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 a National Institute of Water and Atmospheric Research Ltd. (NIWA), P.O. Box 893 Nelson, New Zealand School of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box 600 Wellington, New Zealand c National Institute of Water and Atmospheric Research Ltd. (NIWA), P.O. Box 14 901 Kilbirnie, Wellington, New Zealand b

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 (r 2 = 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 (3365 F 812%, 95% CI) and Mahanga Bay (2749 F 1136%, 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

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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 Secondary metabolites from marine organisms are a rich source of novel chemical compounds (Faulkner, 2001), many of which are biologically active. Since the first pharmaceutically important compounds were isolated from a sponge (Bergmann and Feeney, 1951), the marine environment has become a major resource for collection and screening of organisms for discovery of new drugs (Blunt et al., 2004; Mendola, 2003). Of all marine fauna, sponges are considered to be the best source of novel bioactive compounds (Ireland et al., 1993; Munro et al., 1999). Many sponges have been shown to contain compounds with promising antiviral (Bergmann and Burke, 1955; Perry et al., 1988), anti-microbial (Cariello et al., 1982; Green et al., 1985; McCaffrey and Endean, 1985), anti-inflammatory (Potts et al., 1992; Schmidt and Faulkner, 1996) and anti-tumour (Gunasekera et al., 1990; Litaudon et al., 1997) activity. Supply of natural compounds from wild sponges can be a major factor limiting pharmaceutical development (e.g., Dumdei et al., 1998; Pomponi, 2001; Sennet, 2001). Target compounds often occur in the sponge in trace amounts and continuous supply from wild populations cannot provide enough for pre-clinical studies (Dumdei et al., 1998). Of the supply options available, wild harvest is often not ecologically sustainable (Battershill and Page, 1996). Chemical synthesis is an alternative; however, the molecules are often complex and their multi-step synthesis is seldom amenable to industrial processes (Sennet, 2001). For compounds localized in sponge microbial associates (e.g., Bewley et al., 1996; Unson et al., 1994), isolation and culture in marine bioreactors could lead to an industrially feasible supply option (Pomponi, 2001). Nevertheless, a significant proportion of biologically active compounds are localized in the host sponge cells themselves (e.g., Salomon et al., 2001; Thompson et al., 1983; Uriz et al., 1996). In vitro culture of cell lines of bioactive sponges sug-

gests that production of bioactive metabolites may be a viable option in the future for industrial supply (Pomponi, 1999). However, culture optimisation is still only in its primary stages and further experimentation is needed to improve in vitro conditions specific to sponge cells (Rinkevich, 1999). In-sea and land-based aquaculture systems remain the most cost-effective medium-term production methods for supply of biologically active compounds from marine organisms. Favourable growth rates have been achieved in experimental closed systems (e.g., Belarbi et al., 2003; Duckworth et al., 2003; Osinga et al., 1999, 2003) that enable greater control of environmental conditions, but these systems have yet to be trailed on a commercial scale. In contrast, in-sea systems for supply of anticancer compounds can be economically viable at a larger scale. For instance, production of Bryostatin 1, from the bryozoan Bugula neritina, and ectinascidin 743, from the ascidian Ectinascidia turbinata, have proved cost-effective (Mendola, 2003), and aquaculture trials on biologically active sponges have also demonstrated relatively high growth rates and retention of biological activity in culture (e.g., Battershill and Page, 1996; Duckworth and Battershill, 2003a; Mu¨ller et al., 1999; Munro et al., 1999). The New Zealand endemic marine sponge Mycale hentscheli Bergquist and Fromont (Demospongiae: Poecilosclerida: Mycalidae) is a soft fleshy sponge of massive to encrusting morphology that inhabits subtidal reefs from 5 to 30 m. This sponge contains three classes of biologically active compounds with pharmaceutical potential: mycalamide A, pateamine and peloruside A. The mycalamides (A–D) were the first group of novel cytotoxic compounds to be isolated from M. hentscheli. They have been reported to be potent protein synthesis inhibitors and have recently been found to cause apoptosis (Hood et al., 2001). Pateamine, a biochemically unrelated macrolide isolated from M. hentscheli, is also a potent eukaryotic cytotoxin active in the sub-nanomolar range (North-

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cote et al., 1991). It has subsequently been found to have immunosuppressive and apoptotic properties (Hood et al., 2001; Romo et al., 1998). The most recently discovered and potentially valuable cytotoxic metabolite isolated is peloruside A. This compound was isolated from specimens of M. hentscheli collected from Pelorus Sound on the north coast of the South Island (West et al., 2000). Peloruside A is structurally unrelated to either the mycalamides or pateamine and yet is also a potent eukaryotic cytotoxin. Recently, we have found that peloruside A is a microtubule stabiliser with potency and mode of action similar to the naturally derived anticancer drug TaxolR. Pre-clinical 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 similar profiles of toxicity to a range of organisms and cell lines, and are therefore impossible to distinguish using biological assay-based analytical techniques. However, in a recent study, we developed an analytical method using 1H NMR to quantify the individual compounds in M. hentscheli sponges (Page et al., 2005). Chemical synthesis has been completed for mycalamide A (Trost et al., 2004), pateamine (Pattenden et

al., 2004) and is in progress for peloruside A (Liu and Zhou, 2004); however, immediate sustainable supply is critical for continuing research. In the short- to medium-term aquaculture is the only reliable method of supply. The primary objective of our study was to determine the aquaculture potential for supply of biologically active target compounds from M. hentscheli by: (1) quantifying survival and growth rates of explants in different environmental conditions and (2) determining the influence of environment and explant identity on metabolite biosynthesis of M. hentscheli sponges in aquaculture.

2. Methods 2.1. Site descriptions Capsize Point is situated in Pelorus Sound at the northern end of the South Island of New Zealand (Fig. 1). It is a 40 km long fiord-like estuary fed by the Pelorus River, and discharges into greater Cook Strait. The hydrographic environment in the main channel at Capsize Point is influenced by two major physical

Fig. 1. Map of New Zealand showing location of aquaculture sites.

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factors: (1) nitrogen-depleted phytoplankton-enriched Pelorus River water, which characterizes the near surface low salinity field, and (2) tidally induced bottom intrusions of oceanic water (Gibbs et al., 2002). There is an almost continuous salinity or thermally induced density stratification, or both at between 7 and 10 m. Tidally induced flows at the density discontinuity can range from 10 to 70 cm s 1 (Gibbs et al., 1991), causing a shear zone where high turbulence occurs above and below the pycnocline. The upper and lower water columns are often decoupled so that nitrogen generated from the sediments may not be available to the upper water column and phytoplankton settling from the upper water column may not reach the sediments directly below. Summer thermal stratification coincides with the bottom of the euphotic zone and, consequently, phytoplankton biomass accumulates at the thermocline producing a mid-water chlorophyll maximum at this site. The second site, Mahanga Bay, is situated on the southwest side of Wellington Harbour at the southern end of the North Island of New Zealand, and is separated from Pelorus Sound by Cook Strait (distance 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 stratification (Heath, 1977). The tidal current at Mahanga Bay is comparatively slow (average ~ 3.8 cm s 1) (C. Woods, unpublished data); approximately 5% of the harbour water is renewed with each tide 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 studied; however, plankton is likely to be well mixed throughout euphotic zone and blooms are known to occur during spring and autumn (Morrison Cassie, 1960). 2.2. Harvest of explants and experimental design Ten large M. hentscheli sponges from a wild population at Capsize Point (Fig. 1), Pelorus Sound (41805.12VS, 173855.96VE) were selected as donor sponges and were tagged with weighted plastic labels

259

placed adjacent (approximately 30 cm) to each sponge. Two 27 cm3 size pieces were removed as explants for culture from each of the 10 sponges. Each explant was made so as to retain (on one of its sides) a section of the exopinacoderm of the donor sponge. This method of making explants has been found to optimise healing and survival of explants. Three additional replicate pieces (~ 30 g) for chemical analysis were chosen randomly from the centre of each donor sponge, so as to contain representative proportions of cortex and exopinacoderm. The samples were frozen and transported to the laboratory to determine the initial concentration of each of three compounds: mycalamide A, pateamine and peloruside A within each donor (Fig. 2). On removal, explants for culture were placed into separate 600  300 mm seawater filled plastic bags for transportation to aquaculture sites. On 28 August, 2001 at Capsize Point (Capsize explants), one explant was removed from each donor (1–10) and inserted in situ into bottom-weighted mesh arrays suspended by a sub-surface buoy, see Duckworth and Battershill, (2003b, Fig. 2). Explants were cultured at 7 m adjacent to donor sponges; this depth

Replicate chemistry samples

Mahanga Bay

Explants

Capsize Point 10 donor sponges

Capsize Point

Capsize Point 0 Months

8 Months

Fig. 2. Schematic diagram of the experimental design; squares represent three replicate chemistry samples removed from 10 donor sponges at the start of the experiment, and from each of 10 explants suspended vertically in mesh at Mahanga Bay and at Capsize Point.

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was representative of the mean depth of donor sponges at Capsize Point. The second explant was removed 1 month later (27 September 2001) from each of the same donors (1–10). These explants were transported to Mahanga Bay, Wellington Harbour (41817.07VE, 174850VS), held overnight in an open aquarium system at 12 8C and seeded into arrays at 7 m the following morning (Mahanga explants). The initial weight of explants was estimated by measuring the standard wet weight of fifty representative 27 cm3 explants left to drain on a paper towel for 5 min. Duckworth et al. (1997) demonstrated that blotted wet weight was a consistently reliable method for estimating biomass of a range of sponges, one of which (Raspalia agminata) has similar morphology to M. hentscheli. Size and mortality of explants was monitored monthly. Capsize explants were grown for 250 days and Mahanga explants for 214 days. Explants were harvested from both sites on the first week of May 2002, weighed and three ~ 30 g replicate samples taken from each for chemical analysis. At the same time, three replicate samples were removed from surviving donor sponges for chemical comparison. Daily water temperatures were recorded at 7 m depth at each site using HOBO loggers (Onset Computer Corp.).

2.3. Measurement of survival and growth Explant survival and growth was monitored monthly in situ using an underwater Hi-8 digital video camera. Total increase in size was determined by comparing the estimated wet weight of explants at the start of the trial with wet weight of explants harvested from arrays at the end of the trial. A linear relationship between explant surface area (cm2) and wet weight was used to accurately estimate growth in the field (Fig. 3). Surface area data for small explants was derived from 26 sponges of known wet weight strung on a monofilament line and, for large sponges, from standard wet weights of explants harvested from the aquaculture arrays at the end of the experiment. The underwater video recorded explants rotated through 3608 against a 1 cm background grid fixed to a frame. In the laboratory, images were captured from video footage using Sony DV image capture software (Sony Inc.) and analysed using SigmaScan Pro image analysis software (SPSS Inc.) to calculate mean surface area from a front and side image of each explant. Similar methods have been used to monitor irregularly shaped organisms (Handley et al., 2003a; Lobsiger and Manuel, 1999). Growth is expressed as percent of original explant size.

1200 1100

wt =1.03sa - 1.18

1000

= 0.93 n = 45

Wet weight (g)

900 800 700 600 500 400 300 200 100 0 0

100

200

300

400

500

600

700

800

900

1000

2

Mean surface area (cm ) 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.

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Following removal of explants and chemistry samples, the initial size of each donor sponge was 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 5 mm. This information was used for two purposes: principally, to relocate donor sponges for chemical re-sampling at the end of the trial and, secondly, to determine any relative changes in size and mortality that occurred among donors. Measurements were summed for donor sponges that fragmented during the study. 2.4. Chemical analysis A total of 103 frozen samples were dried, weighed and extracted in MeOH following methods described by Page et al. (2005). Ten replications of our analytical protocol on a single homogenous dry sponge sample gave reproducibility of F 5%. The concentration (Ag/g dry weight) of mycalamide A, pateamine and peloruside A was determined from analysis of 1H NMR spectra for each of three replicate samples taken initially from donor sponges, and from surviving Mahanga and Capsize explants and donor sponges at the end of the culture trial (Fig. 2). 2.5. Data analysis A two-factor mixed model analysis of variance (ANOVA) was used to compare concentrations of mycalamide A, pateamine and peloruside A among treatments (donor sponges, Capsize explants and Mahanga explants), and between sponges within treatments after culture. Repeated measures ANOVA were used to compare compound concentrations between donor sponges at the start of the experiment, and explants at each site and donor sponges after culture. Data were transformed prior to analysis if variances proved heterogeneous. To ensure tests were fully

261

orthogonal, only data for explants and donors that survived to the end of the trial were compared.

3. Results 3.1. Growth Explants at both sites exhibited very rapid growth rates. Capsize Point explants grew from an average estimated wet weight of 16.3 g to 548.6 g (3365%) in 250 days. Mahanga explants grew from 16.3 g to 448.8 g (2749%) in 214 days (Table 1). The wet weight of explants estimated from the relationship with surface area (Fig. 3) was not significantly different (T 18 = 0.13, P = 0.89) to the actual weight sampled from arrays at the end of the trial (Table 1). A comparison of normalized daily incremental growth rates shows that explants grew at approximately the same rate at each site (Table 1). The growth of explants was more variable at Mahanga Bay than at Capsize Point. Explant growth increased at a relatively constant rate over the first 5 months (Fig. 4). Mahanga explants regressed in size during February, whereas Pelorus explant growth rates continued to increase over the period of the study. Peak water temperatures at both sites occurred in early February. Maximum growth rates occurred in March and decreased over the last month of the trial. Donor sponges experienced negative growth after removal of explants and chemistry samples (Fig. 5). All but two sponges regressed in size during the first month of the trial and of the six donor sponges that survived only two (D1 and D8) grew. Two donors (D1 and D3) fragmented during the course of the study. Although not quantified, surface fouling was observed on explants at both sites. Hydrozoans, bryozoans and ascidians were predominant fouling taxa on explants at Mahanga Bay, whereas hydrozoans and 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 wet weight (g)

Mean actual wet weight (g)

Mean % increase in wet weight (g)

Mean daily incremental wet weight (g)

N

Mahanga Bay Capsize Point

525.9 F 221.2 486.4 F 77.2

448.1 F185.1 548.6 F 132.5

2749 F 1136 3365 F 812.6

2.0 F 0.8 2.1 F 0.5

9 10

M.J. Page et al. / Aquaculture 250 (2005) 256–269 20

5000 Capsize Point

4000

Mahanga Bay Capsize Point °C

18

Mahanga Bay °C

3000 16 2000 14 1000

Temperature °C

% Change in surface area (cm2 ± 95% C.I.)

262

12 0

Aug01

Oct01

Dec01

Feb02

Apr02

10 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 observed during summer months from December through to February and appeared heaviest on explants at Mahanga Bay in February. The degree of fouling diminished in the following 2 months as fouling species either died off and/or were overgrown by explants.

explants survived after 250 and 214 days, respectively. All wild donor sponges initially survived excision of explants and chemistry samples. However, four (D2, D4, D6 and D7) died during summer March 2002 (Fig. 5). Three of the four that died showed evidence of disease and necrosis in the previous month.

3.2. Survival

3.3. Chemistry

Survival of explants seeded into arrays was high: 100% of Capsize Point and 90% of Mahanga

Mycalamide A occurred in relatively high concentrations in donor sponges and explants throughout the

Fig. 5. Growth and survival of the 10 wild donor sponges at Capsize Point.

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trial (Fig. 6). Concentrations varied significantly among individuals, but were not consistent over time for donor sponges (Table 2), and were not significantly different among treatments (donor sponges, Capsize explants and Mahanga explants) at the end of the trial (Table 3). Pateamine was more variable among donors and explants than mycalamide A (Fig. 6). Significant variation occurred among individuals (Table 2); however, pateamine concentrations in Capsize explants at the end of the trial in May were significantly lower than in donors at the start (Table 2). Although time was not a significant factor in the ANOVA for pateamine in donors and Mahanga explants, concentrations were generally lower at the end of the trial Mycalamide A 600

sponges

(Fig. 6). These differences were not, however, consistent among all sponges within treatments (i.e. the timesponge interaction term was significant, Table 2). There was high variability among individuals within treatments, but this was not consistent between treatments, nor was there any significant difference among treatments at the end of the trial (Table 3). Peloruside A was highly variable among individuals and differences among individuals were not consistent over time (Table 2). Peloruside A was present in 50% (5/10) of wild donor sponges (D1, D2, D3, D6 and D8) and was retained only by those donors (with the exception D2, which died), and their explants at Capsize Point at similar concentrations Pateamine

Peloruside A 300

800

Donor

600

400

263

200

400 200

100

Capsize pt explants

Concentration µg/g ± 95% C.I.

200 0

0

0 1 2 * 3 4* 5* 6 7 * 8 9 10

0 1 2* 3 4* 5* 6 7* 8 9 10 800

600

300

600 400

200 400

200

100

200

0

0 0 1 2 3 4 5 6 7 8 9 10

600

0 1 2 3 4 5 6 7 8 9 10

800

1 2 3 4 5 6 7 8 9 10 300

Mahanga explants

1 2 * 3 4* 5* 6 7* 8 9 10

600

400

200 400

200

100

200 0 0 1 2 3 4* 5 6 7 8 9 10

0

0 1 2 3 4* 5 6 7 8 9 10

1 2 3 4 * 5 6 7 8 9 10

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).

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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

(a) Donor (start) vs. donor (end) Sponge 5 Time 1 Sponge  time 5 Error 24 Total (adjusted) 35

Pateamine

MS

F

29,179.4 26,039.2 17,692.1 4426.0

6.6*** 1.5 4.0**

4.9*** 2.6 0.8

(b) Donor (start) vs. explant (Capsize Point) Sponge 9 25,133.4 Time 1 10,650.7 Sponge  time 9 4150.0 Error 40 5170.1 Total (adjusted) 59 (c) Donor (start) vs. explant (Mahanga) Spongea 7 14,122.9 Time 1 10,111.1 Sponge  time 7 8316.4 Error 32 5327.6 Total (adjusted) 47

MS

2.65* 0.74 0.18

Peloruside A F

MS

F

8.65 0.87 0.46 0.16

54.1*** 1.9 2.9*

25,761.7 45,582.3 8711.1 945.8

27.2*** 5.2 9.4***

5.80 1.43 0.12 0.01

60.0*** 11.8** 1.3

99,576.3 283,837.9 50,857.9 10,248.1

9.7*** 5.6 4.9***

4.56 0.15 0.14 0.09

1462 6147.2 1462 358.4

48.5*** 1.3 1.5

4.1** 4.2 4.1**

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 b 0.05. ** P b 0.01. *** P b 0.001.

after 250 days growth in culture arrays (Fig 6). Whereas, Mahanga explants from the same wild donors had no detectable peloruside A after 215 days growth in culture arrays (Fig. 6).

Table 3 A two-factor mixed model analysis of variance comparing concentrations among treatments (donor sponges, Capsize explants and Mahanga explants) and within sponges harvested in May 2002 Factor

df Mycalamide A MS

Treatment 2 30,932.0 Sponge 4 12,857.6 TS 8 11,022.6 Error 30 5755.6 Total 44 (adjusted)

Pateamine

Peloruside A

F

MS F

MS

2.8 2.2 1.9

1.1 8.9 1.2 0.3

F

0.9 50,979.6 4.2 34.9* 36,399.8 62.5* 4.8* 12,200.3 20.9* 582.8

GLM ANOVA was used to analyse data. To meet assumptions, pateamine data was log transformed. * P b 0.001.

4. Discussion 4.1. Growth M. hentscheli explants exhibited phenomenal growth rates of 4688% year 1 at Mahanga Bay and 4912% year 1 at Capsize Point. These growth rates compare very favourably with aquaculture studies on other species (Table 4). For instance, bath-sponges (Dictyoceratida) that invest in a dense spongin (collagen) skeleton appear to have much slower growth rates (73–150%) than their non-dictyoceratid counterparts (Handley et al., 2003b). A possible explanation is that dictyoceratid sponges emphasise a protein (spongin) skeleton that may require more energy to produce, and therefore take longer to grow (Kelly et al., 2004). Much higher growth rates have been achieved for fleshy non-dictyoceratid species. For instance, Duckworth (2000) measured growth rates of 950–740% over 6 months for Latrunculia brevis

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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. Hippospongia lachne Spongia argaricina Spongia officinalis Spongia (Heterofibria) officinalis Geodia cydonium Lissodendoryx sp. Latrunculia wellingtonensis Polymastia croceus Mycale hentscheli

Dictyoceratida Dictyoceratida Dictyoceratida Dictyoceratida Dictyoceratida Choristida Poecilosclerida Hadromerida Hadromerida Poecilosclerida

~ 100 ~ 100 90 150 73 380 5000 700 360 4912

(Moore, 1910) (Crawshay, 1939) (Verdenal and Vacelet, 1990) (Verdenal and Vacelet, 1990) (Kelly et al., 2004) (Mu¨ller et al., 1999) (Battershill and Page, 1996) (Duckworth and Battershill, 2003b) (Duckworth and Battershill, 2003b) 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 5000% growth was recorded for individual explants of the deepwater sponge species Lissodendoryx sp. (Battershill and Page, 1996). Ecologically, M. hentscheli appears to have an r-selected life history favouring rapid growth and short life span. Fast-growing fleshy species such as M. hentscheli that are adapted for opportunistic colonization of new substrates are most likely to be ideal candidates for translocation and introduction into existing aquaculture systems. The rapid growth of M. hentscheli explants suspended in arrays may also be correlated with increased water flow rate and therefore food, away from benthicboundary layer effects (Freche´tte and Bourget, 1985; Leichter and Witman, 1997; Palumbi, 1984). Furthermore, explants introduced into mesh arrays may have initially experienced low interspecific competition for food and space, enhancing their chance for survival and growth. Explant growth rates were not significantly different between Capsize Point and Mahanga Bay, which shows that both sites had favourable environmental conditions for rapid growth. A regression in size of explants at Mahanga Bay during February was coincident to a 2 8C drop in water temperature because of an incursion of cold oceanic water into Wellington Harbour from Cook Strait during a severe storm event (Carter et al., 2002). Regression in sponge size in relation to temperature down-shocks has been reported for commercial sponges in the Gulf of Mexico (Storr, 1964). M. hentscheli growth rates were relatively slow during winter, but increased as water temperatures rose in spring and summer. For newly

seeded explants, initial energy may have been directed towards healing of cut surfaces and reorganization of damaged canal systems (Mu¨ller et al., 1999), and their subsequent growth influenced by higher temperatures and availability of food in spring (Barthel, 1986; Fell and Lewandrowski, 1981; Turon et al., 1998). 4.2. Survival Explant survival, sustained growth and continued biosynthesis of desired target compounds is critical for guaranteed supply of biologically active compounds from sponges in aquaculture (Munro et al., 1999). The success of sponge aquaculture is highly dependent on seasonality with respect to initiating cultures (Duckworth et al., 2004). In our study, seeding M. hentscheli explants in winter when water temperatures were low ensured high survival (90– 100%). Low mortality has been documented for a number of other sponge species in aquaculture (Duckworth, 2000; Kelly et al., 2004; Verdenal and Vacelet, 1990), and is considered to be positively correlated with low water temperature, reducing metabolic stress, promoting rapid pinacoderm healing and decreasing the probability of infection (Burlando et al., 1992; Vacelet et al., 1994). High survival of M. hentscheli explants can also be attributed to the morphology of this species. Duckworth and Battershill (2003b) concluded that dmeshT arrays were the least invasive method for farming soft fleshy sponges such as Latrunculia wellingtonensis and M. hentscheli. They found these species have the ability to grow rapidly through mesh in aquaculture arrays, effective-

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ly maximizing the surface area available for feeding and respiration. High survival of M. hentscheli translocated explants further demonstrates the suitability of this species to commercial-scale aquaculture production. Explants can be selected for chemical attributes, translocated and seeded on commercial long-line systems similar to those used for mussel culture (Jenkins et al., 1985). Furthermore, sites can be chosen for optimal environmental conditions for growth and biosynthesis of target metabolites. While survival of wild donor sponges was high after initial removal of explants and chemistry samples, most regressed in size or exhibited poor growth, and 4 of 10 died during summer. Larvae in M. hentscheli have been observed at Capsize Point during spring (November–December). As sponge growth can be negatively correlated to the onset of reproductive activity (Barthel, 1986; Elvin, 1979), it is possible that energy directed towards reproduction in wild donor sponges was used for somatic growth in explants. For instance, Reiswig (1973) discovered reproductive competence in a tropical species of Mycale was size-dependent, small sponges grew up to seven times their original size during 1 year, but this growth rate decreased to 60% with the onset of reproductive competence. Further ecological studies are needed to investigate our hypothesis as no undamaged sponges were monitored for growth, and wild donor sponges and explants in our study were not examined for reproductive condition. 4.3. Chemistry Wild parent M. hentscheli sponges and explants cultured in their native environment at Capsize Point retained peloruside A, whereas genetically identical explants translocated to Mahanga Bay did not. This result suggests that environmental conditions in different habitats may in some way affect the biosynthesis of peloruside A and that compound biosynthesis may not necessarily be genetically determined. Chemical variability in relation to changes in environment has also been demonstrated for genetically identical fragments of the sponge Rhopaloeides odorabile (Thompson et al., 1987). They concluded that quantitative and qualitative changes in diterpenes were an adaptive response to surface fouling of algae or lightinduced stress, as evidenced by poor growth rates.

Survival and growth rates of M. hentscheli explants at Mahanga Bay were high and no different from genetically identical explants at Capsize Point; this result demonstrates that differences in peloruside A were unlikely to be correlated with stress or lack of food. However, qualitative differences in fouling organisms in our study may explain changes in compound profiles between sites. Mycalamide A, pateamine and peloruside A are complex, highly cytotoxic compounds that almost certainly play a role in chemical defence against surface fouling, competition and predation. They may have multiple roles in the organism as suggested by Becerro et al. (1997) for the sponge Crambe crambe, and therefore vary according to their ecological role (Fagerstro¨m et al., 1987). The different compounds in M. hentscheli may, for example, be active against different fouling organisms and so absent where there is no target organism present. This hypothesis further explains the variation measured among donor sponges; compounds may be expressed or produced if sponges are challenged by competitors or predators. An alternative explanation for chemical variability in explants and among donors is that compounds are localized in and produced by symbiotic microorganisms (e.g., Faulkner et al., 1999; Unson and Faulkner, 1993). M. hentscheli is known to harbour a diverse population of microbial associates that varies among individuals and between geographic locations (Anderson et al., 2004). Changes in environmental conditions (including transportation in plastic bags and overnight storage in aquaria) may have altered microbial populations and therefore compounds, in explants moved to Mahanga Bay. Furthermore, Mahanga explants were excised from donors for culture a month later than Pelorus explants. The physiological state of donor sponges may have changed in that time, influencing our results. However, previous pilot studies (unpublished data) where a single M. hentscheli individual has been repeatedly sampled from for chemistry have shown consistency in chemical composition over several years.

5. Conclusions Our research demonstrates in-sea aquaculture of M. hentscheli is viable for short- to medium-term supply

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of target compounds for drug development. Our results further support the hypothesis that environmental conditions can be critical for the biosynthesis of compounds. We suggest that sponges such as M. hentscheli, which are opportunistic occupiers of space, can have phenomenal growth rates and have the potential to yield massive increases in biomass over a single growing season. Our trial shows that the 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 native environment to yield the compound. To our knowledge, few studies have used analytical techniques to quantify the effect of environment on biosynthesis of target compounds from sponges in aquaculture. Furthermore, our results suggest that, in order to maximize target compound yield in aquaculture, we can assay wild sponges and select for explants to establish peloruside A-containing cultivars. Preliminary results from a bulk culture trial support our experimental findings; we were able to pre-select ~ 7 kg of cultured explants from a line containing a total of ~ 34 kg of explants and extract ~ 50 mg of peloruside A for a predicted yield of ~ 40 mg. Annual production of peloruside A required for drug development and supply is difficult to predict. Scaling-up farming structures can lead to increased competition from fouling organisms, significantly reducing growth and survival of farmed sponges (Hadas et al., 2005). However, a conservative estimate based on preliminary trials demonstrates that 6.2 mt of explants would need to be grown for an annual production of 10 kg of peloruside A. Further manipulative experiments are needed to identify environmental factors specific to peloruside A production and analysis of sequential generations of explants is essential to determine the inter-generational fidelity of peloruside A production from cultivars.

Acknowledgements We thank Steven Brown and Rob Keysers for their field support under often trying conditions. Many thanks to Phil James and Chris Woods, Mahanga Bay Aquaculture Research Centre (NIWA), who contributed significantly to monitoring and harvesting explants in Wellington, and Anna Tovey for analysing

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videos. Many thanks to Drs. Michelle Kelly (NIWA, Auckland), Russell Cole (NIWA Nelson), Chris Battershill (Australian Institute of Marine Sciences, Townsville) and to the anonymous reviewers, for sharing their ideas and commenting on our manuscript. This research was funded by Foundation for Science Research and Technology (FRST) research contracts CO1809 and COIX0207 with NIWA.

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