Experimental manipulation of sponge/bacterial symbiont community composition with antibiotics:...

R E S EA RCH AR T I C L E

Experimental manipulation of sponge/bacterial symbiontcommunity composition with antibiotics: sponge cell

aggregates as a unique tool to study animal/microorganismsymbiosis

Crystal Richardson, Malcolm Hill, Carolyn Marks, Laura Runyen-Janecky & April Hill

Department of Biology, University of Richmond, Richmond, VA, USA

Correspondence: April Hill, Department of

Biology, Univeristy of Richmond,

Richmond, VA 23173, USA. Tel.: +1 804 484

1591; fax: +1 804 289 8233; e-mail:

[email protected]

Present addresses: Crystal Richardson,

Department of Cell Biology, School of

Medicine, University of Virginia,

Charlottesville, VA, 22908, USA;

Carolyn Marks, Center for Nanoscale

Systems, Harvard University, 11 Oxford

Street, Cambridge, MA, 02138, USA.

Received 14 December 2011; revised 24

February 2012; accepted 7 March 2012.

DOI: 10.1111/j.1574-6941.2012.01365.x

Editor: Gary King

Keywords

microbial ecology; bacterial diversity; host;

antibiotics.

Abstract

Marine sponges can harbor dense and diverse bacterial communities, yet we

have a limited understanding of important aspects of this symbiosis. We devel-

oped an experimental methodology that permits manipulating the composition

of the microbial community. Specifically, we evaluated sponge cell aggregates

(SCA) from Clathria prolifera that had been treated with different classes of

antibiotics to determine whether this system might offer novel experimental

approaches to the study of sponge/bacterial symbioses. Microscopic analysis of

the SCA demonstrated that two distinct morphological types of microbiota

existed on the external surface vs. the internal regions of the SCA. Denaturing

gradient gel electrophoresis and sequence analysis of 16S rRNA gene clone

libraries indicated that we were unable to create entirely aposymbiotic SCA

but that different classes of antibiotics produced distinctive shifts in the SCA-

associated bacterial community. After exposure to antibiotics, some bacterial

species were ‘revealed’, thus uncovering novel components of the sponge-associated

community. The antibiotic treatments used here had little discernible effect on

the formation of SCA or subsequent development of the adult. The experimen-

tal approach we describe offers empirical options for studying the role symbio-

nts play in sponge growth and development and for ascertaining relationships

among bacterial species in communities residing in sponges.

Introduction

Over 100 years ago, Wilson (1907a, b) created a solution

of adult sponge cells by passing Clathria (Clathria) prolif-

era (Ellis & Solander, 1786), known then as Microciona

prolifera, through a fine sieve and demonstrated that the

individual cells would reaggregate to form miniature

sponges with adult features. This groundbreaking work

raised important questions about, and provided a model

system to study, cell adhesion, self/nonself recognition,

immunology, stem cell biology, regeneration, and the ori-

gins of multicellularity, among a host of other questions.

Many studies have used sponge cell aggregates (SCA) to

examine animal cell differentiation and general aspects

of morphogenesis (Wilson, 1911; Huxley, 1912; Galtsoff,

1925a, b; Wilson & Penney, 1930; Spiegel, 1954; Humph-

reys et al., 1960; Curtis, 1962; Humphreys, 1963; Mosc-

ona, 1963, 1968; Borojevic & Levi, 1964; Loewenstein,

1967; MacLennan & Dodd, 1967; MacLennan, 1974;

Buscema et al., 1980; Van de Vyver & Buscema, 1981;

Amano & Hori, 1996). Other studies have used SCA to

study cell motility (Gaino et al., 1985), allorecognition

(Kaye & Reiswig, 1985; Custodio et al., 2004), immune

systems in sponges (Muller & Muller, 2003), secondary

metabolite production (Sun et al., 2007), membrane

structure (Muller, 1982), and phylogenetic trends in

aggregation capability (Holmes & Blanch, 2007). More

recently, researchers have employed the modern tools of

molecular genetics and SCA to re-evaluate some of the

questions that intrigued biologists of the early 20th cen-

tury including general aspects of the control of cell prolif-

eration and growth (Custodio et al., 1998; Muller et al.,

FEMS Microbiol Ecol && (2012) 1–12 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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2003, 2004; Le Pennec et al., 2003; Adell & Muller, 2005;

Pozzolini et al., 2005; Valisano et al., 2006, 2007). Funay-

ama (2010) provided a recent review of the stem cell sys-

tem in sponges and the utility of SCA to address a variety

of cellular, molecular, and developmental processes.

Sponges harbor exceptionally diverse communities of

microorganisms (Vogel, 2008; Webster et al., 2010). Precise

estimates of intra-host species richness are not yet available,

but sponges clearly host novel bacterial taxa, even entirely

new phyla, many of which are distinct from environmental

bacterioplankton (Fieseler et al., 2004; Hill et al., 2006;

Taylor et al., 2007a, b; Lafi et al., 2009; To Isaacs et al.,

2009; Radwan et al., 2010; Gerce et al., 2011). The ecologi-

cal interactions occurring among these bacteria are poorly

understood, and the structure of the intra-host bacterial

communities is only now beginning to be elucidated.

Schmitt et al. (2011a, b) placed bacterial operational

taxonomic units into core (present in all sponges), variable

(present in a sub-set of hosts), and species-specific (present

in a single host) categories. This provided rough estimates

of relative abundances and addressed some general ques-

tions about evenness within the community. Attempts to

uncover bacterial symbiont community structure beyond

this level of resolution face several practical challenges.

First, defining the types of interactions that occur among

symbionts and between the host and its symbiont is diffi-

cult owing to the complexity of the community harbored

by sponges. Second, identifying individual species that are

rare (or not so rare) is a significant challenge with available

molecular approaches, although next-generation sequenc-

ing offers some promise in this regard (Lee et al., 2010;

Webster et al., 2010; Schmitt et al., 2011a). Finally, it is dif-

ficult to conduct manipulative experiments (e.g. removing

particular symbiont species) to monitor cause-and-effect

relationships among the hosts and symbionts (e.g.,

Lemoine et al., 2007; Webster et al., 2008). For this reason,

our knowledge of basic aspects of sponge–bacterial symbio-

ses remains rudimentary.

To fully understand the complex interactions that

occur among the sponge and its symbiotic partners, we

require systems that permit controlled experiments.

Although the microbial symbionts appear to provide a

variety of important functions to the sponge (Taylor

et al., 2007a, b), teasing apart the materials and services

that are exchanged among the bacterial and animal part-

ners has proven remarkably challenging. For sponges that

harbor phototrophic symbionts, several studies have

shown the specific benefits the animal host receives

(Wilkinson, 1979; Hill, 1996; Thacker, 2005; Weisz et al.,

2010). Microorganisms with other metabolic phenotypes,

however, present significant challenges to understanding

these important symbiotic interactions. Evidence indicates

that sponges benefit from the presence of microbial

associates through enhanced metabolic capability [e.g.

bacteria capable of reduction–oxidation reactions, vitamin

production, nutrient transport, and utilization (Hentschel

et al., 2002; Piel, 2004; Hoffmann et al., 2005; Steger

et al., 2008; Thomas et al., 2010)]. One potential role for

heterotrophic bacterial symbionts is the production of

secondary metabolites that may provide a variety of

important ecological functions [e.g. ameliorating stress

(Dattelbaum et al., 2010), defense from predators and

parasites (Taylor et al., 2007a)]. Hochmuth et al. (2010)

found that the putative sponge-specialist bacterial lineage

Poribacteria may produce methyl-branched fatty acids,

and Thomas et al. (2010) found that metabolic interac-

tions are shared between the host Cymbastella concentrica

and its symbiont community, which supports previous

work examining physiological integration between the

partners (e.g. Arillo et al., 1993; Regoli et al., 2004).

These and other studies point to the clear role that

symbionts have on the sponge phenotype.

In contrast to the influence that symbionts have on

host phenotype, almost nothing is known of the role that

bacterial symbionts might play in normal developmental

processes of the host. Vertical transmission of bacterial

associates from mother sponges to their offspring indi-

cates that developmentally, ecologically, and evolution-

arily important relationships exist between these

symbiotic partners (Kaye, 1991; Usher et al., 2001, 2005;

Ereskovsky et al., 2005; Enticknap et al., 2006; Schmitt

et al., 2007, 2008; Sharp et al., 2007). In most cases, how-

ever, the microbial community and the animal develop-

mental system are studied as separate domains. Based on

the supposed metabolic integration, the transmission of

symbionts among generation, and the density of bacteria

harbored by many sponges, it seems likely that sponge

development may also be influenced by symbionts. An

ideal system would be one that permits examination of

developmental and symbiotic domains simultaneously,

and as indicated by Taylor et al. (2007a), a major goal of

work with sponge microbial symbioses is defining the

interactions between the host and its symbionts.

While SCA provide a fascinating system to study

aspects of animal development, they also provide an

opportunity to manipulate bacterial symbioses in a con-

trolled manner. Thus, SCA may serve as a useful tool and

a model system to understand nuanced aspects of animal/

bacterial symbioses. We used a battery of antibiotics with

distinct modes of action during the creation of SCA and

then monitored the effects that the different antibiotics

had on microbial community structure and sponge devel-

opment (see also Sipkema et al., 2003). As a proof-

of-concept, we had three primary aims. The first was to

determine whether it was possible to create completely

axenic SCA. The second was to evaluate the effects

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2012) 1–12Published by Blackwell Publishing Ltd. All rights reserved

2 C. Richardson et al.

different classes of antibiotics had on the types of bacteria

present in the SCA. The final aim was to assess whether

removal of bacterial associates (through antibiotic treat-

ment) had any discernible effect on sponge development.

Materials and methods

Clathria prolifera was collected at dawn from pilings at

the Virginia Institute of Marine Science (37°14.819′N; 76°30.052′W). SCA were created following the protocol pre-

sented in Custodio et al. (1998) with some modifications.

The freshly collected C. prolifera individuals were cut into

1-mm3 pieces within 4 h of collection. The sponge pieces

were placed in 50-mL conical tubes with Ca- and Mg-free

seawater with EDTA (CMFSW-E). The tubes were rocked

on an orbital shaker at 110 r.p.m. for up to 1 h. The first

wash was discarded. The second and third washes were

passed through a 40-lm nylon mesh, and the dissociated

cells were pelleted (500 g for 5 min). The sponge cell pel-

lets were re-suspended in sterile-filtered seawater with

antibiotics (see below). Six millilitres of the cell suspen-

sions were plated in 60-mm Petri dishes at densities of

107 cells mL�1. Petri dishes were placed on an orbital

shaker at 110 r.p.m. and 25 °C (i.e. RT). Within 12 h,

SCA began to form. One-third of the solution in each

Petri dish was replaced with fresh media daily. Once the

SCA reached a diameter of 0.3 mm, they were transferred

to new plates containing sterile-filtered seawater with

antibiotics.

Four antibiotic treatments were used to assess the effect

of antibiotics on microbial communities present within

the SCA. The first antibiotic treatment involved the use

of a combination of streptomycin (an aminoglycoside)

and penicillin (a b-Lactam cell-wall inhibitor) each at

concentrations of 50 lg mL�1. Three other treatments

involved the use of single antibiotics. Nalidixic acid,

which is a quinolone that inhibits DNA synthesis through

inactivation of DNA gyrase, was added at a concentration

of 50 lg mL�1 (Wolfson & Hooper, 1985). Trimethor-

pim inhibits tetrahydrofolic acid synthesis, and thus DNA

replication, and was added at 25 lg mL�1. Gentamicin,

administered at a concentration of 20 lg mL�1, has

broad effects although the precise mode of action is

poorly understood. We attempted to create SCA without

antibiotics as a procedural control, but aggregates did not

form either owing to high levels of biological contamina-

tion (e.g. bacteria, fungi) or because micro-predators

often proliferated. As is common in many cell-culture

systems, we were unable to create viable SCA without

antibiotics and thus were unable to employ that control.

To visualize the microbial communities present after

exposure to antibiotics, several SCA were sampled from

each treatment for histological work. We employed a

modified vacuum-assisted microwave (Pelco BiowaveTM)

protocol to process tissues for subsequent transmission

electron microscopy (Giberson & Demaree, 2001). Pri-

mary fixation occurred in 2.5% Glutaraldehyde and 1%

paraformaldehyde in seawater. After a wash with seawa-

ter, the SCA were placed under vacuum in a 1% Osmium

tetroxide solution in water for secondary fixation. After

washing with water, an en bloc stain with 1% uranyl ace-

tate was performed (also under vacuum). Tissues were

dehydrated (50%, 70%, 90%, and three times at 100% for

40 s each) before transferring them to a series of propyl-

ene oxide-Epon 812 (EM bed-812; Electron Microscopy

Science) solutions (1 : 1, 1 : 2, and then 1 : 4 (v/v) for

30 min each). Three final incubations in full strength

Epon 812 (30 min each) preceded the final embedding

step, which occurred at 60 °C for 18 h. SCA were sec-

tioned with an ultramicrotome (Leica Ultracut UCT) and

examined using a JEOL 1010 TEM with an Advanced

Microscopy Techniques XR-100 Digital CCD.

We also employed molecular tools to analyze the

microbial communities present in SCA after exposure to

antibiotics. Genomic DNA was extracted from randomly

selected SCA taken from each treatment using the Ultra-

Clean® Soil DNA Isolation Kit (MO BIO Laboratories).

We also extracted DNA from tissue from freshly collected

sponges, using the same methods, to compare natural

symbiont communities to those from the antibiotic treat-

ments. Metagenomic DNA was used as template for PCR

employing the universal 16S rRNA gene primer pairs

1055F and 1406R-GC (Wang et al., 2007). Negative con-

trols were included for each 16S rRNA gene amplification

reaction. The cycle profile included an initial denaturing

step at 95 °C for 2 min; 35 cycles of 95 °C for 1 min;

63 °C for 30 s, and 72 °C for 1 min; and a final exten-

sion step at 72 °C for 5 min. The final concentrations for

primers, MgCl2, and dNTP were 10 pmoles, 3, and

2 mM, respectively. One unit of TakaraTM Taq DNA

polymerase was added as was 10 ng of DNA.

PCR products were examined via Denaturing Gradient

Gel Electrophoresis (DGGE) using the Bio-Rad DCodeTM

Universal Mutation detection system (Bio-Rad Labora-

tories, Inc.) using a 30–70% denaturing gradient in a

10% (w/v) polyacrylamide gel in 19 TAE. Electrophoresis

was performed for 17 h at 70 V and 60 °C. The gels were

stained for 30 min in 19 TAE spiked with 1.5 lg of ethi-

dium bromide, destained for 25 min in dH2O, and visu-

alized and photographed with a GelDoc System (GelDoc

2000; Bio-Rad Laboratories, Inc.). A selection of bands

unique to each treatment was carefully excised using an

ethanol-sterilized scalpel for subsequent sequencing. DNA

was eluted from the bands overnight in 25 lL of TE buf-

fer. The eluted DNA was re-amplified with primers 1055F

and 1406R-GC using the PCR conditions described


Sponge cell aggregates as a model to study symbiosis 3

previously. PCR products were purified (MinElute Gel

Extraction Kit; Qiagen) and cloned using the TOPO® TA

Cloning Kit following the manufacturer’s instructions. In

addition to the DGGE band sequences, we also sequenced

10 clones produced from the total pool of PCR amplicons

recovered from each of the treatments and the control

sponge. Ten insert-positive colonies were selected from

each plate, and plasmids were sequenced at Virginia

Commonwealth University’s Nucleic Acid Research Facil-

ity. BLAST searches were conducted to compare our results

with the 16S rRNA gene in the NCBI GenBank database

(http://www.ncbi.nlm.nih.gov). All sequences were ana-

lyzed for taxonomic affiliation using the Classifier pro-

gram at the Ribosomal Database Project using default

parameters (Wang et al., 2007).

To assess the effects of antibiotic treatment on sponge

development, two comparisons were performed. The first

involved examining the number of SCA that formed in

each treatment after 1 day. Plates were examined after

24 h to assess the extent of sponge cell reaggregation.

Twenty-five-mm2 sections were examined, and all SCA

were counted. Differences between treatments in SCA for-

mation were analyzed using one-way ANOVA followed by

Tukey’s HSD post hoc test (Zar 2009). The second involved

measuring the percentage of SCA that attached to the bot-

tom of plates after shaking was stopped. For this experi-

ment, individual SCA were transferred to 12-well culture

plates where they were allowed to undergo metamorphosis

and form miniature sponges (i.e. rhagons – Fig. 1). We

measured attachment as the ability of a metamorphosizing

SCA to avoid dislodgement when a stream of approxi-

mately 100 lL of medium was gently released from a pip-

ette toward the SCA. Differences in the percent of SCA

that remained attached to the plate (after arcsine transfor-

mation) were analyzed using one-way ANOVA followed by

Tukey’s HSD post hoc test (Zar, 2009). In addition to the

streptomycin/penicillin, gentamicin, nalidixic acid, and tri-

methoprim treatments, we added another treatment to this

analysis that included a mix of all antibiotics from the

treatments described above at quarter strength. The SCA

produced from the combined antibiotic treatment were

also used to amplify 16S rRNA gene, and the resulting

PCR products were cloned and analyzed as described

previously.

Results

Transmission electron micrographs showed that a mor-

phologically distinct microbiota existed on the external

surface of many SCA compared with the internal regions

of the SCA (Figs 2 and 3). The external bacteria exhibited

a variety of structures that appear to mediate attachment

to the acellular region of SCA surfaces. In contrast to the

bacteria along the outer margin of the SCA, a morpho-

logically simple bacterial community was found in the

inner portions of the SCA (Fig. 2). Regardless of the anti-

biotic regime employed, we were unable to create SCA

that were devoid of bacteria. A number of bacteria with

Fig. 1. Clathria prolifera SCA and metamorphosed sponges derived from SCA. Surface- and trans-illuminated images are shown (left and right

respectively). The red pigmentation in this sponge is primarily owing to an abundance of carotenoids in its cells. After metamorphosis, adult

features were obvious as can be seen in the trans-illuminated image. Abundant clusters of choanocyte chambers are easily identified (dashed

black oval), a well-defined canal system has developed (dashed white lines), and an osculum projecting upwards was often observed in the

miniature adult sponges (black circle).



fimbriae, and other unique structures projecting from

their surfaces, were observed along the external margin of

many SCA (e.g. Fig. 3c and d; Supporting information,

Fig. S1). These fimbriae and projections may be able to

facilitate interactions with the cellular cortex by forming

what appear to be adhesions (Fig. 3d and e).

PCR amplicons derived from bacterial DNA isolated

from SCA were identified in each antibiotic treatment

using DGGE (Fig. 4). This finding indicates that antibi-

otic treatments did not totally cure SCA of bacteria, as

was also observed in the electron micrographs (Figs 2

and 3). Analysis of sequences from the excised DGGE

bands (Fig. 4) and the pool of PCR product that we

cloned indicated that taxonomically distinct microbiota

persisted within the host depending on the antibiotic

treatment used (Fig. 5; Table S1). Of particular note were

the major differences we observed between sponges

collected from the wild and the SCA reared under differ-

ent antibiotic treatments (Fig. 5). While cyanobacteria

were found to dominate the isolates we recovered from

wild sponges (WS, Fig. 5), taxonomically distinct bacteria

were detected in the other treatments. For example, gen-

tamicin (G, Fig. 5) had a high percentage of taxa from

the Phylum Bacteroidetes, which was not the case for any

of the other antibiotic treatments; the only type of micro-

organisms detected in trimethoprim-treated SCA came

Fig. 2. Representative transmission electron micrographs from Clathria prolifera SCA produced with the streptomycin/penicillin treatment.

Despite treatment with antibiotics, remnant populations of bacteria were observed in each treatment. In many replicates, a morphologically

variable microbiota was observed on the external surface of the SCA. These organisms were often affixed to or associated with the extracellular

material surrounding the SCA (image on left). The bacteria observed deep within the SCA were always phenotypically nondescript and did not

appear to be localized in any predictable pattern within the SCA.

(a) (b) (c)

(d) (e) (f)

Fig. 3. Representative bacteria associated with the external surface of Clathria prolifera SCA from the streptomycin/penicillin treatment are

shown (a–d). A close up of the point of attachment in (d) is shown in (e). For comparative purposes, bacteria found in the inner portions of the

SCA are shown in (f). Scale bar in a–e are 100 lm. Scale bar in (f) is 500 lm.



from the Phylum Proteobacteria. Each of the antibiotic

treatments differed from one another in taxonomic com-

position of the SCA-associated microbiota (Fig. 5; Table

S1), which was likely related to the distinct modes of

action that the different antibiotics had on SCA, and the

relative susceptibilities of the sponge-associated bacteria

to the antibiotics.

Most of the sequences we obtained from antibiotic

treated SCA shared highest affinities with samples from

environmental sources. That is, BLAST searches recovered

symbiont-specialist lineages, and thus putative sponge-

specialist symbionts, in less than 20% of the cases. In the

S-/P-treated SCA, we identified two symbiont-affiliated

sequences: a Vibrio-like sequence (accession JQ004822;

band 2, Fig. 4) showed affinity to bacteria associated with

sponges from Florida (e.g. Mycale and Ircinia), and a

Planctomyces-like sequence (accession JQ004818) was sim-

ilar to sequences derived from a Haliclona host in the

northwest Pacific. Three sequences from the gentamicin-

treated SCA retrieved sponge-affiliated bacterial taxa from

GenBank during BLAST searches. The first was Marinoscil-

lum sp. (accession JQ004825), the second was Roseivirga

sp. (accession JQ004830), and the third was a Labrenzia

species from DGGE band 7 (accession JQ004834; Fig. 4).

Three of the sequences we analyzed from the nalidixic

acid-treated SCA recovered sponge-/symbiont-associated

bacteria from GenBank [Crocinitomix (accession JQ004841);

Seohaeicola (accession JQ004849); and Pseudovibrio (accession

Fig. 4. DGGE results comparing microbial community characteristics

for each antibiotic treatment. C = control sponge collected from the

native habitat, S/P = streptomycin/penicillin, N = nalidixic acid,

G = gentomicin, and T = trimethoprim. DNA was extracted, cloned,

and sequenced from bands excised from the gel (identified with

numbers).

Fig. 5. Comparison of broad taxonomic representation of bacteria identified in SCA after each of the antibiotic treatments. The first row

includes a taxonomic breakdown at the rank of Phylum. The second row includes a taxonomic breakdown at the rank of Class. The numbers

within some of the wedges represent the number of families that were recovered. Any wedge without a number is represented by a single

family. S/P = streptomycin/penicillin, N = nalidixic acid, G = gentomicin, T = trimethoprim, M = mixed antibiotic treatment, and WS = wild-

caught sponges.



JQ004843)]. None of the sequences identified in the

trimethoprim or mixed antibiotic treatments, nor in the

wild-caught sponge, included bacteria from putative sym-

biont lineages. The remaining sequences shared highest

identity with bacteria from environmental sources and a

variety of biofilms (Table S1). While we found different

numbers of bacterial genera in the different antibiotic

treatments (as predicted in Classifier; S/P = 7; G = 11;

N = 11; T = 5; M = 6), it is unclear whether these repre-

sent significant differences.

One-way ANOVAs indicated that significant differences

existed among antibiotic treatments for SCA production

(Fig. 6a; F5,24 = 11.43, P < 0.001). Tukey’s HSD test indi-

cated that significantly lower numbers of SCA were pro-

duced in each of the antibiotic treatments compared with

the streptomycin/penicillin treatment, which resulted in

the highest number of SCA (Fig. 6a). The different antibi-

otic treatments did not differ in their effect on SCA abil-

ity to attach to the substratum (Fig. 6b; F5,12 = 1.76,

P = 0.21).

Discussion

Our primary goal with this study was to assess the utility

of SCA exposed to different types of antibiotics as a

model to study animal/bacterial symbioses in general and

sponge/bacterial interactions in particular. While we were

incapable of creating axenic SCA, the shifts we observed

in community structure were intriguing. Several observa-

tions from our work indicate that the approach we

describe may be very useful for future endeavors to tease

apart nuanced aspects of host/symbiont interactions as

well as interactions among members of the bacterial com-

munity.

One area that will likely be of broad interest involves

the distinct morphological types of bacteria that appear

on the surface vs. inner regions of the SCA. Superficially,

many of the externally located bacteria were often

phenotypically similar to bacterial pathogens attached to

mucosal membranes in animals [e.g. fimbriae of entero-

pathogenic and uropathogenic Escherichia coli (Giron

et al., 1993, and Johnson, 1991, respectively)] and share

characteristics of bacteria forming biofilms (Flemming &

Wingender, 2010; Pamp et al., 2007). The findings we

present differ from work with sponge larvae that found

an absence of bacteria in the outer ciliated region of the

larva (Schmitt et al., 2007). The spatial segregation that

we observed may involve phenomena that are unique to

SCA, but SCA appear to provide an opportunity to exam-

ine processes involved in biofilm formation. A majority,

but not all, of the SCA had bacteria along the external

margins. It is unclear whether the host has any control

over the composition of the internal pools of bacteria

that we observed, but SCA also provide a model system

to explore any host-influenced community selection (if,

indeed, these communities are distinct). Bacteria with

certain structures may be able to attach to the outside of

the SCA (which may be a prelude to entry into a host

cell/SCA), whereas bacteria without those structures may

remain in the inner regions of SCA. It is also possible

that once internalized, the bacteria turn off expression of

attachment factors because they no longer need them.

The data we report could not elucidate the taxonomic

composition of these two communities, but future work

could examine whether different types of bacteria reside

in these two distinct habitats (perhaps with FISH). Along

(a)

(b)

Fig. 6. Effects of antibiotics on Clathria prolifera SCA formation (a)

and SCA metamorphosis and attachment to the substratum (b). (a)

The S/P treatment resulted in the production of a significantly

greater number of SCA. None of the other treatments differed in

the number of SCA produced, but all were lower than S/P. (b) None

of the antibiotics affected the ability of C. prolifera SCA to attach to

the substratum. S/P = streptomycin/penicillin, N = nalidixic acid, G =

gentomicin, T = trimethoprim, and M = mixed antibiotic treatment.

Any treatment connected by a line did not differ at P < 0.05.



these lines, our data indicate that one can enrich for rare

bacterial species, many of which are not a conspicuous

part of the flora associated with wild-caught sponges.

This permits at least two types of analyses. The first is

that antimicrobial treatment may permit detection of

unusual components of bacterial taxonomic diversity. For

example, our detection of three species from the order

Oceanospirillales (Halomonas, Marinomonas, and Salinico-

la) indicates this might be a valuable tool to uncover

novel diversity, which has not been commonly encoun-

tered in sponges (Sfanos et al., 2005; Li & Liu, 2006;

Kaesler et al., 2008). Many of the sponge-derived 16S

rRNA gene sequences we uncovered are closely related to

seawater, biofilm, and sediment sequences and thus may

not come from symbiosis-specific lineages. Thus, the

second type of analysis that SCA permit includes an

analysis of whether the microbial species affiliated with

nonsymbiotic evolutionary lineages (i.e. affiliated with

environmental sources) represent transient interactions,

commensalisms, or more intimate ecologic interactions

(e.g. mutualisms).

In contrast, some of the microorganisms we encoun-

tered appear to come from symbiont-specializing lineages.

For example, Crocinitomix from the nalidix acid treat-

ment (accession JQ004841), Labrenzia and Marinoscillum

from the gentamicin treatment [accession JQ004834 &

JQ004825, respectively), and Vibrio from the streptomy-

cin/penicillin treatment (accession JQ004813)] all may

belong to symbiosis-specialist lineages. SCA offer the pos-

sibility of contrasting the conditions that favor one type

of host/symbiont interaction over another (e.g. the degree

of specificity or the level of reciprocity between partners).

In this context, SCA holds the potential to uncover the

ecological factors that release rare species from the con-

straints on their prevalence in sponge tissue under normal

circumstances.

Another favorable aspect of SCA work is that longer-

term growth experiments could begin to partition the

effects particular suites of bacteria have on host perfor-

mance. Sponges are one of the most prolific producers of

novel natural products (Blunt et al., 2011), but the gene-

sis of many of the compounds ultimately rests with

microbial associates (Taylor et al., 2007b). For example,

we know that tissue from C. prolifera contains abundant

and diverse carotenoids (Fig. 1), some of which have bac-

terial origins (Dattelbaum et al., 2010). By monitoring

the chemical profiles of the rhagons, we may be able to

parse the effects that some of the sponge-associated bacte-

ria have on host phenotype. This type of work should be

especially applicable to any sponges capable of SCA for-

mation and would be particularly attractive for sponges

that produce pharmaceutically interesting compounds

(Taylor et al., 2007b).

The shift in banding patterns that we observed in our

DGGE analysis and the changes in the composition of the

metagenome uncovered in our sequence analysis were

compelling. Our data clearly indicate that it is possible to

change the composition of the sponge-associated bacterial

community. The observed shift had no affect on one

aspect of host development (i.e. attachment to the sub-

stratum), which was surprising given that we anticipated

the loss of bacterial associates to compromise sponge per-

formance in some noticeable manner (see Sipkema et al.,

2003). Other antibiotic regimes might produce different

outcomes, and additional work in this area is clearly

required before definitive statements can be made about

the effects symbionts have on sponge development. Given

the long-standing interest in linking symbiont and host

performance, however, SCA offer useful experimental

approaches to teasing apart symbiont contributions to

phenotypic features of the holobiont.

Gerce et al. (2011) recently identified differences

between the bacterial communities associated with the

surfaces of eight Mediterranean sponges and those found

within the mesohyl. Their analysis of DGGE banding pat-

terns identified two main clusters that could be distin-

guished as containing samples from the mesohyl or

surface of their sponges. SCA may provide an experimen-

tal mechanism to explore how these distinctions arise

during sponge development given our observations of

unique microbiota associated with the external surfaces

of SCA. As indicated previously, the spatial segregation of

bacterial associates that occurred in our experiments indi-

cates that the host may have some selective capacity. If

this is so, SCA provide an unparalleled opportunity to

examine aspects of host:symbiont specificity.

Recently, To Isaacs et al. (2009) found that wild-caught

C. prolifera harbored a dense and diverse microbiota

dominated by cyanobacteria, actinobacteria, and spiro-

chetes. Those authors did not find a strong congruence

between DGGE profiles and the clone libraries, whereas

our dominant DGGE band (band 1 in Fig. 4) matched

the abundance of this sequence type in the clones we ana-

lyzed that were derived from the total pool of PCR

amplicons recovered from the SCAs. Of particular impor-

tance for the work reported here, however, was To Isaacs

et al.’s (2009) documentation of a shift in bacterial com-

munity composition upon rearing in aquaria. Similarly,

Taylor et al. (2005) reported significant geographic

variability in the composition of microbial communities

harbored by C. concentrica. Combined with our findings,

these data raise important questions about the nature of

the association between C. prolifera and the bacteria har-

bored in and on its tissues. Furthermore, To Isaacs et al.

(2009) documented the loss of actinobacteria in aquaria-

reared sponges, but we only recovered this type of



bacteria when SCA were exposed to a mix of antibiotics.

Thus, aquaculture procedures looking to produce particu-

lar bacterially derived bioactive compounds (Hill, 2004)

may consider our findings particularly intriguing.

The diversity of microbial communities, and the chal-

lenges working with fastidious bacteria, present significant

empirical challenges to ecologists interested in under-

standing how the communities are structured. For exam-

ple, recent work has indicated that the symbiont

communities found in high and low microbial abundance

sponges (Weisz et al., 2008) may have distinct character-

istics (e.g. Schlappy et al., 2010; Erwin et al., 2011; Gerce

et al., 2011; Schmitt et al., 2011b). The SCA we produced

using different antibiotics opens the possibility of explor-

ing nuanced aspects of the bacterial communities har-

bored by sponges. While C. prolifera continues to be a

model to study aspects of developmental and cellular

biology, we introduce this sponge, and SCA, as a new

model for examining microbial symbioses. Employing

antibiotics permits a classic ‘species removal’ type of eco-

logical experiment that has been difficult to perform to

date. Although the antibiotics we used represent blunt

instruments and more precise tools for manipulating

symbiotic bacterial community remain to be developed,

our study demonstrates the practical utility of SCA to

enhance manipulative studies of microbial symbioses.

Acknowledgements

Grants from the National Science Foundation to A.H.

and M.H. (OCE-0647119 & DEB-0829763) funded this

work. Students in the HHMI-supported Integrative

Quantitative Science course at the University of Rich-

mond contributed all of the cloned metagenomic

sequences to the project. We thank three anonymous

reviewers for helpful comments and suggestions for

improving the manuscript.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Electron micrographs of bacteria with unusual

morphological characteristics from each antibiotic treat-

ment.

Table S1. Aggregate data on DNA sequences isolated

from clone libraries and DGGE bands.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

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