Aquaculture Trials for the Production of Biologically Active Metabolites in the New Zealand Sponge Mycale Hentscheli (Demospongiae: Poecilosclerida)

Aquaculture 250 (2005) 256–269 www.elsevier.com/locate/aqua-online

Aquaculture trials for the production of biologically active metabolites in the New Zealand sponge Mycale hentscheli (Demospongiae: Poecilosclerida)

Michael J. Pagea,*, Peter T. Northcoteb, Victoria L. Webbc, Steven Mackeyb, Sean J. Handleya

aNational Institute of Water and Atmospheric Research Ltd. (NIWA), P.O. Box 893 Nelson, New Zealand bSchool of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box 600 Wellington, New Zealand cNational Institute of Water and Atmospheric Research Ltd. (NIWA), P.O. Box 14 901 Kilbirnie, Wellington, New Zealand Received 11 February 2004; received in revised form 26 April 2005; accepted 29 April 2005

Abstract

Genetically identical explants of the New Zealand marine sponge Mycale hentscheli were cultured in two different habitats at 7 m depth using subsurface mesh arrays to determine the effect of environment on survival, growth and biosynthesis of the biologically active secondary metabolites, mycalamide A, pateamine and peloruside A. Two 27 cm3 explants were excised from each of 10 wild donor sponges at Capsize Point, Pelorus Sound. One explant from each donor sponge was grown in arrays next to the wild donor sponge population for 250 days, while the second explant from each donor was translocated and grown at 7 m at Mahanga Bay, Wellington Harbour for 214 days. Growth rate measured by surface area and survival of explants was monitored in situ using a digital video camera. Explant surface area correlated positively with blotted wet weight (r2 =0.93). The mean concentration of each of the three compounds was determined analytically from 1H NMR spectra of replicate 30-g samples from each of 10 donor sponges at the start of the trial, and compared to mean concentrations in donors and explants at the end of the trial. Phenomenal growth rates were achieved for explants both at Capsize Point (3365F812%, 95% CI) and Mahanga Bay (2749F1136%, 95% CI). Explant survival was high: 100% at Capsize Point and 90% at Mahanga Bay. Wild donor sponges regressed in size and experienced 40% mortality by the end of the trial. Mycalamide A was present in relatively high concentrations in donors and explants throughout the trial. Pateamine was more variable among individuals and was present at lower concentrations in Capsize explants at the end of the trial. Peloruside A was highly variable among wild donor sponges. Only 50% of donors contained detectable concentrations of peloruside A, and only those sponges and their explants grown in their native environment at Capsize Point continued to biosynthesise peloruside A. No explants at Mahanga Bay contained peloruside A after 214 days in culture, indicating the production of this compound may be environmentally controlled. Our results demonstrate that in-sea aquaculture of M. hentscheli is a viable method for supply of mycalamide A, pateamine and peloruside A, and that

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

0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.04.069 M.J. Page et al. / Aquaculture 250 (2005) 256–269 257 environmental conditions may be critical for the biosynthesis of peloruside A. Furthermore, results show the potential to establish cultivars to maximize peloruside A yield. D 2005 Elsevier B.V. All rights reserved.

Keywords: Mycale hentscheli; Sponge; Secondary metabolites; Culture; Drug supply

1. Introduction gests that production of bioactive metabolites may be a viable option in the future for industrial supply Secondary metabolites from marine organisms are (Pomponi, 1999). However, culture optimisation is a rich source of novel chemical compounds (Faulkner, still only in its primary stages and further experimen- 2001), many of which are biologically active. Since tation is needed to improve in vitro conditions specific the first pharmaceutically important compounds were to sponge cells (Rinkevich, 1999). isolated from a sponge (Bergmann and Feeney, 1951), In-sea and land-based aquaculture systems remain the marine environment has become a major resource the most cost-effective medium-term production for collection and screening of organisms for discov- methods for supply of biologically active compounds ery of new drugs (Blunt et al., 2004; Mendola, 2003). from marine organisms. Favourable growth rates have Of all marine fauna, sponges are considered to be the been achieved in experimental closed systems (e.g., best source of novel bioactive compounds (Ireland et Belarbi et al., 2003; Duckworth et al., 2003; Osinga et al., 1993; Munro et al., 1999). Many sponges have al., 1999, 2003) that enable greater control of envi- been shown to contain compounds with promising ronmental conditions, but these systems have yet to be antiviral (Bergmann and Burke, 1955; Perry et al., trailed on a commercial scale. In contrast, in-sea 1988), anti-microbial (Cariello et al., 1982; Green et systems for supply of anticancer compounds can be al., 1985; McCaffrey and Endean, 1985), anti-inflam- economically viable at a larger scale. For instance, matory (Potts et al., 1992; Schmidt and Faulkner, production of Bryostatin 1, from the bryozoan Bugula 1996) and anti-tumour (Gunasekera et al., 1990; neritina, and ectinascidin 743, from the ascidian Ecti- Litaudon et al., 1997) activity. nascidia turbinata, have proved cost-effective (Men- Supply of natural compounds from wild sponges dola, 2003), and aquaculture trials on biologically can be a major factor limiting pharmaceutical devel- active sponges have also demonstrated relatively opment (e.g., Dumdei et al., 1998; Pomponi, 2001; high growth rates and retention of biological activity Sennet, 2001). Target compounds often occur in the in culture (e.g., Battershill and Page, 1996; Duck- sponge in trace amounts and continuous supply from worth and Battershill, 2003a; Mu¨ller et al., 1999; wild populations cannot provide enough for pre-clin- Munro et al., 1999). ical studies (Dumdei et al., 1998). Of the supply The New Zealand endemic marine sponge Mycale options available, wild harvest is often not ecologi- hentscheli Bergquist and Fromont (Demospongiae: cally sustainable (Battershill and Page, 1996). Chem- Poecilosclerida: Mycalidae) is a soft fleshy sponge ical synthesis is an alternative; however, the molecules of massive to encrusting morphology that inhabits are often complex and their multi-step synthesis is subtidal reefs from 5 to 30 m. This sponge contains seldom amenable to industrial processes (Sennet, three classes of biologically active compounds with 2001). For compounds localized in sponge microbial pharmaceutical potential: mycalamide A, pateamine associates (e.g., Bewley et al., 1996; Unson et al., and peloruside A. The mycalamides (A–D) were the 1994), isolation and culture in marine bioreactors first group of novel cytotoxic compounds to be iso- could lead to an industrially feasible supply option lated from M. hentscheli. They have been reported to (Pomponi, 2001). Nevertheless, a significant propor- be potent protein synthesis inhibitors and have recent- tion of biologically active compounds are localized in ly been found to cause apoptosis (Hood et al., 2001). the host sponge cells themselves (e.g., Salomon et al., Pateamine, a biochemically unrelated macrolide iso- 2001; Thompson et al., 1983; Uriz et al., 1996). In lated from M. hentscheli, is also a potent eukaryotic vitro culture of cell lines of bioactive sponges sug- cytotoxin active in the sub-nanomolar range (North- 258 M.J. Page et al. / Aquaculture 250 (2005) 256–269 cote et al., 1991). It has subsequently been found to al., 2004) and is in progress for peloruside A (Liu and have immunosuppressive and apoptotic properties Zhou, 2004); however, immediate sustainable supply (Hood et al., 2001; Romo et al., 1998). The most is critical for continuing research. In the short- to recently discovered and potentially valuable cytotoxic medium-term aquaculture is the only reliable method metabolite isolated is peloruside A. This compound of supply. was isolated from specimens of M. hentscheli collect- The primary objective of our study was to deter- ed from Pelorus Sound on the north coast of the South mine the aquaculture potential for supply of biologi- Island (West et al., 2000). Peloruside A is structurally cally active target compounds from M. hentscheli by: unrelated to either the mycalamides or pateamine and (1) quantifying survival and growth rates of explants yet is also a potent eukaryotic cytotoxin. Recently, we in different environmental conditions and (2) deter- have found that peloruside A is a microtubule stabi- mining the influence of environment and explant liser with potency and mode of action similar to the identity on metabolite biosynthesis of M. hentscheli naturally derived anticancer drug TaxolR. Pre-clinical sponges in aquaculture. trials are currently being conducted on peloruside A for development as a new anti-tumour drug. All three compounds derived from M. hentscheli have very 2. Methods similar profiles of toxicity to a range of organisms and cell lines, and are therefore impossible to distin- 2.1. Site descriptions guish using biological assay-based analytical techniques. However, in a recent study, we developed an Capsize Point is situated in Pelorus Sound at the analytical method using 1H NMR to quantify the northern end of the South Island of New Zealand (Fig. individual compounds in M. hentscheli sponges 1). It is a 40 km long fiord-like estuary fed by the (Page et al., 2005). Pelorus River, and discharges into greater Cook Strait. Chemical synthesis has been completed for myca- The hydrographic environment in the main channel at lamide A (Trost et al., 2004), pateamine (Pattenden et Capsize Point is influenced by two major physical

Fig. 1. Map of New Zealand showing location of aquaculture sites. M.J. Page et al. / Aquaculture 250 (2005) 256–269 259 factors: (1) nitrogen-depleted phytoplankton-enriched placed adjacent (approximately 30 cm) to each Pelorus River water, which characterizes the near sponge. Two 27 cm3 size pieces were removed as surface low salinity field, and (2) tidally induced explants for culture from each of the 10 sponges. bottom intrusions of oceanic water (Gibbs et al., Each explant was made so as to retain (on one of its 2002). There is an almost continuous salinity or ther- sides) a section of the exopinacoderm of the donor mally induced density stratification, or both at be- sponge. This method of making explants has been tween 7 and 10 m. Tidally induced flows at the found to optimise healing and survival of explants. density discontinuity can range from 10 to 70 cm Three additional replicate pieces (~30 g) for chemical sÀ 1 (Gibbs et al., 1991), causing a shear zone where analysis were chosen randomly from the centre of high turbulence occurs above and below the pycno- each donor sponge, so as to contain representative cline. The upper and lower water columns are often proportions of cortex and exopinacoderm. The sam- decoupled so that nitrogen generated from the sedi- ples were frozen and transported to the laboratory to ments may not be available to the upper water column determine the initial concentration of each of three and phytoplankton settling from the upper water col- compounds: mycalamide A, pateamine and peloruside umn may not reach the sediments directly below. A within each donor (Fig. 2). Summer thermal stratification coincides with the bot- On removal, explants for culture were placed into tom of the euphotic zone and, consequently, phyto- separate 600Â300 mm seawater filled plastic bags plankton biomass accumulates at the thermocline for transportation to aquaculture sites. On 28 August, producing a mid-water chlorophyll maximum at this 2001 at Capsize Point (Capsize explants), one ex- site. plant was removed from each donor (1–10) and The second site, Mahanga Bay, is situated on the inserted in situ into bottom-weighted mesh arrays southwest side of Wellington Harbour at the southern suspended by a sub-surface buoy, see Duckworth end of the North Island of New Zealand, and is and Battershill, (2003b, Fig. 2). Explants were cul- separated from Pelorus Sound by Cook Strait (dis- tured at 7 m adjacent to donor sponges; this depth tance approximately 40 km, see Fig. 1). Wellington harbour water is generally isothermal: strong winds, common to the region and keep the water column well mixed. However, higher temperatures during summer, calm weather (December to March) and intrusions of freshwater from the Hutt River can lead to weak Replicate chemistry stratification (Heath, 1977). The tidal current at samples Mahanga Bay is comparatively slow (average ~3.8 Mahanga Bay cm sÀ 1) (C. Woods, unpublished data); approximately 5% of the harbour water is renewed with each tide Explants giving any given body of water a residence time of at least 10 days. Spatial and temporal patterns of phytoplankton productivity within the harbour are not well Capsize Point studied; however, plankton is likely to be well mixed 10 donor sponges Capsize Point throughout euphotic zone and blooms are known to occur during spring and autumn (Morrison Cassie, 1960). Capsize Point

0 8 2.2. Harvest of explants and experimental design Months Months

Ten large M. hentscheli sponges from a wild pop- Fig. 2. Schematic diagram of the experimental design; squares represent three replicate chemistry samples removed from 10 ulation at Capsize Point (Fig. 1), Pelorus Sound donor sponges at the start of the experiment, and from each of 10 (41805.12VS, 173855.96VE) were selected as donor explants suspended vertically in mesh at Mahanga Bay and at sponges and were tagged with weighted plastic labels Capsize Point. 260 M.J. Page et al. / Aquaculture 250 (2005) 256–269 was representative of the mean depth of donor 2.3. Measurement of survival and growth sponges at Capsize Point. The second explant was removed 1 month later (27 September 2001) from Explant survival and growth was monitored each of the same donors (1–10). These explants were monthly in situ using an underwater Hi-8 digital transported to Mahanga Bay, Wellington Harbour video camera. Total increase in size was determined (41817.07VE, 174850VS), held overnight in an open by comparing the estimated wet weight of explants at aquarium system at 12 8C and seeded into arrays at 7 the start of the trial with wet weight of explants m the following morning (Mahanga explants). The harvested from arrays at the end of the trial. A linear initial weight of explants was estimated by measur- relationship between explant surface area (cm2) and ing the standard wet weight of fifty representative 27 wet weight was used to accurately estimate growth in cm3 explants left to drain on a paper towel for 5 min. the field (Fig. 3). Surface area data for small explants Duckworth et al. (1997) demonstrated that blotted was derived from 26 sponges of known wet weight wet weight was a consistently reliable method for strung on a monofilament line and, for large sponges, estimating biomass of a range of sponges, one of from standard wet weights of explants harvested from which (Raspalia agminata) has similar morphology the aquaculture arrays at the end of the experiment. to M. hentscheli. Size and mortality of explants was The underwater video recorded explants rotated monitored monthly. Capsize explants were grown for through 3608 against a 1 cm background grid fixed 250 days and Mahanga explants for 214 days. to a frame. In the laboratory, images were captured Explants were harvested from both sites on the from video footage using Sony DV image capture first week of May 2002, weighed and three ~30 g software (Sony Inc.) and analysed using SigmaScan replicate samples taken from each for chemical anal- Pro image analysis software (SPSS Inc.) to calculate ysis. At the same time, three replicate samples were mean surface area from a front and side image of each removed from surviving donor sponges for chemical explant. Similar methods have been used to monitor comparison. Daily water temperatures were recorded irregularly shaped organisms (Handley et al., 2003a; at 7 m depth at each site using HOBO loggers Lobsiger and Manuel, 1999). Growth is expressed as (Onset Computer Corp.). percent of original explant size.

1200

1100

1000 wt =1.03sa - 1.18

900 = 0.93 n = 45 800

700

600

500

Wet weight (g) 400

300

200

100

0 0 100 200 300 400 500 600 700 800 900 1000 Mean surface area (cm2)

Fig. 3. Linear regression of mean sponge surface area measured from video images of sponges versus wet weight. Circles represent data from 26 sponges strung on a monofilament line and squares from final measurements taken from explants growing in arrays at Mahanga Bay and Capsize Point at the end of the experiment. M.J. Page et al. / Aquaculture 250 (2005) 256–269 261

Following removal of explants and chemistry orthogonal, only data for explants and donors that samples, the initial size of each donor sponge was survived to the end of the trial were compared. recorded, and survival and growth monitored monthly in situ using a clear plastic ruler to measure sponge length, width and height with to the nearest 3. Results 5 mm. This information was used for two purposes: principally, to relocate donor sponges for chemical 3.1. Growth re-sampling at the end of the trial and, secondly, to determine any relative changes in size and mortality Explants at both sites exhibited very rapid growth that occurred among donors. Measurements were rates. Capsize Point explants grew from an average summed for donor sponges that fragmented during estimated wet weight of 16.3 g to 548.6 g (3365%) in the study. 250 days. Mahanga explants grew from 16.3 g to 448.8 g (2749%) in 214 days (Table 1). The wet 2.4. Chemical analysis weight of explants estimated from the relationship with surface area (Fig. 3) was not significantly differ- A total of 103 frozen samples were dried, weighed ent (T18 =0.13, P =0.89) to the actual weight sampled and extracted in MeOH following methods described from arrays at the end of the trial (Table 1). A com- by Page et al. (2005). Ten replications of our analyt- parison of normalized daily incremental growth rates ical protocol on a single homogenous dry sponge shows that explants grew at approximately the same sample gave reproducibility of F5%. The concentra- rate at each site (Table 1). The growth of explants was tion (Ag/g dry weight) of mycalamide A, pateamine more variable at Mahanga Bay than at Capsize Point. and peloruside A was determined from analysis of 1H Explant growth increased at a relatively constant NMR spectra for each of three replicate samples taken rate over the first 5 months (Fig. 4). Mahanga explants initially from donor sponges, and from surviving regressed in size during February, whereas Pelorus Mahanga and Capsize explants and donor sponges explant growth rates continued to increase over the at the end of the culture trial (Fig. 2). period of the study. Peak water temperatures at both sites occurred in early February. Maximum growth 2.5. Data analysis rates occurred in March and decreased over the last month of the trial. A two-factor mixed model analysis of variance Donor sponges experienced negative growth after (ANOVA) was used to compare concentrations of removal of explants and chemistry samples (Fig. 5). mycalamide A, pateamine and peloruside A among All but two sponges regressed in size during the first treatments (donor sponges, Capsize explants and month of the trial and of the six donor sponges that Mahanga explants), and between sponges within treat- survived only two (D1 and D8) grew. Two donors (D1 ments after culture. Repeated measures ANOVA were and D3) fragmented during the course of the study. used to compare compound concentrations between Although not quantified, surface fouling was ob- donor sponges at the start of the experiment, and served on explants at both sites. Hydrozoans, bryozo- explants at each site and donor sponges after culture. ans and ascidians were predominant fouling taxa on Data were transformed prior to analysis if variances explants at Mahanga Bay, whereas hydrozoans and proved heterogeneous. To ensure tests were fully filamentous red algae dominated surface fouling on

Table 1 Mean and 95% confidence interval of the estimated wet weight of explants calculated from linear regression of surface area, the actual wet weight recovered, percent increase in wet weight and daily incremental growth rate for each site Site Mean estimated Mean actual Mean % increase in Mean daily incremental N wet weight (g) wet weight (g) wet weight (g) wet weight (g) Mahanga Bay 525.9F221.2 448.1F185.1 2749F1136 2.0F0.8 9 Capsize Point 486.4F77.2 548.6F132.5 3365F812.6 2.1F0.5 10 262 M.J. Page et al. / Aquaculture 250 (2005) 256–269

5000 20

Capsize Point Mahanga Bay 4000 Capsize Point °C 18

95% C.I.) Mahanga Bay °C ±

3000 C ° 16

2000 erature 14 p

1000 Tem

12 0

% Change in surface area (cm 10 Aug01 Oct01 Dec01 Feb02 Apr02 Jun02

Fig. 4. Growth of explants from 10 donor sponges at Capsize Point and Mahanga Bay. Growth is represented as a percent of the original explant size. The mean daily temperature at each site is shown on the same graph. explants at Capsize Point. Generally, fouling was explants survived after 250 and 214 days, respec- observed during summer months from December tively. All wild donor sponges initially survived through to February and appeared heaviest on excision of explants and chemistry samples. Howev- explants at Mahanga Bay in February. The degree of er, four (D2, D4, D6 and D7) died during summer fouling diminished in the following 2 months as March 2002 (Fig. 5). Three of the four that died fouling species either died off and/or were overgrown showed evidence of disease and necrosis in the by explants. previous month.

3.2. Survival 3.3. Chemistry

Survival of explants seeded into arrays was high: Mycalamide A occurred in relatively high concen- 100% of Capsize Point and 90% of Mahanga trations in donor sponges and explants throughout the

Fig. 5. Growth and survival of the 10 wild donor sponges at Capsize Point. M.J. Page et al. / Aquaculture 250 (2005) 256–269 263 trial (Fig. 6). Concentrations varied significantly (Fig. 6). These differences were not, however, con- among individuals, but were not consistent over sistent among all sponges within treatments (i.e. the time for donor sponges (Table 2), and were not sig- timeÂsponge interaction term was significant, Table nificantly different among treatments (donor sponges, 2). There was high variability among individuals Capsize explants and Mahanga explants) at the end of within treatments, but this was not consistent be- the trial (Table 3). tween treatments, nor was there any significant dif- Pateamine was more variable among donors and ference among treatments at the end of the trial explants than mycalamide A (Fig. 6). Significant (Table 3). variation occurred among individuals (Table 2); how- Peloruside A was highly variable among indivi- ever, pateamine concentrations in Capsize explants at duals and differences among individuals were not the end of the trial in May were significantly lower consistent over time (Table 2). Peloruside A was than in donors at the start (Table 2). Although time present in 50% (5/10) of wild donor sponges (D1, was not a significant factor in the ANOVA for D2, D3, D6 and D8) and was retained only by those pateamine in donors and Mahanga explants, concen- donors (with the exception D2, which died), and their trations were generally lower at the end of the trial explants at Capsize Point at similar concentrations

Mycalamide A Pateamine Peloruside A 600 300 Donor 800 sponges 600 400 200 400 200 100 200

0 0 0 012345678910* * * * 12345* * * 6 789* 10 12345* **610 789 *

600 800 300

Capsize pt 600

g/g ± 95% C.I. 400 200

explants µ 400

200 100 200

0 0 0

Concentration 023457891 61012345610 789 12345610 7 89 600 800 300 Mahanga explants 600 400 200 400 200 100 200

0 0 0 023457891 * 61012345* 61012345789 789 * 610

Sponge

Time 0 Time 8 * Dead

Fig. 6. The mean concentration of three compounds: mycalamide A, pateamine and peloruside A of three replicate chemistry samples taken from 10 donor sponges at the start of the aquaculture study (T0) compared to concentrations in explants and donor after 8 months (T8) of growth (points plotted without 95% confidence intervals represent either zero values or points where only one replicate chemistry sample was taken from small explants). 264 M.J. Page et al. / Aquaculture 250 (2005) 256–269

Table 2 Repeated measures analysis of variance table comparing initial concentrations of mycalamide A, pateamine and peloruside A in donor sponges at the start of the aquaculture trial with concentrations in donors, Capsize and Mahanga explants harvested at the end of the trial in May 2002 Factor df Mycalamide A Pateamine Peloruside A MS F MS F MS F (a) Donor (start) vs. donor (end) Sponge 5 29,179.4 6.6*** 8.65 54.1*** 25,761.7 27.2*** Time 1 26,039.2 1.5 0.87 1.9 45,582.3 5.2 SpongeÂtime 5 17,692.1 4.0** 0.46 2.9* 8711.1 9.4*** Error 24 4426.0 0.16 945.8 Total (adjusted) 35

(b) Donor (start) vs. explant (Capsize Point) Sponge 9 25,133.4 4.9*** 5.80 60.0*** 4.56 48.5*** Time 1 10,650.7 2.6 1.43 11.8** 0.15 1.3 SpongeÂtime 9 4150.0 0.8 0.12 1.3 0.14 1.5 Error 40 5170.1 0.01 0.09 Total (adjusted) 59

(c) Donor (start) vs. explant (Mahanga) Spongea 7 14,122.9 2.65* 99,576.3 9.7*** 1462 4.1** Time 1 10,111.1 0.74 283,837.9 5.6 6147.2 4.2 SpongeÂtime 7 8316.4 0.18 50,857.9 4.9*** 1462 4.1** Error 32 5327.6 10,248.1 358.4 Total (adjusted) 47 Data was log transformed to meet assumptions. a Mahanga explant 3 was too small for replicate chemical analysis and therefore excluded from the ANOVA. * P b0.05. ** P b0.01. *** P b0.001. after 250 days growth in culture arrays (Fig 6). 4. Discussion Whereas, Mahanga explants from the same wild donors had no detectable peloruside A after 215 4.1. Growth days growth in culture arrays (Fig. 6). M. hentscheli explants exhibited phenomenal growth rates of 4688% yearÀ 1 at Mahanga Bay and À 1 Table 3 4912% year at Capsize Point. These growth rates A two-factor mixed model analysis of variance comparing concen- compare very favourably with aquaculture studies on trations among treatments (donor sponges, Capsize explants and other species (Table 4). For instance, bath-sponges Mahanga explants) and within sponges harvested in May 2002 (Dictyoceratida) that invest in a dense spongin (colla- Factor df Mycalamide A Pateamine Peloruside A gen) skeleton appear to have much slower growth MS F MS F MS F rates (73–150%) than their non-dictyoceratid counter- Treatment 2 30,932.0 2.8 1.1 0.9 50,979.6 4.2 parts (Handley et al., 2003b). A possible explanation Sponge 4 12,857.6 2.2 8.9 34.9* 36,399.8 62.5* is that dictyoceratid sponges emphasise a protein TÂS 8 11,022.6 1.9 1.2 4.8* 12,200.3 20.9* (spongin) skeleton that may require more energy to Error 30 5755.6 0.3 582.8 produce, and therefore take longer to grow (Kelly et Total 44 (adjusted) al., 2004). Much higher growth rates have been GLM ANOVA was used to analyse data. To meet assumptions, achieved for fleshy non-dictyoceratid species. For pateamine data was log transformed. instance, Duckworth (2000) measured growth rates * P b0.001. of 950–740% over 6 months for Latrunculia brevis M.J. Page et al. / Aquaculture 250 (2005) 256–269 265

Table 4 Comparison of growth rates, normalized as mean percentage increase per year, between experimentally farmed sponges Species Order % Growth Study Hippospongia and Spongia spp. Dictyoceratida ~100 (Moore, 1910) Hippospongia lachne Dictyoceratida ~100 (Crawshay, 1939) Spongia argaricina Dictyoceratida 90 (Verdenal and Vacelet, 1990) Spongia officinalis Dictyoceratida 150 (Verdenal and Vacelet, 1990) Spongia (Heterofibria) officinalis Dictyoceratida 73 (Kelly et al., 2004) Geodia cydonium Choristida 380 (Mu¨ller et al., 1999) Lissodendoryx sp. Poecilosclerida 5000 (Battershill and Page, 1996) Latrunculia wellingtonensis Hadromerida 700 (Duckworth and Battershill, 2003b) Polymastia croceus Hadromerida 360 (Duckworth and Battershill, 2003b) Mycale hentscheli Poecilosclerida 4912 This study Table adapted from Duckworth and Battershill (2003a). Data represent highest % mean growth obtained in each study irrespective of method used. and Polymastia croceus explants, respectively, and up seeded explants, initial energy may have been directed 5000% growth was recorded for individual explants of towards healing of cut surfaces and reorganization of the deepwater sponge species Lissodendoryx sp. (Bat- damaged canal systems (Mu¨ller et al., 1999), and their tershill and Page, 1996). Ecologically, M. hentscheli subsequent growth influenced by higher temperatures appears to have an r-selected life history favouring and availability of food in spring (Barthel, 1986; Fell rapid growth and short life span. Fast-growing fleshy and Lewandrowski, 1981; Turon et al., 1998). species such as M. hentscheli that are adapted for opportunistic colonization of new substrates are 4.2. Survival most likely to be ideal candidates for translocation and introduction into existing aquaculture systems. Explant survival, sustained growth and continued The rapid growth of M. hentscheli explants suspended biosynthesis of desired target compounds is critical in arrays may also be correlated with increased water for guaranteed supply of biologically active com- flow rate and therefore food, away from benthic- pounds from sponges in aquaculture (Munro et al., boundary layer effects (Freche´tte and Bourget, 1985; 1999). The success of sponge aquaculture is highly Leichter and Witman, 1997; Palumbi, 1984). Further- dependent on seasonality with respect to initiating more, explants introduced into mesh arrays may have cultures (Duckworth et al., 2004). In our study, seed- initially experienced low interspecific competition for ing M. hentscheli explants in winter when water food and space, enhancing their chance for survival temperatures were low ensured high survival (90– and growth. 100%). Low mortality has been documented for a Explant growth rates were not significantly differ- number of other sponge species in aquaculture ent between Capsize Point and Mahanga Bay, which (Duckworth, 2000; Kelly et al., 2004; Verdenal and shows that both sites had favourable environmental Vacelet, 1990), and is considered to be positively conditions for rapid growth. A regression in size of correlated with low water temperature, reducing met- explants at Mahanga Bay during February was coin- abolic stress, promoting rapid pinacoderm healing cident to a 2 8C drop in water temperature because of and decreasing the probability of infection (Burlando an incursion of cold oceanic water into Wellington et al., 1992; Vacelet et al., 1994). High survival of M. Harbour from Cook Strait during a severe storm event hentscheli explants can also be attributed to the mor- (Carter et al., 2002). Regression in sponge size in phology of this species. Duckworth and Battershill relation to temperature down-shocks has been (2003b) concluded that dmeshT arrays were the least reported for commercial sponges in the Gulf of Mex- invasive method for farming soft fleshy sponges such ico (Storr, 1964). M. hentscheli growth rates were as Latrunculia wellingtonensis and M. hentscheli. relatively slow during winter, but increased as water They found these species have the ability to grow temperatures rose in spring and summer. For newly rapidly through mesh in aquaculture arrays, effective- 266 M.J. Page et al. / Aquaculture 250 (2005) 256–269 ly maximizing the surface area available for feeding Survival and growth rates of M. hentscheli explants and respiration. High survival of M. hentscheli trans- at Mahanga Bay were high and no different from located explants further demonstrates the suitability of genetically identical explants at Capsize Point; this this species to commercial-scale aquaculture produc- result demonstrates that differences in peloruside A tion. Explants can be selected for chemical attributes, were unlikely to be correlated with stress or lack of translocated and seeded on commercial long-line sys- food. However, qualitative differences in fouling tems similar to those used for mussel culture (Jenkins organisms in our study may explain changes in com- et al., 1985). Furthermore, sites can be chosen for pound profiles between sites. Mycalamide A, patea- optimal environmental conditions for growth and bio- mine and peloruside A are complex, highly cytotoxic synthesis of target metabolites. compounds that almost certainly play a role in chem- While survival of wild donor sponges was high ical defence against surface fouling, competition and after initial removal of explants and chemistry sam- predation. They may have multiple roles in the organ- ples, most regressed in size or exhibited poor growth, ism as suggested by Becerro et al. (1997) for the and 4 of 10 died during summer. Larvae in M. sponge Crambe crambe, and therefore vary according hentscheli have been observed at Capsize Point during to their ecological role (Fagerstro¨m et al., 1987). The spring (November–December). As sponge growth can different compounds in M. hentscheli may, for exam- be negatively correlated to the onset of reproductive ple, be active against different fouling organisms and activity (Barthel, 1986; Elvin, 1979), it is possible that so absent where there is no target organism present. energy directed towards reproduction in wild donor This hypothesis further explains the variation mea- sponges was used for somatic growth in explants. For sured among donor sponges; compounds may be instance, Reiswig (1973) discovered reproductive expressed or produced if sponges are challenged by competence in a tropical species of Mycale was competitors or predators. size-dependent, small sponges grew up to seven An alternative explanation for chemical variability times their original size during 1 year, but this growth in explants and among donors is that compounds are rate decreased to 60% with the onset of reproductive localized in and produced by symbiotic microorgan- competence. Further ecological studies are needed to isms (e.g., Faulkner et al., 1999; Unson and Faulkner, investigate our hypothesis as no undamaged sponges 1993). M. hentscheli is known to harbour a diverse were monitored for growth, and wild donor sponges population of microbial associates that varies among and explants in our study were not examined for individuals and between geographic locations (Ander- reproductive condition. son et al., 2004). Changes in environmental conditions (including transportation in plastic bags and overnight 4.3. Chemistry storage in aquaria) may have altered microbial populations and therefore compounds, in explants moved Wild parent M. hentscheli sponges and explants to Mahanga Bay. Furthermore, Mahanga explants cultured in their native environment at Capsize Point were excised from donors for culture a month later retained peloruside A, whereas genetically identical than Pelorus explants. The physiological state of explants translocated to Mahanga Bay did not. This donor sponges may have changed in that time, influ- result suggests that environmental conditions in dif- encing our results. However, previous pilot studies ferent habitats may in some way affect the biosynthe- (unpublished data) where a single M. hentscheli indi- sis of peloruside A and that compound biosynthesis vidual has been repeatedly sampled from for chemis- may not necessarily be genetically determined. Chem- try have shown consistency in chemical composition ical variability in relation to changes in environment over several years. has also been demonstrated for genetically identical fragments of the sponge Rhopaloeides odorabile (Thompson et al., 1987). They concluded that quan- 5. Conclusions titative and qualitative changes in diterpenes were an adaptive response to surface fouling of algae or light- Our research demonstrates in-sea aquaculture of M. induced stress, as evidenced by poor growth rates. hentscheli is viable for short- to medium-term supply M.J. Page et al. / Aquaculture 250 (2005) 256–269 267 of target compounds for drug development. Our videos. Many thanks to Drs. Michelle Kelly (NIWA, results further support the hypothesis that environ- Auckland), Russell Cole (NIWA Nelson), Chris Bat- mental conditions can be critical for the biosynthesis tershill (Australian Institute of Marine Sciences, of compounds. We suggest that sponges such as M. Townsville) and to the anonymous reviewers, for hentscheli, which are opportunistic occupiers of sharing their ideas and commenting on our manu- space, can have phenomenal growth rates and have script. This research was funded by Foundation for the potential to yield massive increases in biomass Science Research and Technology (FRST) research over a single growing season. Our trial shows that the contracts CO1809 and COIX0207 with NIWA. target compound, peloruside A, is found only in a small proportion of wild sponges and that only explants from these sponges can be cultured in their References native environment to yield the compound. To our knowledge, few studies have used analytical techni- Anderson, S.A., Maas, E.W., Page, M.J., Webb, V.L., 2004. Mo- ques to quantify the effect of environment on biosyn- lecular approaches to examine the spatial and temporal variabil- thesis of target compounds from sponges in ity in bacterial communities associated with the New Zealand marine sponge Mycale (Carmia) hentscheli. International Soci- aquaculture. Furthermore, our results suggest that, in ety for Microbial Ecology (ISME), Cancun, Mexico. order to maximize target compound yield in aquacul- Barthel, D., 1986. On the ecophysiology of the sponge Halichon- ture, we can assay wild sponges and select for dria panicea in Kiel Bight: I. Substrate specificity, growth and explants to establish peloruside A-containing culti- reproduction. Mar. Ecol., Prog. Ser. 32, 291–298. vars. Preliminary results from a bulk culture trial Battershill, C.N., Page, M.J., 1996. Sponge aquaculture for drug production. Aquac. Update, 5–6. support our experimental findings; we were able to Becerro, M.A., Turon, X., Uriz, M.J., 1997. Multiple functions for pre-select ~7 kg of cultured explants from a line secondary metabolites in encrusting marine invertebrates. containing a total of ~34 kg of explants and extract J. Chem. Ecol. 23, 1527–1547. ~50 mg of peloruside A for a predicted yield of ~40 Belarbi, E.H., Ramı´rez Domıńguez, M., Ceroń Garcıá, M.C., mg. 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