Variation of antimicrobial activity of the sponge Aplysina fistularis (Pallas, 1766) and its relation to associated fauna

Journal of Experimental Marine Biology and Ecology, 223 (1998) 1–18

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Variation of antimicrobial activity of the sponge Aplysina fistularis (Pallas, 1766) and its relation to associated fauna 1 ,a , 2 ,b ´ ´ Miguel Betancourt-Lozano *, Fernando Gonzalez-Farias , Barbara a a c ´ ´ Gonzalez-Acosta , Alejandra Garcıa-Gasca , Jose´ Rolando Bastida-Zavala a

´ Experimental, Centro Interdisciplinario de Ciencias Marinas /IPN, Apdo. Laboratorio de Biologıa ´ Postal 592, La Paz, B.C.S., C. P. 23000, Mexico b ´ en Alimentacion ´ y Desarrollo, A.C., Unidad Mazatlan ´ , Apdo. Postal 711, Centro de Investigacion ´ , Sin., C. P. 82000, Mexico ´ Mazatlan c ´ e Investigacion ´ Ambiental, A.C., Apdo. Postal 1 -278, Morelia, Mich., C. P. 58000, Mexico ´ Consultorıa Received 16 June 1995; received in revised form 15 May 1997; accepted 3 June 1997

Abstract The sponge Aplysina fistularis (Pallas, 1766) was studied to determine if it produces different levels of antibiotic activity during an annual cycle, and to try to establish some ecological relations of this phenomenon. Monthly collections of sponges were made at Isla Espiritu Santo, Baja California Sur, Mexico from May 1989 to June 1990. Bioassays of antimicrobial susceptibility were carried out and densities of the associated organisms were recorded. Antibiotic activity levels of the sponge correlated with inquilism level. Because antimicrobial activity was always found, we believe that metabolites are produced continuously, although the sponge is probably able to change the substance production mechanism in the presence of a particular stimulus. The massive reproduction of the associated invertebrates and the arrival of large quantities of organisms over a short time could be important stress factors for the sponge and a stimulus that modifies the production of secondary metabolites.  1998 Elsevier Science B.V. Keywords: Antibiosis; Aplysina fistularis; Associated fauna; Chemical ecology; Metabolites; Sponges

*Corresponding author. 1 ´ en Alimentacion ´ y Desarrollo, A.C., Unidad Mazatlan, ´ Apdo. Postal Present address: Centro de Investigacion ´ Sin., C. P. 82000, Mexico. ´ 711, Mazatlan, 2 ´ Estacion ´ Mazatlan, ´ Universidad Nacional Present address: Instituto de Ciencias del Mar y Limnologıa, ´ ´ ´ Sin., C. P. 82000, Mexico ´ Autonoma de Mexico. Apdo. Postal 811, Mazatlan, 0022-0981 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0022-0981( 97 )00153-6

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1. Introduction Experimental evidence suggests that there are qualitative and quantitative variations in secondary metabolites produced by some organisms (Christophersen, 1991). There is a tendency to explain these variations ecologically, with environmental factors influencing the biochemical profile of the organisms. There are many examples of marine invertebrates with chemical defenses (Bakus et al., 1986; Bobzin and Faulkner, 1992; Pawlik, 1992). The synthesis and storage of these substances favors the survival of such organisms in a complex environment (Thompson et al., 1983), and gives such species a selective advantage due to the genetic transmission of the capacity for chemical defense synthesis (Kittredge et al., 1974; Mieres, 1989). In marine sponges, the presence of antimicrobial substances has been well documented (Green, 1977b; Bergquist and Bedford, 1978; Bakus et al., 1985; Faulkner, 1990). It has been proposed that antibiotic substances of sponges inactivate the bacteria retained by its filtration system for subsequent phagocytosis and digestion (Burkholder, 1973). Green (1977a) suggested antibiotics found in sponges can be used as offensive or defensive mechanisms; offensive when involved in feeding, and defensive to prevent infections caused by pathogenic microorganisms. Antibiotics in sponges might also be used as antifoulants, preventing the attachment of bacteria (Becerro et al., 1994), or invertebrates (McCaffrey and Endean, 1985; Thompson, 1985; Goto et al., 1993). Researchers have tried to determine whether antibiotic activity of sponges varies temporally. Hypotheses involving temperature as the main factor affecting this variation have been proposed. Nigrelli et al. (1959) found that the production of ecthyonine in Microciona prolifera decreases in cold months. They suggest that this compound plays a role in the biochemical ecology of the sponge, and its variation could result from either the natural metabolic cycle of the sponge or changes in environmental conditions. Bakus et al. (1985) and Green et al. (1985), in studies of sponges along the Mexican coasts (Pacific, Gulf of Mexico, and Caribbean), suggested that there is a seasonal variation in activity levels of the antimicrobial properties of sponges. They found a high number of sponges showing antibiotic activity during the ‘warm season’, with the opposite true in the ‘cold season’. McCaffrey and Endean (1985) considered the antibiotic properties of some sponges related to antifouling activity and proposed some species grow in the virtual absence of surface organisms because of inhibition of larval settlement. Thompson (1985) showed that exudates of the sponge Aplysina fistularis can reduce larval settlement, inhibit metamorphosis of veliger larvae in gastropods, and modify the behavior of adult snails. Antimicrobial compounds can have additional antipredatory and antifouling effects which suggest that the increase in predation and fouling in the ‘warm season’ can favor adaptive responses in sponges (Green et al., 1985). However some antifouling compounds do not have antimicrobial properties (Bakus et al., 1985). The specificity of secondary metabolites to receptor systems, and the possibility that the production of metabolites result from natural selection (Christophersen, 1991), makes plausible the appearance of organisms tolerant to bioactive substances. The appearance of such tolerant organisms could be a survival problem for a chemically-

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defended organism. Tolerant organisms can live on a host as parasites, micropredators, or commensals, and may weaken the general health of the host. Christophersen’s hypothesis Christophersen (1991) of the dynamic character in the pattern of the secondary metabolites, which depends on the physiological or environmental conditions and the interaction of the metabolites with receptor systems, can explain survival of chemically-defended organisms. The present study shows temporal variation in antimicrobial activity of the marine sponge Aplysina fistularis (Pallas, 1776) under natural conditions, and discusses some ecological relationships of this phenomenon.

2. Methodology The sponge Aplysina fistularis was selected because of its antipredatory, antifouling, and antimicrobial properties. Species of the genus Aplysina produce substances with different bioactive properties (Faulkner, 1977; Walker et al., 1985; Michel-Reynoso, 1986; Cruz, 1988; Keer, 1988; Mieres, 1989). Sponges of uniform size (height 10 to 15 cm) were collected from rocks exposed to light. Two kinds of A. fistularis samples were taken each month. Four sponges were collected for the antibiosis test (except in June 1989), and two for the taxonomic determination of the associated fauna (except in June and August 1989). Since the collecting site was limited in area, we considered six the maximum number to be sampled without affecting the natural density of the sponge population. The sponges were collected by SCUBA diving at 5 m depth from May 1989 to June 1990. Water temperature was measured at the surface and at 5 m depth.

2.1. Sampling station The sampling station was located at Isla Espiritu Santo - La Partida, in Bahia de La Paz, Baja California Sur, Mexico (Fig. 1), between 248 249–248 369 N and 1108 179–1108 259 W. Preliminary SCUBA diving indicated that there was a suitable density of healthy sponges (health based on turgescence, color, and non-exposed fibers).

2.2. Antibiosis tests Sponges collected for antibiosis tests were carefully separated from the substrate, preventing the release of bioactive substances through damage (Walker et al., 1985). The water excess from the sponges was drained (without squeezing) and associated macroorganisms were removed. The sponges were kept in plastic bags and transported on ice to the laboratory where they were frozen at 2 48C. To detect antibiosis activity, six marine bacterial strains were used. Four of them were obtained from the bacteria collection at the Centro de Investigaciones Biologicas del Noroeste (CIBNOR) (Bacillus megaterium, Flavobacterium sp., Vibrio sp. and Paracoccus sp.) and the other two were isolated in the Laboratory of Experimental Biology at the Centro Interdisciplinario de Ciencias Marinas (CICIMAR) (Pseudomonas vesicularis

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Fig. 1. Map of Mexico and the Peninsula of Baja California. The arrow indicates the sampling site.

and Pseudomonas cepacia). Both institutes are in Baja California Sur, Mexico. The strains were maintained in culture tubes with Marine Agar 2216 (Zobell, 1941) under refrigeration (48C) with periodic culture transfers. At the beginning of the bioassays, a liquid inoculum was prepared for each strain and incubated at 358C. The density of bacteria cultures was monitored with a spectrophotometer (Bausch and Lomb – Spectronic 21). For each species, petri dishes (10 cm diameter) were prepared using the spread plate method (APHA-AWWA-WPCF, 1985) with 10 ml of Marine Agar 2216 and 1 ml of inoculum (density approximately 1 3 10 8 cells / ml). From each sponge, cylindrical fragments of 1 cm diameter and 0.5 cm height were obtained. This process was done with a ‘core extractor’ using a syringe with the conical anterior part cut off. All the fragments were of the same size and shape. Each month, two fragments from each sponge were tested for antibiotic activity against each strain (see discussion for the reason of using the sponge fragment approach). Sponge fragments were placed with the exopinacoderm in contact with the marine agar. Petri dishes were incubated at 358C. After 24 h, inhibition halo diameters (cm) were measured. For each bacterial strain, a single-classification analysis of variance and a Tukey’s multiple comparisons test (Sokal and Rohlf, 1995) were used to compare the antibiotic activity between the months. DIFCO sensidisks with chloramphenicol (30 mg), nitrofuranthonin (300 mg), novobiocin (30 mg), and streptomycin (10 mg) were used as controls.

2.3. Taxonomic determination of the associated fauna Each sponge and its associated organisms sampled for taxonomic work were

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introduced in situ into labelled plastic bags. In the laboratory, these samples were placed in glass jars and preserved in ethanol 70%. Gosner (1971); Smith and Carlton (1975), and Brusca (1980) were used for the ´ ´ identification of invertebrates; Caso (1961) for ophiurids; De Leon-Gonzalez et al. ´ (1989) and Bastida-Zavala (1993) for polychaetes; and Garcıa-Madrigal (1993) for brachyurans. In the laboratory, each sponge was cut into thin slices (0.5–1 cm) which were examined under the stereomicroscope. The associated organisms were separated with forceps and those retained on a 500 mm sieve (macrofauna) were used for the taxonomic work (Parsons et al., 1984). After obtaining the wet weight of all the associated organisms, they were separated into taxonomic groups and identified to the lowest level possible. For each group, the wet weight and number of individuals was recorded as well as the presence of ovigerous females or reproductive structures. The sponge slices, free of associated organisms, were dried (908C) to constant weight. Density relations were calculated for each group: 1. abundance 5 number of organisms / dry weight of sponge 2. biomass 5 wet weight of organisms / dry weight of sponge This procedure standardized levels of associated fauna independent of sponge size. To select principal groups of associated fauna, and to simplify the analysis, incidental groups present at less than 10% of the total abundance were excluded. The ecological index IVB (Importance Value; Zabi, 1984) was calculated monthly for each group (parameters are expressed as the percentage of the total for all groups): IVB 5 A 1 F 1 D 1 B, where: A5relative abundance(%), F 5relative dominance(%) and B5relative biomass(%)

frequency(%),

D5relative

3. Results

3.1. Temperature The water temperature variation at the sampling site is shown in Fig. 2. We found no differences between the temperature at the surface and at 5 m depth. The highest temperature recorded (308C) was in September 1989, and the lowest in February 1990 (158C). From this month there were steady temperature increments till June.

3.2. Antibiotic activity All the tested bacterial strains were sensitive to the antibiotic activity of A. fistularis which is presented as average inhibition halos of four sponges (Fig. 3). Antibiotic activity on Vibrio sp. showed significantly higher values in April, May, and June 1990 (Fig. 3a), but no clear variations during previous months.

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Fig. 2. Variation of water temperature (8C) at the sampling site, from May 1989 to June 1990 (June 1989 value is missing).

Peak antibiotic activities for Pseudomonas vesicularis occurred in April and May 1990 (Fig. 3b), with uniform levels in other months. Pseudomonas cepacia showed a similar response, with only one significantly higher value in May 1990 (Fig. 3c). Flavobacterium sp. was the most sensitive bacteria to the antibiotic action of A. fistularis (Fig. 3d); however, statistical analysis did not detect peak levels of antibiotic sensitivity. Paracoccus sp. (Fig. 3e) and Bacillus megaterium (Fig. 3f) showed similar responses through time, without detectable statistical differences. The sensitivity of the different bacterial strains to commercial antibiotics did not show variations over time.

3.3. Associated fauna A total of 43 functional groups were recognized of which nine were considered principal representing 90.59% of the total abundance (Table 1). We use the term inquiline to define organisms of the principal groups, while incidental groups are those showing lower frequency. A monthly summation of importance values of principal groups was made to evaluate the degree of inquilism in the sponge over time (Table 2). Inquilism levels were low from May 1989 to January 1990, with values varying between 13 and 20. Inquilism increased from February to May 1990, and dropped in June. The frequency of incidental groups peaked in September 1989 and May 1990 (Fig. 4). The highest frequency of reproductive events of principal groups occurred in March, April and May 1990 (Fig. 5a). September 1989 and May 1990 showed the highest frequency for incidental groups (Fig. 5b).

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Fig. 3. Antibiotic activity (average inhibition halos, and standard deviations) of A. fistularis (n54) on the bacterial strains during the study period (no samples were taken in June 1989): (a) Vibrio sp., (b) Pseudomonas vesicularis, (c) Pseudomonas cepacia, (d) Flavobacterium sp. (e) Paracoccus sp. and (f) Bacillus megaterium. (* denotes significantly different values).

3.4. Relation between antibiosis and associated fauna The relationship between antibiosis activity of sponges on some bacterial strains and the inquilism level of the principal groups associated with A. fistularis is presented in Fig. 6. ANOVA-multiple comparison tests showed that sponge antibiosis against three bacteria strains (Vibrio sp., Pseudomonas cepacia and Pseudomonas vesicularis) had significantly higher values in May 1990. The inquilism level had also its highest value in May 1990. Antibiosis levels of the sponges on Vibrio sp. correlated with the sum of the importance values IVB (Fig. 7a). Pseudomonas vesicularis (Fig. 7b), and Pseudomonas cepacia (Fig. 7c) showed similar effects.

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Table 1 Groups of organisms associated with Aplysina fistularis and their relative abundance during the study period (P.G.5Principal group) Groups

Phylum CNIDARIA Class ANTHOZOA (5HEXACORALLIA) Order ACTINIARIA Phylum MOLLUSCA Class GASTROPODA Subclass OPISTHOBRANCHIA Order NOTASPIDEA (Tylodina) Class BIVALVIA Phylum ANNELIDA Class POLYCHAETA Order ORBINIDA Family ORBINIDAE Order SPIONIDA Family SPIONIDAE Family CIRRATULIDAE Order CAPITELLIDA Family CAPITELLIDAE Family MALDANIDAE Order PHYLLODOCIDA Family PHYLLODOCIDAE Family POLYNOIDAE Family CHRYSOPETALIDAE Family HESIONIDAE Family PILARGIDAE Family SYLLIDAE Family NEREIDIDAE Order EUNICIDA Family EUNICIDAE Family LUMBRINERIDAE Family OENONIDAE Family DORVILLEIDAE Order FLABELLIGERIDA Family FLABELLIGERIDAE Order TEREBELLIDA Family TEREBELLIDAE Order SABELLIDA Family SABELLIDAE Family SERPULIDAE Family SPIRORBIDAE

Relative abundance

1.512

Groups

P.G.

0.143 1.192

0.036 0.054 0.077 0.372 0.054 0.164 0.166 0.022 0.022 0.079 28.118 0.404 0.522 0.056 3.471 0.144 0.030 0.464 0.192 0.446 0.129

P.G.

P.G.

Phylum ARTHROPODA Subphylum CHELICERIFORMES Class PYCNOGONIDA Subphylum CRUSTACEA Class MAXILLOPODA Subclass COPEPODA Order HARPACTICOIDA Subclass CIRRIPEDIA Order THORACICA Suborder BALANOMORPHA Class MALACOSTRACA Order DECAPODA Infraorder CARIDEA Family PALAEMONIDAE Family ALPHEIDAE Infraorder ANOMURA Family PORCELLANIDAE Infraorder BRACHYURA Family MAJIDAE Family XANTHIDAE Family GRAPSIDAE Order CUMACEA Order TANAIDACEA Suborder MONOKONOPHORA Suborder DIKONOPHORA Order ISOPODA Suborder FLABELLIFERA Suborder ASELLOTA Order AMPHIPODA Suborder GAMMARIDEA Suborder CAPRELLIDEA Phylum ECHINODERMATA Class ESTELLAROIDEA Subclass OPHIUROIDEA Order OPHIURIDA Family OPHIOCHITONIDAE Family OPHIOCOMIDAE

Relative abundance

0.304

7.997

P.G.

0.115

0.765 0.380 0.086 0.876 0.031 0.057 0.793 8.036 3.546

P.G. P.G.

0.437 14.362

P.G.

16.970 0.582

P.G.

6.577 0.053

P.G.

4. Discussion Ecologically, it is important to evaluate the spatial, temporal, quantitative, and qualitative variations in the production of secondary metabolites (Paul and Fenical,

Principal Groups

May ’89

Jul ’89

Sep ’89

Oct ’89

Nov ’89

Dec ’89

Jan ’90

Feb ’90

Mar ’90

Apr’90

May ’90

Jun ’90

GAMMARIDEA MONOKONOPHORA DIKONOPHORA ASELLOTA HARPACTICOIDA SYLLIDAE OENONIDAE OPHIOCHITONIDAE ACTINIAARIA Total of Importance values

6.79 0.00 1.59 6.11 0.00 0.00 0.00 0.00 0.00 14.50

2.22 1.94 0.78 1.46 1.10 4.68 0.00 2.41 1.86 16.44

2.48 1.80 0.96 1.75 1.06 7.92 0.66 3.13 0.00 19.75

1.64 2.29 0.00 1.11 1.40 2.87 0.00 4.06 0.00 13.38

1.75 0.92 0.93 1.19 1.66 5.59 1.33 2.89 0.00 16.26

1.49 1.07 0.00 4.73 2.93 1.41 0.00 0.00 0.00 11.62

1.29 0.77 0.58 3.70 0.75 2.55 6.20 1.20 0.00 17.05

1.51 1.08 0.93 5.06 7.30 6.37 0.60 2.82 0.00 25.67

2.83 7.17 1.32 4.54 1.41 7.41 0.93 0.77 0.55 26.93

2.22 1.38 1.33 2.50 1.62 10.33 3.29 6.10 2.68 31.45

15.19 1.82 2.12 4.50 1.49 9.97 2.68 6.25 0.83 44.86

1.85 1.64 1.09 2.27 1.48 4.13 1.20 6.28 0.00 19.96

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Table 2 Importance values (IVB) for principal groups of associated organisms, during the study period (June and August 1989 samples were not taken)

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Fig. 4. Frequency of incidental groups during the study period (June and August 1989 samples were not taken).

1986; Pawlik et al., 1986; Kernan et al., 1988; Sammarco and Coll, 1990; Steinberg and Paul, 1990; Meyer and Paul, 1992). Additional variation may relate variation in the extraction methods or solvents used (Bergquist and Bedford, 1978; Thompson et al., 1983). Because bioactive compounds vary in chemical polarities, some require multiple solvents for extraction. The use of non-appropiated solvents results in a negative record of bioactivity. Other factors which may influence the bioactivity are the collection of samples from different depths (Thompson, 1985) and handling and preservation of samples (Rinehart, 1988). Conventional bioassays may not always detect small quantities of compounds or particular substances produced. In general, the contribution from associated microorganisms to metabolite production in sponges is unknown (Thompson et al., 1983; McCaffrey and Endean, 1985), though the production of secondary metabolites by symbiotic cyanobacteria has been demonstrated (Unson and Faulkner, 1993). In our study, the sponge fragment assay was selected based on A. fistularis characteristics. The sponge has a rubber-like consistency that yields well-shaped and uniform cylindrical fragments, in order to place the sponge exopinacoderm in contact with the test bacteria. Thompson et al. (1983) have reported metabolite-containing globular cells under the exopinacoderm of A. fistularis. Green et al. (1985) suggested that variation in metabolite production by sponges could vary with seasonal differences, and believed that the increase of antimicrobial agents during the warm season may have several explanations. They pointed out that the temperature increase has an indirect effect, because it stimulates bacterial populations, so antibacterial incidence could be an adaptive response by the sponge to increase in

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Fig. 5. Frequency of reproductive events in: (a) Principal groups, and (b) Incidental groups (June and August 1989 samples were not taken).

pathogenic bacteria. The authors did not exclude the direct effect of temperature on the metabolic rate of sponges, since high temperatures can increase this, which may stimulate antimicrobial metabolite production (Q 10 effect). The proportion of active sponges in the tropics is higher than at temperate latitudes (Bakus and Green, 1974; Green et al., 1985; Lozano, 1988). Another explanation is that antimicrobial compounds with a broad spectrum have additional bioactivities, such as antipredation or antifouling. An increase in predation and / or fouling during the ‘warm season’ may also cause adaptative responses from the sponges (Green et al., 1985). However, Bergquist and Bedford (1978) found that temperate sponges show a high proportion of active extracts, relative to tropical species, and suggested that antimicrobial activity of sponges is common, not being directly related to latitude or habitat. In our study, the antimicrobial activity of A. fistularis did not show a direct relation with temperature. However, temperature could trigger the incidence and reproduction of most sponge symbionts, and we found a significant correlation between inquilism and the antibiosis shown by some strains. There are several kinds of associations between the sponge and its inhabitants, ranging

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Fig. 6. Relation between antibiosis and inquilism. Antibiosis is represented by inhibition values from Vibrio sp., Pseudomonas vesicularis and P. cepacia (standard deviations are shown in Fig. 3a–c). Inquilism is represented by the monthly IVB of associated principal groups.

from simple temporal contact to metabolic dependence on the host (Vernberg, 1974). The host represents the symbiont’s environment in which the ecological community interacts (Westinga and Hoetjes, 1981). Some organisms have a commensal relation with A. fistularis, e.g. Alpheida and Paleomonida. Although they were not found in large quantities, at least one individual was always present (including ovigerous females) and many times the sponge inner surface had ‘imprinted’ the morphology of the symbiont (pers. obs.), indicating that the sponge is their permanent refuge. Brittlestars occupied sponge interstices during the whole study, with high densities in April and May 1990 (though they may enter and leave the sponge depending on the time of the day). Mutualism also occurs between sponges and brittlestars, where the symbiont is a non-selective consumer of detritus on the sponge surface. This ‘cleaning service’ improves the sponge filtration capacity (Hendler, 1984). The same kind of association may be present with small crustaceans, such as gammarids, copepods, isopods, tanaidacea, and some polychaetes, that clean the detritus on the inhalant surface of the sponge (pers. obs.). Micropredation by some crustaceans on sponges has been reported (Oshel and Steele, 1985). Majid, xanthid, grapsid and porcellanid crabs can be commensals of A. fistularis, and predators of the associated microcommunity (Wurzian, 1977). Some of these crabs burrow in the sponge surface exhibiting Aplysina-like camouflages (pers. obs.) that make them inconspicuous for potential preys or predators. Polychaetes frequently associate with sponges (Dauer, 1974). Motile polychaetes, as the syllid Branchiosyllis oculata, eat soft parts of some sponges (Pawlik, 1983) apparently using the proboscis tooth to perforate the body wall of the prey (Day, 1967). Our study noted several syllid polychaetes. Characteristics of this group are parental care of the eggs, direct development, and multiple mechanisms of asexual reproduction such as stolons, gemmations, or epitoky (Wilson, 1991) which can explain its demographic

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Fig. 7. Inquilism–antibiosis correlation. Inhibition values for: (a) Vibrio sp. (R50.792, n512), (b) Pseudomonas vesicularis (R50.889, n512), (c) Pseudomonas cepacia (R50.796, n512).

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explosion in May 1990 in A. fistularis. Syllids can be cannibals, parasites, detritivores, and herbivores. They generally occur in sponge interstices because of their small size (Fauchald and Jumars, 1979). These species are probably not necessarily obligate symbionts, but use the sponge as a refuge, a reproduction area, and even as a food source. Oenonids are another important polychaete group having a conic prostomium and a robust mandibular apparatus that cuts by friction, characteristics corresponding to their digging habits (Colbath, 1989). We observed them to perforate galleries in A. fistularis. However, they were not found in all months and perhaps are facultative parasites or micropredators which apparently use the sponge as a reproduction area when conditions are appropriate. None of the groups considered as encrusting epibionts were successful in A. fistularis colonization. Serpulids, with opportunistic larvae and builders of calcium carbonate tubes, were infrequent. Only one barnacle was found and few juvenile bivalves. However, even if the abundance and biomass in incidental groups are minor, their simultaneous appearance could have ecological importance. Some sponges grow almost free of surface organisms, which indicates that there is an inhibition of larval settlement (McCaffrey and Endean, 1985; Thompson, 1985). The relation between antifouling compounds and antimicrobials may be due to microfouling inhibition (e.g. bacteria) preventing the succession of organisms. However, some compounds are exclusively antifouling and lack antimicrobial properties (Bakus et al., 1985). Further studies are necessary to evaluate the inhibition of bacterial attachment and fouling, the metabolites involved, and the temporal variation of these processes. A special case is Tylodina sp., an opisthobranch which is the only one reported to practice direct micropredation on the surface of A. fistularis (Thompson, 1985). It can also deposit its eggs on the sponge surface (pers. obs.). This mollusk has a bright yellow color that may come from its diet of Aplysina sponges (Ros, 1977) which are yellow inside. Pigmentation can play a warning function (aposemasis) to potential predators (Ros, 1977). Chemical defenses in opisthobranchs may result from their diet of sponges (Faulkner and Ghiselin, 1983; Pawlik et al., 1988). Evidence indicates that Tylodina has these characteristics since inhibition halos occur in antibiosis tests carried out with fragments of Tylodina collected from the A. fistularis in our sampling site (unpublished data). Furthermore, Tylodina can live quite a distance from the sponges (pers. obs.). In laboratory experiments, the nudibranch Diaulula sandiegensis shows a chemical recognition of the sponge Haliclona permolis (Elvin, 1977). The same behavior occurs in Tylodina with A. fistularis exudates (unpubl. data). More brominated compounds have been extracted from Aplysina than from any other ´ 1990). These compounds may have a common sponges (Cruz, 1988; Arrieta-Guzman, biosynthetic origin related to 3,5-dibromotyrosine (Tymiak and Rinehart, 1981). Cruz (1988) reviewed the chemistry of Aplysina and found that besides the tyrosine-derived halogenated compounds, steroids, carotenoids and nitrogenous compounds have also been isolated, some of them bioactive. Two of the most studied compounds from Aplysina sponges are aerotyonin and homoaerothionin (Moody et al., 1972). Individual variations of these metabolites have been recorded (Walker et al., 1985). Aplysina sponges can release them as a protection against other organisms growing on their surface, or to prevent tissue damage by mobile

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animals (Thompson, 1985). Under natural or artificial stress, release rate can be significantly increased and high levels can be maintained over a long period (Walker et al., 1985). This relates to the presence of these compounds in globular cells, located under the exopinacoderm and in the channels of the endopinacoderm of A. fistularis. Some of these cells degrade, releasing their bioactive substances and, depending on the conditions, can provoke antimicrobial, antifouling, or antipredatory effects (Thompson et al., 1983). Production of metabolites by A. fistularis is continuous, because antibiotic activity occurred in all samples. The sponge can probably modify the mechanisms of bioactive substance production in the face of a particular stimulus (Christophersen, 1991). When inquilism levels were low, the recorded antibiosis in most of the strains did not show considerable variation. However in March, April, and May 1990, when densities of associated organisms increased, the sponge antibiosis on Vibrio sp., Pseudomonas vesicularis and Pseudomonas cepacia reached its highest levels. Christophersen (1991) suggested that metabolite expression could result from a stimulus that interacts with specific receptor systems. He pointed out that one organism can produce different metabolite patterns (quantitative or qualitative) depending on the situation. When the sponge is unstressed, it may use only indispensable chemical defenses, expressed as a continuous release of broad bioactive compounds. However, excessive symbiont reproduction, and arrival of several organisms at the sponge in a short period, could be an important stress factor for the sponge, resulting in a stimulus to adapt to this new ecological situation through modification of the pattern of secondary metabolites. Tissue damage by inquilism may also provoke a bacterial invasion with consequent release of metabolites (Bakus, pers. com.). This agrees with Christophersen (1991) concerning the stimulus-response mechanism and the dynamic character of the secondary metabolite pattern in organisms with chemical defenses. Rasping and predatory fishes could generate strong selective pressures which favor the appearance and persistence of chemical defenses in some tropical benthic invertebrates (Bakus, 1964; Bakus and Green, 1974). Lesions by fish were not observed in sponge specimens used in this study, however fishes also reject A. fistularis fragments in feeding experiments (pers. obs.). Insects develop tolerance to repetitive use of pesticides in agricultural fields, and resistant pathogenic bacteria appear due to indiscriminate use of antibiotics. Similarly, chemically-defended organisms can not afford to persistently use the same chemical defenses to control the fluctuating symbiont populations. Further research in this field should be done in order to identify specific metabolite mechanisms for chemicalbiological control.

Acknowledgements This research was supported by the Instituto Politecnico Nacional (Mexico) through the Programs PIFI and COFAA. Our gratitude to the Centro de Investigaciones Biologicas de B.C.S. (CIB), to Professor G. Bakus for his comments and criticism, and to Dr. E. Glazier (CICIMAR), and Dr. A. van der Heiden (CIAD) for his help on the

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´ J. Caraveo and D. Herrero for their help on the English manuscript. We thank S. Garcıa, taxonomic work and for their comments.

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