long term culture of sponge explants

Long-term culture of sponge explants: conditions enhancing survivaland growth, and assessment of bioactivity

So`nia de Caralt, Gemma Agell, Mara-J. Uriz *

Centre dEstudis Avancats de Blanes (CEAB), CSIC, Acces a la Cala Sant Francesc, 14, 17300 Blanes, Girona, Spain

Abstract

Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasinginterest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easilyproduce sponge aggregates after dissociation (primmorphs), others show a great capacity to regenerate after fragmentation(explants). Corticium candelabrum is a Mediterranean bacteriosponge that can undergo asexual reproduction. We have takenadvantage of this capability and cultured C. candelabrum explants under several experimental conditions. To find the bestconditions for obtaining functional explants, we assayed a range of conditions, including seasons of collection, culture temperature,filtered versus filtered-sterile seawater, addition of antibiotics and proportion of ectosome. We monitored the changes in shape andultrastructure during the formation of explants. After 24 h, TEM images showed the aquiferous system disarranged, in particular atthe sponge periphery. From 2 to 4 weeks later, the aquiferous system regenerated, and fragments became functional sponges(explants). Explants were cultured under two regimes: in vitro and in a closed aquarium system. Antibiotics were only added to thein vitro culture to assess their effect on the symbiotic bacteria, which remained healthy despite the presence of antibiotics. Two foodregimens (marine bacteria and green algae) were assayed for their ability to satisfy the metabolic requirements of explants. Wemonitored explant survival and growth. Explants showed a high long-term survival rate (close to 100%). Growth rates were higher inthe closed aquarium system, without antibiotic addition, and fed with algae. Explants cultures were hardly contaminated becausemanipulation was reduced to a minimum and we used sterilized seawater. C. candelabrum produces bioactive molecules, which mayplay a defensive role in the sponge and may have pharmaceutical interest. The bioactivity of the explants was similar to that of wildsponges.# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Sponges; Explant culture; Bioactivity; Corticium candelabrum ; Bacteriosponge; Mediterranean Sea

1. Introduction

Sponges represent the lowest metazoan phylum. Theyare sessile organisms that pump and process largeamounts of water through a system of conducts andchambers (aquiferous system), and efficiently retainparticles such as phytoplankton, heterotrophic bacteria,heterotrophic eukaryotes and detritus, and also appearto take up dissolved materials [1]. Sponges have severalcell types with different functions. Two zones can bedistinguished in sponges: the choanosome or innerregion, formed by channels and choanocyte chambers,and the ectosome or peripheral region, which is mainly

formed by pseudoepithelial cells (pinacocytes), spheru-lous cells, collencytes and collagen fibrils [2]. Cellplasticity allows sponges to regenerate and adapt theirshape to the environmental conditions [3!/5].Sponges usually produce bioactive compounds, which

have biological and ecological roles in nature [6], such aspredator deterrence [7], antifouling activity [8,9] or inter-species competition [10]. Numerous studies have shownthe pharmaceutical and commercial interest of thesebioactive compounds, with a variety of properties suchas cytotoxic, antitumour [11], antibiotic, anti-inflamma-tory and antiviral activities [12]. Collection of sufficientamounts of bioactive sponges for commercial produc-tion of their secondary metabolites may have an adverseimpact on the environment [12]. On the other hand,chemical synthesis of the metabolites is not feasible in allcases [13]. Consequently, sponge culture has been

* Corresponding author.E-mail address: [email protected] (M.-J. Uriz).

Biomolecular Engineering 20 (2003) 339!/347www.elsevier.com/locate/geneanabioeng

1389-0344/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S1389-0344(03)00045-5

undertaken as a promising alternative. Some authorsattempted to culture sponge cells [13,14] and aggregates[15!/18] but problems of microbial and protozoancontamination and poor cell growth [13] preventedsuccessful culture. Thus alternative systems have alsobeen assayed. In vitro sponge cultivation appearspromising, since most functional aspects are maintainedand it can be performed under controlled conditions[19].We cultured sponge explants obtained by fragmenta-

tion of wild sponges. We used Corticium candelabrumSchmidt, 1862, a common Mediterranean species thatcan undergo asexual reproduction, has symbiotic bac-teria, and is bioactive. These characteristics make C.candelabrum suitable for collection and providing ex-plants. We monitored the morphological and ultrastruc-tural changes undergone by sponge fragments beforethey become functional, and measured pre-explantsurvival and explant growth under several treatments.Antibiotics are necessary to maintain the culture axenic,but they may harm the symbiotic bacteria. To monitorthe effect of antibiotics on the symbiotic bacteria, wecompared the bacteria populations in wild sponges andexplants after the addition of antibiotics, by TEM.Explants bioactivity was also measured to verify that thetarget compounds were still being produced.

2. Material and methods

2.1. Sponge explant formation

Individuals of C. candelabrum were collected off theBlanes sublittoral (NE Iberian Peninsula, western Med-iterranean), placed in bowls underwater and transportedto the laboratory immediately. In the laboratory, we cutthe individuals into 3 mm2 fragments with a sterilerazor. Cuttings were placed in sterile 6-well plates (oneto two fragments per well) with a sterile pipette. Two 6-well plates were placed in each plastic container filledwith 2 l of filtered seawater sterilised by autoclave, sothat 12!/24 sponge fragments were immersed in 2 l ofseawater.The sponges did not come into contact with the air,

manipulation was reduced to a minimum and watertemperature was maintained at 14!/15 8C.The water in the containers was changed daily during

the first week, and twice a week thereafter. A mixture ofantibiotics (100 IU ml"1 of penicillin plus 100 mg ml"1

of streptomycin [16]), was added to the culture at eachchange of water during the first 2 weeks.

2.2. Morphological and ultrastructural changes duringexplant formation (pre-explants)

Morphological changes during the first hours wererecorded by a video camera connected to a Leika-WildM-10 stereomicroscope and a Macintosh PC. Subse-quent pictures were taken at different time intervals witha Wild camera connected to the stereomicroscope.Pictures were digitalised for analysis.Ultrastructural changes of both sponge cells and

bacteria were monitored on 0 and 24 h-old pre-explants,and 2-month-old explants. For TEM observation,samples (2 mm3) were fixed in a cocktail consisting of1% OsO4 and 2% glutaraldehyde in 0.45 M Sodiumacetate buffer (pH 6.4) with 10% sucrose [1], embeddedin Spurr resin, thin-sectioned, and stained with uranylacetate and lead citrate.

2.3. Pre-explant survival

We monitored pre-explant survival under differenttreatments: season of collection of parental sponges(winter!/spring vs. summer!/autumn), culture tempera-ture (14 vs. 20 8C), addition of antibiotics, filtered versusfiltered!/sterilised seawater, and several ectosome/choa-nosome ratios. The same general conditions (filtered!/sterilised seawater, 14 8C and antibiotic addition) weremaintained in all the experiments except for the treat-ment that was to be assayed in each experiment.Pre-explant survival (N#/20!/91) from samples col-

lected every month for 1 year was monitored on days 0,3, 8, 21, 30, 60, and 240. We compared mean survival ofpre-explants from adult sponges collected either duringwinter!/spring months (seawater T, 12!/15 8C) orsummer!/autumn months (seawater T, 19!/24 8C).Fragments of sponges collected in winter were used to

assay the best temperature conditions in the cultures forpre-explant survival. Fragments were placed at either14 8C (N#/20) or 20 8C (N#/12). Survival was mon-itored at days 0, 6, 9, and 60.For testing whether, besides adding antibiotics, ster-

ilised seawater improved survival, we placed fragmentsin filtered (0.7 mm pore diameter) seawater and filtered!/sterilised seawater (N#/25). We monitored survival ondays 0, 3, 7, 26, and 90.To assay the effect of antibiotics on pre-explant

survival, 100 IU ml"1 of penicillin and 100 mg ml"1

of streptomycin were added to a set of samples (N#/20)while another set (N#/24) did not receive antibiotics.Fragments came from sponges collected in the sameseason. Survival was monitored on days 0, 3, 9, 21, and90.Fragments with a different proportion of ectosome/

choanosome: 30!/50, 10!/30, and 0!/10% of ectosomewere cultured, and survival was monitored on days 0, 3,7, 26, and 90.

S. de Caralt et al. / Biomolecular Engineering 20 (2003) 339!/347340

2.4. Explants growth

When fragments became functional explants (active,filter-feeding sponges), we monitored explant growthunder different treatments: in vitro with antibioticsversus in a closed aquarium system without antibiotics,and in vitro using bacteria versus algae as a food source.The in vitro system (N#/20) consisted of plastic

containers filled with 2 l of filtered sterile seawater towhich antibiotics were added (see above). Containerswere placed in culture chambers at a constant tempera-ture of 14 8C in the dark to prevent algal contamination.The closed aquarium system consisted of several con-nected aquaria (30 l each) through which water floweddriven by a pump. Water was filtered mechanically andthen sterilised by ozone and ultraviolet light. Watertemperature was 14 8C and darkness was applied.A mixture of heterotrophic Mediterranean bacteria

(ca.1 mm in size) grown on marine broth was added as afood to the two systems. Explants were fed regularlytwice a week by adding bacteria up to a concentration of106 cells ml"1 in the container (to mimic the naturalbacteria concentration in the sea [20!/22]. Seawater waschanged and antibiotics were added 12 h after foodsupply. In the closed aquarium system, explants werealso fed twice a week with the same bacterial concentra-tion. Water flow was stopped before food supply andthen restored 24 h later.In both systems water chemical parameters were

monitored during the culture. Water samples werecollected before feeding the explants to analyse nutrients(silicates, phosphates, nitrites and nitrates) and totalorganic carbon and nitrogen concentrations. Nutrientswere analysed by colorimetric techniques (autoanalyserTechnicon). For the analysis of particulate organicmatter (C:N), a known volume of seawater was passedthrough a 0.2 mm diameter, GF/F glass fibre filter,previously exposed to hydrochloric acid vapour for 48 hin order to eliminate any inorganic material. Filterscontaining the organic matter were dried and analysedwith a C:H:N autoanalyser Eager 200 [23].To test the effect of food on explant growth, in vitro

explants were placed in containers at a constanttemperature of 14 8C. A set of explants (N#/22) wasfed with a mixture of marine bacteria at a concentrationof 106 cells ml"1 and another set (N#/21) was fed with aconcentration of 105 cells ml"1 of the algae Chlorella sp.(3 mm in size) grown in F/2 medium. These twoconcentrations mimic the natural sea concentration ofbacteria and phytoplankton, respectively. The explantswere fed twice a week. We changed the water 12 h afterfood supply.Explant growth under different treatments was mon-

itored by digital camera (SPOT cooled colour), and theexplant areas were calculated with an image analysisprogram (NIH Image, public domain). Since the ex-

plants mainly grew in two dimensions, changes in areaare a good estimate of changes in biomass [24,25]. Fromthe changes in area over time, a growth rate GRt wascomputed from the formula:

GRt#(At"At"1)=At"1;

where At and At"1 are the areas at time t and at theprevious time, respectively. This growth rate is thechange in area relative to area at the previous time [24].

2.5. Bioactivity

The toxicity of individuals of C. candelabrum col-lected from their natural habitat and explants living formore than 6 months in the in vitro cultures wasmeasured by the standardized Microtox assay [26,27].

2.6. Statistical methods

Survival was assessed as the percentage of fragmentsthat were alive on a given day related to the initialnumber of fragments. Mean differences in survivalbetween seasons of collection were compared by a t-test at each monitored day. Differences in survival forthe remaining treatments were analysed by two-sampleand multiple-sample (ectosome/choanosome ratio, threelevels) Gehans Wilcoxon tests. When necessary forparametric analysis, normality and homogeneity ofdata were determined by a K!/S and Lilliefors test andLevenes test, respectively.Differences in growth rates between treatments and in

toxicity between wild and cultured sponges were ana-lysed by t-test. When the two culture systems (in vitroand in aquaria closed system) were compared, data werelog transformed to meet the assumptions of this para-metric test.

3. Results

3.1. Morphological and ultrastructural changes duringformation of explants

During the first 2 h, cuttings maintained the samemorphology as an adult (Fig. 1A). Cells then movedtowards the sponge periphery and spread on thesubstrate (Fig. 1B). Cell spreading increased pre-explantarea but this did not imply pre-explant growth. Inparallel, the border of the cuttings started to heal. From1 to 2 weeks later, we could differentiate totallydisorganized fragments (Fig. 1C) from partially orga-nized fragments (Fig. 1D). The latter showed an almosthealed central mass, similar in colour and appearance towild sponges, surrounded by a layer of dispersed cells.Fragments aged 2!/4 weeks, which were totally disorga-nized, died leaving a thin spicule layer (Fig. 1E). The

S. de Caralt et al. / Biomolecular Engineering 20 (2003) 339!/347 341

partially organized fragments became compact andglobular in shape (Fig. 1F). These fragments can beconsidered functional explants since they were similar toadults in shape and showed active water exchange. One-to 2-month-old explants adhered to the substrate bybody extensions (Fig. 1G). During the process weobserved spontaneous formation of primmorphs (Fig.1H).The ultrastructure of fragments at time 0 (recently

collected wild sponges) showed well-structured conductsand choanocyte chambers (Fig. 2A). Choanocytes (thebasic cell type of sponges, Fig. 2B) displayed theirtypical shape and elements (choana and flagellum, Fig.

2C). Several cell types other than choanocytes were alsoobserved: endopinacocytes (pseudoepithelial cells, Fig.2D), spherulous cells (Fig. 2E), which may contain thebioactive compounds [28,29], and collencytes (cells thatsegregate collagen) (Fig. 2F!/G). Spicules with an axial

Fig. 1. Morphological changes of fragments during explant formation(C. candelabrum ). (A) Newly cut fragments; (B) after 2 h, peripheralcells spread on the substrate; (C) totally disorganised fragments 2weeks later; (D) partially organised fragments with a healed centralmass; (E) spicules layer from totally disorganised fragments; (F)functional explant 4 weeks later; (G) Functional explant showingbody extensions for adhering to the substrate; (H) spontaneouslyformed primmorph.

Fig. 2. TEM images of ultrastructural aspects of wild sponges (C.candelabrum ). (A) Well organised conducts (arrow) and choanocytechambers (cc); (B) typical elongate in shape choanocytes (arrows) withchoana (ch) and flagellum (f); (C) transversal section of choanocyteflagella (f) showing microtubules triplets (m) joined by glycocalyx andchoana (ch); rough collagen fibrils (cf) are also visible; (D) endopina-cocytes (arrows) lining a conduct (co). (E) Spherulous cell (s)surrounded by symbiotic bacteria (b); (F) collencyte containingcollagen fibrils (arrow); (G) detail of a collencyte containing collagenfibrils (arrows). (H) Transversal section of a spicule showing anirregular central axial filament (arrow).


filament (protein filament), which indicates recentspicule formation [30], were also present (Fig. 2H).TEM images of 24 h-old fragments showed disorga-

nized conducts and choanocyte chambers, mainly at theperiphery (Fig. 3A). Choanocytes were rounded andlacked choana and flagellum (Fig. 3B). No other celltypes could be distinguished.TEM images of 2-month-old explants showed a

similar ultrastructural pattern to wild sponges. Con-ducts and choanocyte chambers were reorganized (Fig.4A). Choanocytes recovered their typical morphologywith flagellum and choana (Fig. 4B). We also observedcollencytes (Fig. 4C) and spicules with an axial filament(Fig. 4D).Symbiotic bacteria were abundant in samples from

wild sponges. Six different bacterial morphologies couldbe differentiated (Fig. 5A). Symbiotic bacteria degener-ated in 24 h-old fragments, giving rise to brokenmembranes (Fig. 5B). Only three morphological typesof bacteria could be distinguished and they were lessabundant than in wild sponges. TEM images of 2-months old explants showed the same bacterial types atsimilar abundance to wild sponges (Fig. 5C) despite theantibiotic added.

3.2. Pre-explant survival

Pre-explant survival was affected by the differentculture conditions assayed: season of collection, watertemperature, filtered versus filtered!/sterilised seawater,antibiotic addition, and ectosome ratio.Mean survival during the first days was higher for

pre-explants obtained from individuals collected inwinter!/spring (Fig. 6) but differences were not signifi-cant until day 21 (t -test, PB/0.05) (45.85 and 15.66% forpre-explants collected in winter!/spring and summer!/autumn, respectively). These survival rates were main-tained until the end of the experiment (ca. 12 months).

Water temperature determined differential survival inthe cultures (Fig. 7). Survival was significantly higher(Gehans Wilcoxon test, PB/0.005) in pre-explantscultured at the lower temperature (14 8C). All fragmentscultured at 20 8C died before day 9.Although survival was slightly higher for fragments

cultured in filtered!/sterilised seawater (Fig. 8), differ-ences with respect to those cultured in only filteredseawater were not significant (Gehans Wilcoxon test,P#/0.36).Antibiotic addition significantly improved pre-ex-

plant survival (Gehans Wilcoxon test, PB/0.05).Although differences were detected from day 3 (100 vs.83.3% of survival for pre-explants with and withoutantibiotics, respectively), differences were particularlyhigh after day 9, with a survival of 33.3% for explantswithout antibiotics versus 60% for explants with anti-biotics (Fig. 9).The amount of ectosome relative to the choanosome

of the fragments appeared to affect survival: the higherthe ectosome proportion, the higher the survival. Frag-ments with 30!/50% of ectosome and with 10!/30% hadsignificantly higher survival (Gehans Wilcoxon test,PB/0.05) than explants with only a 1!/10% of ectosome(Fig. 10). Differences became evident on day 3 (only12% of survival for fragments with 1!/10% of ectosomevs. 75% for the other two).

3.3. Explant growth

Explants in the two culture systems behaved differ-ently (t-test, PB/0.05). In the closed aquarium systemexplants grew at a mean rate of 0.058 per month. Incontrast, in the in vitro system with antibiotics, explantsdecreased their area at a mean rate of "/0.235 for thesame period (Fig. 11). Nutrients and total carbon in thewater were in general higher in the closed aquariumsystem than in the in vitro system. Silicate, nitratephosphate and nitrite values ranged between 15.87 and

Fig. 3. TEM images of 24-h-old fragments. (A) Disorganised choanocyte chamber; (B) rounded choanocyte (arrow) without choana or flagellum.


61.85, 135.75 and 495.5, 4.45 and 11, 0.43 and 3.17 mMl"1, respectively in the closed aquarium system, andbetween 1.83 and 61.7, 14.62 and 76.25, 3.34 and 9.65,0.40 and 2.73 mM l"1, respectively in the in vitro system.Total carbon ranged between 1095 and 4935 mg ml"1both in vitro and in the closed aquarium system (Fig.12).

When different food was assayed, explants fed withalgae (Chlorella sp.) showed a significantly highergrowth rate (t-test, PB/0.05) than those fed withbacteria. These differences were not significant at month1 (mean growth rate 0.065 and 0.164, for algae andbacteria food, respectively). However, at month 5explants fed with Chlorella sp. had a higher growth

Fig. 4. TEM images of 2-month-old functional explants. (A) Well-organised choanocyte chamber (cc); (B) typical in shape choanocyte with choana(ch) and flagellum (f); (C) collencyte surrounded by collagen (cf); (D) transverse section of two spicules with axial filament (arrows).

Fig. 5. TEM images of the sponge symbiotic bacteria. (A) Healthy bacteria in wild sponges; (B) broken bacteria in 24-h-old fragments; (C) healthybacteria in 2-month-old explants.


rate (PB/0.05) than explants fed with a bacteria mixture(mean growth rate 0.038 and "/0.1 per month, respec-tively).

3.4. Bioactivity

No significant differences (t-test, P!/0.05) in toxicity(Microtox, EC50) were found between cultured ex-

plants and wild sponges. This indicates that explantscontinue to synthesize the bioactive compounds.

4. Discussion

Sponges are characterised by their plasticity andcapacity to regenerate complete animals from fragments[3]. However, species such as C. candelabrum regeneratefaster than others and even perform asexual reproduc-

Fig. 6. Time course of pre-explant survival from sponges collected inwinter!/spring and in summer!/autumn.

Fig. 7. Time course of pre-explant survival cultured at two differenttemperatures.

Fig. 8. Time course of pre-explant survival cultured in filtered andfiltered!/sterilised seawater (both with antibiotic addition).

Fig. 9. Time course of pre-explant survival cultured with and withoutantibiotic addition.

Fig. 10. Time course of pre-explant survival from fragments withdifferent ectosome/choanosome proportions.

Fig. 11. Explants growth rate per 3 months in vitro and in the closedaquarium system.


tion by propagula, thus being particularly suitable forexplant production.The morphological and ultrastructural changes dur-

ing explant formation from sponge fragments indicatethat, after an initial phase of disorganisation and celltransformations, cell differentiation and rearrangementoccur within a period of 2!/4 weeks. During this periodfragments do not grow but lose biomass, since the cellsthat spread on the substrate die. Dead cells wereremoved to reduce the risk of contamination.The sponge symbiotic bacteria underwent a similar

process: disintegration followed by recovery, likely bycell division. The healthy appearance of the bacteria inthe explants despite the addition of antibiotics wasunexpected. Although symbiotic bacteria might beresistant to the wide-spectrum antibiotic used, theirhealthy state may be due to isolation from the externalmedium and from the sponge cells by a collagen coat(see Fig. 2), which may protect them.Among the several conditions assayed during explant

formation, we found that fragments from spongescollected in winter and spring, cultured at 14 8C, withfiltered sterilised seawater, antibiotic addition and ahigh ectosome/choanosome ratio survived longest.The higher survival of fragments from individuals

collected during the colder months of the year could berelated to the biological cycle of C. candelabrum, whichreleases larvae in early summer (personal observation)and, as a result, its aquiferous system become disar-ranged [31]. Fragments from sponges with disarrangedconducts and chambers may be less able to rearrangeinto functional explants.Lower temperature, antibiotic addition and water

sterilisation diminish bacterial proliferation. Conse-quently, all these conditions promote explant survivalduring the critical first steps of the process in which acertain degree of cell death is inevitable and fragmentsare most sensitive to infections.As previously reported for Chondrosia reniformis [32],

a high ectosome proportion seems to favour healing of

the cut faces. The ectosome is mainly formed bypinacocytes, which are pseudoepithelial cells involvedin isolating the sponge from the external medium. Thesecells and the collencytes, which secrete the collagen(structural protein) and are particularly abundant in theectosomal region, play an important role in healing [33].Growth rates were higher in the aquaria closed system

without antibiotics than in vitro with antibiotics,although we fed both cultures in the same way. Thehigher nutrients and total carbon concentrations in theformer (see Section 3) and a negative effect of antibioticson sponge growth in the latter might account for thedifferences.Previous authors disagree about the optimal food

source for the growth of sponges in vitro. Some chosemarine algae [34,35], others tried bacteria [36,37] andothers chose a mixture of algae and bacteria [38]. Thefew studies dealing with sponge feeding in situ reportedthat sponges predominantly retain small organisms (B/4mm) such as heterotrophic bacteria, cyanobacteria andprochlorophytes [39!/41]. Moreover, in semi-enclosedsystems, fragments of Halichondria panicea (Pallas,1766) were grown successfully with Chlorella sp. [34].Furthermore, the study by [42] on Crambe crambe andDysidea avara demonstrated that although there weredifferences in filtering abilities with particle size, timeand species, the most efficiently retained particles weresmaller than 4 mm. On the basis of these results, weselected heterotrophic bacteria (1 mm) and the algaChlorella sp. (3 mm) as the two alternative food sources.Our study showed that growth was higher in the culturesfed with Chorella sp. than with bacteria, possibly due tothe higher carbon content of the former.Although the growth rates found in our cultures

appear to be low even when we grew explants withChlorella , the values correspond to the growth ratesfound for wild populations of C. candelabrum in winter("/0.1!/0.1 per month), when sea T is similar to the T inour cultures (ca. 14 8C). Higher temperatures areexpected to enhance explant growth, as occurs for wildsponges in summer (0.3!/0.7 per month, authors, currentresearch) and are here proposed for culturing thisspecies once the explants are functional and the risk ofbacterial contamination has been reduced.The characteristic bioactivity of C. candelabrum was

maintained in explants cultured for more than 6 months.Some bioactive compounds originally isolated frominvertebrates such as sponges have been subsequentlylocalized in microbial associates [43]. However, wecannot state yet whether the bioactivity of C. candelab-rum is due to the sponge cells or to the symbioticbacteria, since both healthy bacteria populations andbioactivity were maintained in the cultured explants.Further studies are required to assess the role ofsymbiotic bacteria (if any) in the biosynthesis of thebioactive compounds of C. candelabrum . It is also

Fig. 12. Explant growth rates fed with bacteria and algae.


Kori Mulholland

Kori Mulholland

Kori Mulholland

Kori Mulholland

Kori Mulholland

Kori Mulholland

important to establish the relationships between bacteriapopulations and between bacteria and sponge cells, sincecompetition for resources might determine the produc-tion of the toxic metabolites with potential applications.In this preliminary study, we have fixed some of the

favourable conditions for enhancing explant survivaland growth of a Mediterranean bacteriosponge, andshowed that antibiotics do not affect the symbioticbacteria. Temperature and food seem to be decisive forexplants survival and growth. Thus, we propose summertemperatures and continuous algae supply in a closedaquarium system (bioreactor) to improve culture yield.

Acknowledgements

Our special thanks go to Rene Wijffels and his teamfor giving us the opportunity to participate at theMarine Biotechnology Conference in Matalascanas.We also thank X. Turon for helping with the electronmicroscope, E. Cebrian and R. Mart for field work andE. Adell and M. Bardaj for laboratory work. This studywas partially funded by grants INTERREG III AFrance/Spain 2000!/2006 from the EU and GENECOREN 2001-2312-CO3-01/MAR from the CICYT (Span-ish Government).

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22.


Kori Mulholland

Long-term culture of sponge explants: conditions enhancing survival and growth, and assessment of bioactivityIntroductionMaterial and methodsSponge explant formationMorphological and ultrastructural changes during explant formation (pre-explants)Pre-explant survivalExplants growthBioactivityStatistical methods

ResultsMorphological and ultrastructural changes during formation of explantsPre-explant survivalExplant growthBioactivity

DiscussionAcknowledgementsReferences

long term culture of sponge explants

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