (Tziveleka et al., 2003, Blunt et al., 2013). Sponges are an exceptional source of compounds, whose structural characteristics have demonstrated a wide variety of pharmacological activities (Faulkner, 2002), and in some cases, these compounds have reached preclinical and even clinical evaluation. However, this potential is affected by the limited availability of the compounds in organisms as well as being scarce and scattered in the natural environment (Page et al., 2005; Carballo et al., 2009; Duckworth, 2009). Therefore, in the search for an alternative solution for obtaining compounds (natural marine products) with less difficulty, and to bring production of the compounds to a sustainable level, strategies have been proposed such as the chemical synthesis of the metabolite and the culture of the organism under different artificial conditions: growing cell lines in the laboratory (primordial dissociated or disintegrated cells) and the culture of organisms in the sea (Sipkema et al., 2006).

From these strategies, aquaculture at sea, where the natural conditions of the organism’s habitat are used, is considered the most effective method because of its low cost and efficiency (Ruiz et al., 2013). Thus, fragments of natural populations are collected, allowed to recover in the field, and then fixed to a substrate for their development. With this alternative, significant growth rates can be obtained, guaranteeing a constant supply of biomass and therefore of the compounds of interest (Muller et al., 1999; Duckworth and Battershill, 2003a, 2003b; Van Treeck et al., 2003; Page et al., 2005).

Pilot studies of mariculture with sponges have been conducted where at least three criteria for the evaluation of cultivation efficiency have been established: survival, growth, and metabolite concentration; however, results so far indicate that success depends on factors such as species, location, fragment size, season, depth, and epibiosis, among others. Survival rates from 23% (Duckworth and Battershill, 2003a) to 100% (Voogd, 2007) have been obtained, and growth rates have been obtained ranging from weight loss (Duckworth et al., 1997) to an increase of 960% (Duckworth and Battershill, 2003a). In some studies, the manipulation of the sponge increases the concentration of the metabolite of interest (Hadas et al., 2005); in some, the concentration of the metabolite does not change significantly with respect to the concentration of the sponge in natural environments; and in others, the metabolite was either not produced or the concentration decreased significantly (Voogd, 2007). Thus, some sponge cultures have shown to be promising for obtaining biomass and producing metabolites; however, to date, they have not been scaled for industrial production.

The sponge Discodermia dissoluta is known for the production of the compound (+)-discodermolide, a lactone of the polyketide family with strong antitumor, antimitotic, antifungal, and immunosuppressive activity (Gunasekera et al., 1990). This compound was patented by the Harbor Branch Oceanographic Institution -HBOI (Longley et al., 1998, US Patent 5840.750) and licensed by the latter to the pharmaceutical company Novartis Pharma for its development as a commercial drug. During phase I clinical research with human patients suffering from solid tumors, this compound was shown to be efficient, but it also showed some secondary pulmonary toxicity in 3 of 32 patients (Mita et al., 2004). However, preclinical animal research has continued to determine the pharmacophore of the molecule and the viability of some of its synthetic analogs or semi-synthetic derivatives. Nevertheless, the supply continues to be a limiting factor in these studies (Ruiz et al., 2013), due to the low concentration of the compound (0.002% by weight), as well as the low density of the sponge and the great depth of its habitat (> 30 m). In addition, this type of research has used completely synthesized material, making it a very costly and impractical strategy (Smith et al., 2003; Florence et al., 2008).

Unlike the rest of the Caribbean, in the Santa Marta area D. dissoluta is found in shallow waters between 12- and 25-m depth, with a moderate population density of two to five individuals per 50 m2 (Zea, 1987; Ruiz, 2009), which cannot sustain direct exploitation but allows cultivation experiments aimed at obtaining and producing (+)-discodermolide in the medium or long term for use in clinical trials (Ruiz, 2009). For this reason, the present work is the first attempt to study two different strategies of culture systems to evaluate the survival, growth, and production of (+)-discodermolide with environmental variables.

MATERIALS AND METHODS

Fragment collection

The fragments were collected by SCUBVA at a depth of 18-25 m at Morro de Santa Marta (11° 15’01.1’’N and 74°13’47.8’’W) and the Ensenada de Granate (11°17’43.3’’N and 74°11’47.9’’W) near the coast of the city of Santa Marta, Magdalena, Colombia (Figure 1). Once the fragments were identified, the larger ones were cut with a metal knife above the basal zone to allow their regeneration. Subsequently, the fragments were placed in sealed bags and transported in 40-L plastic coolers to the underwater nurseries located 10 m deep at Punta de Betín (PB) and Nenguange (NG) for recovery and adaptation for 16 days (Figure 2).

Figure 1 Location of the sites (Punta de Betín and Nenguange, Santa Marta, Colombian Caribbean), where the experimental culture of D. dissoluta fragments was conducted. 

Figure 2 Nursery for the adaptation and recovery of the fragments collected (depth 10 m); suspended and fixed culture systems installed at 15-m depth to evaluate the growth and survival of fragments of the sponge D. dissoluta. Note the marker (steel washer) used to differentiate each fragment, the “HOBO” (precision electronic thermometers to record temperature and solar radiation), sediment trap for measuring sedimentation rate, and gypsum spheres for water flow. 

Cultivation systems

The culture was evaluated in two ways: a fixed system, composed of a 1-m² quadrant or panel, built in half- inch PVC pipe and fittings, covered with plastic net with 3 cm² mesh (internally reinforced with PVC pipe), which served as a substrate to keep the fragments fixed to the mesh in a specific area constructed with 3-mm polyethylene rope (Figure 2), and a suspended system, based on the same 1-m2 quadrant or panel, composed of a horizontal reinforcement with five lines of 3-mm polyethylene ropes (Figure 2), which served to hang the 18 x 18-cm2 bags made with plastic net with 3-cm² mesh. Four quadrants were built per system (eight in total), installing two fixed and two suspended at a depth of 15 m in the two sites of Punta de Betín (PB) and Nenguange (NG).

Measurement of size (by volume)

After the recovery and adaptation period (16 days), the fragments were collected and placed in sealed bags, which were placed in 40-L plastic coolers (containing sea water), and transferred to the Invemar Marine Bioprospecting Laboratory (LabBIM). For each fragment, the initial and final size (volume) was measured by the displacement of the sea water, with an error of ± 2 mL, using a volumetric device manufactured by Ruiz and Zea (2013), consisting of a 250-mL graduated cylinder adhered to a hermetically sealed 860-mL vessel. In addition, the origin of the explant fragments were classified into fragments coming directly from the natural environment (F0) and fragments from experiments carried out 1.5 months prior, to standardize the experiments in this work (F2). Later, the marking was continued, which consisted of a coded stainless-steel washer, tied with telephone cable to each fragment for the fixed system and in the mesh bags for the suspended one (Figure 2).

To determine the initial biomass of the culture, the following equation was applied:

where:

B I = Initial biomass

E I = Number of initial fragments

W I = Average weight in volume

Explanting and harvesting the fragments

Once measured and marked, the fragments were placed in sealed bags, collected in 40-L plastic coolers, and transferred by SCUBA and placed in each culture system, with a total of 160 fragments-80 in each station and 20 per cultivation system, with two replicates per system-being explanted.

At the time of explant, the fragments to be cultured in the fixed system were placed in the panel on the mesh, and another plastic mesh (2 cm mesh size) was placed on top, while the fragments of the suspended system were introduced into the mesh bags and hung with plastic cable ties to the polyethylene ropes installed in the panel (Figure 2).

The fragments were cultivated for 9 months (March to November 2013) and cleaned every 15 days by generating strong currents of water with the hand, to remove the sedimentation and fouling adhered to the mesh and fragments. At the end of the culture period, fragments were collected by SCUBA, brought to the surface and placed into 40-L plastic coolers and transported from the collection site to the LabBIM to determine their size (displaced volume). At the same time, 12 fragments of the total number harvested or recovered at the end of the culture (three for each system and culture site) were harvested to carry out the (+)-discodermolide extraction, with the samples being preserved at -15 °C until the time of extraction and quantification.

Regarding the percentage of growth of the fragments expressed in volume, the volume gained or lost by the sponge during the culture period was considered. This percentage was calculated as follows:

where:

% C = Percentage growth

Vi = Initial volume

Vf = Final volume

Additionally, the monthly growth was calculated using the following formula:

where,

% Cm = Monthly growth rate

Vi = Initial volume

Vf = Final volume

Δt= Cultivation time in months

Survival was evaluated visually by the color of the fragments, as living sponges have an intense color (red-brown), whereas dead sponges are white, and their skeletons are covered with algae, sponges, and other invertebrates and sediment.

The survival rate was calculated from the total number of fragments seeded relative to the number of dead fragments present in the culture after 8 months, expressed as follows:

Environmental variables

For determination of the influence of the environmental variables on the growth (biomass) and survival of the cultured fragments, the temperature and solar radiation were evaluated with precision electronic thermometers known as “HOBOS” (Onset) (Figure 2), which were programmed to record the information every hour and were replaced monthly by new devices. Salinity was recorded every 15 days in triplicate using a YSI multiparameter probe. The analysis of particulate organic matter was performed out using a modified total solids technique according to Garay et al. (2003). Sedimentation was evaluated using sediment traps according to Huang et al. (2011) with some modifications. Briefly, PVC tubes 3.8 cm in diameter and 30 cm in length were used, and the traps were left for a period of 2 months (Figure 2). Finally, water flow was estimated indirectly through the loss of the gypsum sphere mass (Figure 2) (Nishizaki and Ackerman, 2007). The gypsum used was commercial type IV for dentistry, and 4.5-cm-diameter spheres were obtained by using appropriate molds. The spheres, once compact, were dried at 38 °C for 3 hours, and then, their initial mass was recorded before they were installed at the growing sites. After 48 hours of immersion, the spheres were collected and oven dried to determine weight loss.

Quantification of (+)-discodermolide

For the extraction and quantification, the methodology according to Valderrama et al. (2010) was followed using Agilent 1260 Infinity LC HPLC (Agilent Technologies) and an Agilent 1260 Infinity G1315C DAD detector. For this analysis, the metabolite of wild individuals was first isolated, and a calibration curve for the wild metabolite was constructed using methanol solutions ranging from 1.4 to 118 μg/mL. From this calibration curve, the quantification of the metabolite was performed in the 12 selected fragments.

For the entire process of analyzing the (+) - discodermolide content in the tissues of D. dissoluta, for both the natural environment and culture systems, a pattern of the pure substance supplied by the HBOI was used to verify the presence of the metabolite in the samples processed.

Statistical analysis

A generalized linear model (GLM) was used to determine the performance of the culture, with monthly growth being used as the dependent variable; the site (Nenguange and Punta de Betín), cultivation systems (fixed and suspended), and origin (F0 and F2) as the predictive factors; and the initial size of the cultured fragments, based on the size classes (small: ≤ 20 mL; medium: 21-39 mL; and large: ≥ 40 mL), as covariate. Finally, a simple linear regression was performed between the initial size and growth to determine if the growth depended on the initial size of the fragment.

Likewise, to evaluate mortality, the total number was quantified, and the percentage that corresponded to the four factors (sites, cultivation systems, size classes, and origin) was determined, after which a test of independence of the frequency of live fragments versus dead, determined according to the mentioned factors, was performed using statistical software Statgraphics Plus version 5.1.

Two-way and three-way ANOVAs were also used, taking the cultivation systems, the two sites, and the origin of the fragment (F0 and F2) as the factors and taking the initial explant size as covariate after assumptions of normality and homogeneity of the variances had been tested. Likewise, a simple linear regression was performed between the initial size and growth to determine if the increase in volume depended on the initial volume of the fragments.

Finally, to evaluate the possible relation between mortality in the different culture systems with the environmental variables, a correlation analysis was performed using the Pearson correlation coefficient as a statistical predictor (significance value of p > 0.05) using SPSS software 20.

RESULTS

Survival and growth of fragments

In the first months, high mortality occurred in both cultivation systems (fixed and suspended) (Figure 3). As of April, the fragment survival at Punta de Betín was relatively stable, decreasing slightly at the end of the culture period. However, in Nenguange, this behavior occurred in the suspended system because, in the fixed system, a continuous decrease was observed in the survival, which reached 55% by the end of the experiment (Figure 3). As for the culture system, the fragments that remained suspended showed a slightly higher growth (Figure 3).

Figure 3 Cumulative survival of D. dissoluta fragments grown in two systems: fixed (A) and suspended (B) (NG = Nenguange; PB = Punta de Betín). 

The highest mortalities, which consider the effect of the origin of the fragment, independent of the culture site, ocurred in F0 (extracted directly from the natural environment) relative to those derived from previous experiments (F2) (Figure 4).

Figure 4 Percentage mortality of D. dissoluta fragments grown in two systems (fixed and suspended) according to origin (F0 and F2). 

For the independence tests, only the combined size classes for the cultivation systems showed a variation in mortality frequency (chi-square = 11.6; degrees of freedom = 2; p < 0.05); the value for small size class was 63.26%, followed by the medium with 55.25% and the large ones with 33.36%.

The growth of the surviving fragments at the end of the culture period was variable, with values ranging from -10.9 to 109.4% of the initial size, with an average of 2.1 ± 1.0% of monthly increase (± 1 standard error); 58.94% of the cases showed negative growth, and only 21.85% exceeded 5% monthly growth. Importantly, for the fixed system installed at Punta de Betín, the monthly growth was negative (- 0.968%) (Figure 5).

Figure 5 Mean percentage of monthly growth of D. dissolute fragments grown in two systems (fixed and suspended) and sites (NG = Nenguange; PB = Punta de Betín) at 15 m depth. Bars indicate the standard error. 

For the size classes (small: ≤ 20 mL; medium: 21- 39 mL; and large: ≥ 40 mL), according to the culture system, the fragments in the suspended system showed better results in all three size classes than in the fixed system; two-way ANOVA showed a significant difference (F = 3.44, p = 0.03; small: 7.5 ± 5.0%, n = 23; medium: -0.0 ± 0.6%, n = 60; and large: 2.1 ± 1.3%, n = 68) (Table 1), with the largest growth in the small fragments, with a mean of 13.9% ± 13.8% (n = 8), which was also seen in the results from the linear regression (p = 0.04) but with a very small variability explained by the initial size (R2 = 2.9%).

Table 1 Two-way analysis of variance (ANOVA), comparing the (monthly) percentage of growth of D. dissoluta fragments cultivated in two systems (fixed and suspended) and for three sizes (small: ≤ 20 mL; medium: 21- 39 mL; and large: ≥40 mL), using the initial size as covariate. DF = degrees of freedom; P = probability. 

However, three-way ANOVA, comparing the culture systems (fixed and suspended), sites (NG 15 and PB 15 m), and origin (F0 and F2), where size was the covariable, indicated that the factors did not affect the growth significantly (p > 0.05), nor their interactions (Table 2), probably because when a greater number of variables was evaluated, the distribution of the small fragments was homogeneous, generating a similar result among the evaluated treatments in the end.

Table 2. Three-way analysis of variance (ANOVA) comparing the (monthly) percentage of growth of D. dissoluta fragments cultivated in two systems (fixed and suspended) and at two sites (NG = Nenguange, PB = Punta de Betín) and origins (F0 and F2), using the initial volume as a covariate. DF = degrees of freedom; P = probability. 

Environmental variables

The temperature during the cultivation was similar at both sites, with an average of 27 °C, starting with a temperature of 24 °C in March, with the temperature increasing gradually until November, reaching its highest value in October with 29 °C. For the solar radiation, the behavior was variable, with the highest records in June in Nenguange, with 2.36 watt/ m2, and September in Punta de Betín, with 0.48 watt/ m2 (Figure 6). Salinity decreased from 37 to 35 at Punta de Betín and from 37 to 36 at Nenguange. The organic matter at the two sites was highly variable, ranging between 11.1 to 5.1 mg/L, with Punta de Betín having the highest values of organic matter during much of the study (Figure 6). As for the sedimentation rate, Punta de Betín had the highest value in May, with 421.8 mg/ m2/day; however, subsequently, the sedimentation rate decreased, unlike Nenguange, whose sedimentation rate gradually increased, with the highest value being observed in November of 265.6 mg/m2/day. Finally, the water flow did not show significant variation. The lowest flow was recorded in Nenguange during June with 1 df, and the highest flow was recorded in Punta de Betín during April with 2.7 df (Figure 6).

Figure 6 Environmental variables evaluated during the culture of D. dissoluta fragments (NG = Nenguange; PB = Punta de Betín). Bars indicate the standard deviation. 

The correlation results between the environmental variables and the survival of the fragments were not significant (p > 0.05). A negative correlation was observed between temperature and survival at Punta de Betín for the two cultivation systems (n = 9; p < 0.05, PB15 Suspended C = -0.9 and PB15 Fixed C = -0.9) (Table 3).

Table 3 Pearson correlation analysis between mortality and the environmental variables evaluated in the culture of D. dissoluta fragments (NG = Nenguange, PB = Punta de Betín, S = Suspended, F = Fixed). (p > 0.05). C = correlation value, p = significance, and n = sample size. 

Obtaining (+)-discodermolide

The determination of the content of (+)-discodermolide from the sites and systems showed that in Punta de Betín, the fixed system presented the highest average production of the metabolite, with 132.9 μg/g ash free dry weight; however, no significant differences (p > 0.05) were observed between the amount of metabolite produced by the fragments according to site, nor by the culture system (Figure 7).

Figure 7 Variation in the (+)-discodermolide content of D. dissolute fragments obtained from two culture systems: NF = Nenguange Fixed, NS = Nenguange Suspended, PBF =: Punta de Betin Fixed, PBS = Punta de Betin Suspended. 

DISCUSSION

This study considered the site, type of cultivation, and environmental variables to determine if they affected the growth and survival of the fragments as well as the concentration of the (+)-discodermolide. The high mortalities of the fragments at the beginning of the cultivation were attributed to stress from manipulation outside the water during the weighing, marking, and explanting process, which affected the sponge and its community of associated symbiont bacteria (Webster et al., 2008), causing a weakening of the organism, tissue necrosis, death of the fragment, and energy consumption by those fragments that survived during the healing process (Van Treeck et al., 2003). Although the survival rate for both sites and systems was 55-72%, this rate is not very promising when compared to the percentage (93%) obtained by Ruiz et al. (2013) using suspended culture (pockets) at 18-m depth and other studies performed with Negombata magnifica, with survival rates ranging from 17 to 84% (Hadas et al., 2005). However, adjusting some aspects, such as the time out of water for the above procedures and using only suspended culture, would probably increase the survival of the fragments.

Duckworth (2009) reported that mesh bags have openings that maximize the surface area for feeding and breathing, obtaining high survival rates in several species of sponges. The results support this statement for the fragments grown in pockets, which presented greater survival and higher monthly growth for both sites, indicating that this system allows a greater flow of water and therefore more food and less sedimentation. Additionally, the fragments grown at Punta de Betín in the fixed system corroborate that high sedimentation corresponded with high colonization by other organisms. Another consideration is that because Punta de Betín is exposed to residual discharges from the Gaira and Manzanares rivers (Parra and Zea, 2003; Roa et al., 2007), greater sedimentation and, consequently, more food is expected, which could eventually be negative for the sponge and prevent its growth and production of the metabolite (+)-discodermolide. Despite periodic cleanings with the express purpose of removing sedimentation adhered to the mesh and the fragments, both cultivation systems, especially the fixed, were easily filled with sedimentation covering the mesh.

Other research on sponge cultivation (Polymastia croceus and Latrunculia wellingtonensis), indicate that mesh bags increase the growth potential because the organism may initially have low interspecific competition for food and space (Caralt, 2007). Other authors suggest the advantages of growth in fixed-mesh systems in terms of increased wave protection (Van Treeck et al., 2003; Duckworth, 2009), as well as greater exposure to light, which would favor the translocation of nutrients from photosynthesis performed by symbiotic cyanobacteria (Schirmer et al., 2005). However, this study did not reveal any outstanding behavior of this cultivation system (fixed); in contrast, it favored the growth of fouling organisms, which plugged the mesh and diminished the water flow.

The environmental variables evaluated did not have significant effects (p > 0.05) on the growth, survival, and content of (+)-discodermolide. Additionally, the origin of the fragment was found to be a determining factor for sponge survival, with sponges from the previous experiment (F2) having higher survival than those extracted directly from their natural environment (F0). Duckworth and Battershill (2003a) and Ferrati et al. (2006) observed that the growth rates of Agelas oroides and Petrosia ficiformis are not related to the initial size of the sponge, whereas in other species, explant size is determinant (Reiswig, 1973; Leys and Lauzon, 1998). In our case, the small fragments grew more, a behavior that was also present in the fragments of D. dissoluta in the study by Vásquez (2011), in which the fragments doubled and even quadrupled their volume, suggesting that fragmenting donor sponges into a larger number of small fragments is a very good strategy to achieve better and faster growth.

This study evaluated the best cultivation condition for maximizing the growth of the fragments and, thus, a higher concentration of the metabolite, considering that conditions should be chosen that could be adapted for fragment collection, depth, and site to ensure feasibility of the study. The results are contrasting, with a positive and negative monthly growth rate, which depends first on many variables in terms of growth form and regeneration capacity, and second on attachment methods, the location of the fragments, and the environmental conditions. Culture studies conducted with species such as Geodia cydonium, Latrunculia wellingtonensis, Polymastia croceus, Axinella corrugata, Negombata magnifica, Callyspongia (Euplacella) biru, and Mycale hentscheli (Duckworth et al., 1997; Müller et al., 1999; Duckworth and Battershill, 2003a; Hadas et al., 2005; Page et al., 2005; Voogd, 2007; Duckworth 2009) note the best results were obtained by placing structures in areas with good water circulation, placing the fragments above the substrate to maximize food production and minimize the effects of the substrate, and keeping them in wide mesh bags or securing them with nylon.

Duckworth and Battershill (2003a) and Voogd (2007) note that for the cultivation of these organisms to be feasible or sustainable, a good supply of the metabolite is necessary, in addition to adequate biomass production and high survival. In the case of D. dissoluta, the (+)-discodermolide is in low concentration; therefore, implementing a culture strategy would be effective for this species (Valderrama, 2009). Several studies with other species of sponges and using various methods of analysis show the concentration of compounds to be above or below those found in natural conditions; for example, in Callyspongia (Euplacella) biru, concentrations of the fragments in culture (0.03 and 0.17% by weight) were not significantly lower than those in natural populations (0.06 and 0.22% by weight) (Voogd, 2007), whereas in Latrunculia wellingtonensis, Polymastia croceus (Duckworth and Battershill, 2003a), and Mycale hentscheli (Page et al., 2005), the culture concentrations were higher than the natural population. These results demonstrate the production variability of these compounds, which may depend on the species and the type of test being developed.

CONCLUSIONS

The low variation in the environmental conditions during the culture was not a determining factor for the growth and survival of D. dissoluta. The highest survival rates were obtained in the suspended system, which had increased water flow, food availability, easy cleaning (removal of sediment and fouling), and harvesting of the fragments. Although fragments of the D. dissoluta sponge can heal, survive, and grow, factors such as excessive manipulation lead to stress of the animal, causing weakness, infections, tissue death, and even death of the fragment. This, in turn, has been considered the main cause of the variation in growth, which was between -10.9 to 109.4% for the sites and culture systems. In addition, the origin of the fragments is a determining factor for the survival of the sponge, with sponges from the previous experimental culture having better survival than those extracted directly from the natural environment for the experiment. In terms of (+)-discodermolide, this content was highly variable, but because no significant differences existed between the sites and culture systems, establishing which culture system was the most efficient was difficult.

The use of smaller fast-growing fragments is recommended, with suspended pockets, thereby minimizing the time the fragments are exposed to air and handling during the process of cutting, weighing, and marking the fragments.

ACKNOWLEDGEMENTS

The authors are grateful for the financial support from the Departamento Administrativo de Ciencia, Tecnología e Innovación - Colciencias (Administrative Department of Science, Technology and Innovation) under contract RC No. 327-2011. The authors acknowledge the Instituto de Investigaciones Marinas y Costeras (Institute of Marine and Coastal Research - Contribution N° 1164) “José Benito Vives De Andréis”-Invemar; the Universidad Nacional de Colombia Sede Caribe (National University of Colombia, Caribbean); and the Ministerio de Ambiente y Desarrollo Sostenible -MADS (Ministry of Environment and Sustainable Development), which through the Banco de Proyectos de Inversión Nacional-BPIN (Bank of National Investment Projects) financed the present project PRY-VAR-006-011. Additionally, the authors acknowledge the Línea de Bioprospección Marina del Programa de Valoración y Aprovechamiento de los Recursos Marinos (Marine Bioprospection Division of the Program for the Evaluation and Use of Marine and Coastal Resources), where the project was carried out, and especially thank Carlos Puentes for the quantification of (+)-discodermolide. Sven Zea’s work is contribution 466 of Instituto de Estudios en Ciencias del Mar-CECIMAR, Universidad Nacional de Colombia, Sede Caribe.

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Received: December 29, 2015; Accepted: August 30, 2017

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