acmi.0.000691.v1-html.html

How suitable is freshwater sponge

Ephydatia fluviatilis

(Linnaeus, 1759) for time-

integrated biomonitoring of microbial water quality?

Authors

Allison Cartwright

, James S.G. Dooley

, Chris D. McGonigle

, Joerg Arnscheidt

Ulster University, Cromore Road, Coleraine, Northern Ireland, BT52 1SA

Corresponding contact

–

a.cartwright@ulster.ac.uk

ORCID ID

Allison Cartwright - 0000-0003-2796-4048

James S.G. Dooley - 0000-0002-9459-5572

Chris D. McGonigle

–

0000-0002-0262-0559

Joerg Arnscheidt - 0000-0002-9744-9917

Abstract

Faecal pollution of water by bacteria has a negative effect on water quality and can pose a

potential health hazard. Conventional surveillance of microbial water quality relies on the

analysis of low frequency spot samples and are thus likely to miss episodic or periodic

pollution. This study aimed to investigate the potential of filter feeding sponges for time-

integrated biomonitoring of microbial water quality. Laboratory trials tested the effects of

different ratios of bacterial abundance and the sequence of exposure on bacterial retention by

the freshwater sponge

Ephydatia fluviatilis

(Linnaeus, 1759) to establish their potential to

indicate bacterial exposure.

Gemmule grown sponges were simultaneously exposed to

Escherichia coli

and

Enterococcus

faecalis

but at different ratios (Trial 1) or individually exposed to each of bacterial species

but in different sequential order (Trial 2). The

E. coli

and

E. faecalis

retained in each sponge

was quantified by culture on selective agars. Data analysis was conducted using Kruskal-

Wallis test and/ or Mann Whitney U test to compare between the numbers of bacteria

retained in each treatment. Additionally, the Wilcoxon Matched-Paired Signed-Rank test was

used for comparison of the different bacterial abundance retained within each individual

sponge.

Sponges from all trials retained

E. coli

and

E. faecalis

in small numbers relative to the

exposure (<0.05% Trial 1 and <0.07% Trial 2) but exhibited preference for retention of

coli

. Higher abundance of either bacterial species resulted in significantly lower (p<0.005)

retention of the same species within sponges (Trial 1). An initial exposure to

E. coli

resulted

in significantly higher (p=0.040) retention of both bacterial species than when sponges were

exposed to

E. faecalis

first (Trial 2).

Bacterial retention by sponges was neither representative of bacterial abundance in the

ambient water nor the sequence of exposure. This implies either selective filtration or an

attempt by sponges to prevent infection. However, freshwater sponges may be useful in

biomonitoring as qualitative time-integrated samplers of faecal indicator bacteria.

Keywords

Water quality monitoring, faecal pollution, freshwater sponges,

Ephydatia fluviatilis,

Enterococcus faecalis, E. coli

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Preprint DOI:

Posted on August 17, 2023

https://doi.org/10.1099/acmi.0.000691.v1

© 2023 Crown Copyright. This is an open-access article distributed under the terms of the Creative Commons
Attribution License.

1. Introduction

Freshwater and transitional water bodies can receive high inputs of bacteria including

enterococci and

Escherichia coli

from human and animal waste (Walk

et al.

, 2007; Longo

al.

, 2010; Gao

et al.

, 2013). This bacterial pollution not only compromises the integrity of

aquatic ecosystems but can also have negative effects on human health as sewage contains

pathogenic bacteria and viruses (Cabral, 2010; Longo

et al.

, 2010). Furthermore, Metcalf

al.

(2023) found that faecal indicator bacteria can bind with other anthropogenic pollutants

including glass and microplastics, allowing them to persist in the environment for over 25

days and so they may be present long after a pollution event. Recently, within the United

Kingdom (UK), there has been an increase in public awareness of storm overflow

management which allows wastewater including storm flow and raw sewage to be released

directly into water systems. In 2022, from 13,080 monitored storm overflows, there has been

a total of 301,091 events with the release of untreated wastewater into English water systems

(DEFRA, 2023). All these events contribute to faecal pollution of aquatic systems, and in

extreme cases they cause mass fish mortality in river systems. For example, in 2017 Anglian

Water (UK) released around 6 million litres of raw sewage killing 5,000 fish, resulting in a

fine of £560,170 (Environmental Agency, 2023). Therefore, faecal indicator bacteria are

monitored so that the public can be warned when the level of water contamination presents an

increased risk of exposure to harmful pathogens (Wiedenmann

et al.

, 2006; Ferguson

et al.

2012). Among the bacteria routinely sampled to indicate microbial water quality are

E. coli

and

Enterococcus

spp. (Wiedenmann

et al.

, 2006; Ferguson

et al.

, 2012; Metcalf

et al.

2023). If counts of these, or other faecal indicator bacteria, exceed the acceptable threshold

values set by regulatory agencies the water should not be used for bathing or drinking

purposes until bacterial counts drop to safe levels (Fu

et al.

, 2006; Kay

et al.

, 2006; Harwood

et al.

, 2014).

Regulators conventionally evaluate microbial parameters based on low frequency, single spot

water samples (Kirchner

et al.

, 2004; Briciu-Burghina

et al.

, 2014). While this type of

sampling can show bacterial abundance through culture-based quantification, it will only

indicate the bacterial load at a few random points in time and is thus likely to miss episodic,

periodic or persistent pollution. However, filter feeders such as sponges process waterborne

bacteria and potentially also accumulate bacteria from episodic pollution over an extended

period of time when post event water samples would not detect them anymore. When

sponges are feeding they have the ability to retain bacteria in their mesohyl (Wehrl

et al.

2007; Topçu

et al.

, 2010; Perea-Blázquez

et al.

, 2013). A sponge sampled at any given point

will contain bacteria which have been filtered from the water either awaiting digestion or

have been incorporated as symbionts (Fu

et al.

, 2006; Wehrl

et al.

, 2007). Thus, the

quantification of bacteria retained within sponges could be used as an indication of the filter

organisms’ exposure to waterborne bacteria.

Therefore, this study aimed to investigate the

potential of freshwater sponges to be used for time-integrated biomonitoring of bacteria. We

determined the retention of the faecal indicator bacteria

E. coli

and

Enterococcus faecalis

developing a novel model system, based on in vitro cultivation of

E. fluviatilis

to quantify

bacterial retention by sponges

The experimental approach was to test individual sponges in

laboratory microcosms with regard to the effect of sponge exposure to these bacteria in

different abundance ratios and varied sequence of exposure.

2. Methods

Two laboratory trials were conducted to explore the retention of bacteria by sponges and how

this related to different modes of exposure. Trial 1 tested simultaneous exposure of sponges

E. coli

and

E. faecalis

, but at different abundance ratios. Trial 2 exposed sponges to either

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E. coli

E. faecalis

before removal of sponges for subsequent exposure to the other bacterial

101

species. These trials were conducted to establish if the abundance of

E. coli

and

E. faecalis

102

retained by sponges were a quantitative representation of their numerical ratio in the ambient

103

water laboratory microcosms (trial 1) or whether it responded to the sequence of exposure to

104

different bacteria in laboratory microcosms (trial 2).

105

106

2.1 Culturing of sponges

107

Ephydatia fluviatilis (

Linnaeus, 1759) gemmules were surface sterilised by submersion in 1%

108

for 10 min prior to hatching (modified method from Rasmont, 1970). To allow for

109

movement of the sponges, gemmules were hatched onto a piece of 4 cm

transparency

film

110

(Xerox type 1) in a 6 cm diameter petri dish containing 10 ml UV treated (10 min at 254 nm)

111

mineral water. Culturing in mineral water enabled high hatching rates as it contains silica

112

required for spicule formation. Seven-day-old sponges were used for all experiments with

113

their individual horizontal dimensions (mm

) measured at the start of the trials.

114

115

2.2 Culturing of bacteria

116

The bacteria used for these experiments were

E. coli

GFP (ATCC 25922GFP) and

E. faecalis

117

(MW01105 - Conwell

et al.

2017). Before each experimental addition of bacteria, a 10 ml

118

overnight culture was grown of each bacteria culture separately. Each culture was produced

119

using 10 ml of nutrient broth in a universal tube to which 1 bead coated with the specific

120

bacteria was added from the -80

℃

stock culture. This suspension was mixed and incubated

121

at 37

℃

for 18 h.

122

123

2.3 Bacterial addition

–

(trial 1) ratios of E. coli and E. faecalis

124

Universal tubes with 18 ml of UV treated mineral water had 2 ml of bacterial suspension

125

added from the overnight nutrient broth culture. The bacteria suspension contained different

126

ratios of

E. coli

E. faecalis

. There were 22 replicates conducted over three trials for each of

127

the following

E. coli

E. faecalis

ratios: 10:90; 50:50; 90:10. Sponges on transparency films

128

were placed into the tube, so that they were leaning on the wall, to minimise settling of

129

bacteria on sponge surfaces due to deposition. These tubes were kept at 20 ºC for 24 h.

130

131

2.4 Bacterial addition

–

(trial 2) separate exposure to E. coli and E. faecalis

132

Universal tubes with 19 ml of UV treated mineral water had 1 ml bacteria suspension added

133

(either

E. coli

E. faecalis

) before the sponge was introduced on transparency film and

134

positioned to lean

on the tube’s

interior side wall. There were eight replicates for each

135

exposure conducted over two trials, however one sponge exposed to

E. coli

first died so was

136

excluded. The tubes were incubated at 20 ºC for 24 h before the sponge was removed and

137

washed. The sponge was then placed in a fresh universal tube with mineral water and 1 ml of

138

the bacterial species that the sponge had not been previously exposed to. These were

139

incubated at 20 ºC for a further 24 h.

140

141

2.5 Sample processing (trial 1 and 2)

142

After exposure to bacteria, transparency films with sponges were removed and washed with

143

autoclaved deionised water three times to remove bacteria from the surface before being

144

placed in a fresh tube with 20 ml of UV treated mineral water. These were left for a further

145

24 h to allow the sponges to incorporate the bacteria retained. To end the trial, sponges and

146

transparency film were once again washed as described above before the sponges were lifted

147

and placed into an Eppendorf tube with 1 ml of autoclaved water. Each Eppendorf tube was

148

vortexed for 2 min to extract the bacteria before tenfold serial dilutions to 10

-6

. The vortex

149

action disintegrated the sponge into single cells so any live bacteria could be quantified. The

150

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samples for bacteria analysis were tested for

E. coli

and

E. faecalis

using MacConkey No. 3

151

and Slanetz & Bartley agars respectively. The dilutions were plated onto each selective

152

medium in six 20 µl aliquots and incubated at 37 °C for 48 h before total viable colonies were

153

counted.

154

155

2.6 Data visualisation and analysis

156

Counts were converted to colony forming units (cfu) per mm

of sponge surface for each

157

individual sponge as follows:

158

= N

159

160

Where B

= bacteria in sponge, N = bacteria number per 1 ml, A = sponge area (mm

)

161

162

The bacterial numbers in all sponge replicates were used to calculate the arithmetic means

163

and standard error values for each experimental group, for

E. coli

E. faecalis

. The bacterial

164

counts from sponges for each bacteria type was converted to represent the percentage

165

retained of the original

E. coli

and

E. faecalis

added to the experimental tube. Statistical tests

166

were carried out in SPSS (RRID:SCR_002865) using the bacterial counts from each sponge.

167

Kolmogorov

–

Smirnov tests showed that bacteria retention was not normally distributed and

168

so different treatments were tested for significant differences (p<0.05) with Kruskal Wallis

169

tests and post hoc pairwise comparisons using Bonferroni corrected Mann

–

Whitney U tests

170

(trial 1) or the Mann-Whitney U test (trial 2). To test for significant differences (p<0.05) in

171

the bacteria retained by the individual sponges, Wilcoxon Matched-Paired Signed-Rank tests

172

were conducted for both trials.

173

174

3. Results

175

The

laboratory experiments quantified the sponges’ response to

the simultaneous exposure

176

with

E. coli

and

E. faecalis

at different ratios (trial 1) and to a different sequential order of

177

exposure with an identical abundance of the two bacterial species

(trial 2).

178

179

3.1 Retention of E. coli and E. faecalis in sponges exposed to different ratios of both bacteria

180

(trial 1)

181

The percentage of bacterial retention by sponges was very low regardless of the initial

182

bacterial concentration, and the bacterial retention of sponges never exceeded 0.05% (Figure

183

1) possibly owing to the small size of the sponges (<6 mm

) as they were hatched from a

184

single gemmule and could not merge into larger organisms as occurs in rivers. Considering

185

each bacterial species separately, there was a significant difference between the bacterial

186

retention for both

E. coli

and

E. faecalis

(

E. coli

H=14.25,

=2, p=0.001;

E. faecalis

187

H=13.24,

=2, p=0.001). The relative

E. coli

retention by sponges was significantly higher

188

when it constituted 10% and 50% of the bacterial suspension than when it was 90% (10%-

189

90% U=95 p<0.001; 50%-90% U=136, p=0.005). A similar pattern was observed for

190

faecalis

retention whereby the amount retained was higher at 10% than 50% and was

191

significantly lower when it represented 90% of the assemblage of waterborne bacteria (10%-

192

90% U=405 p=0.001; 50%-90% U=136, p=0.005). Thus, higher relative abundances of both

193

tested bacteria appeared to result in lower retention within the sponge.

194

195

Considering the combined pattern of both bacterial species, retention values for

E. coli

were

196

always higher than for

E. faecalis

in individual sponges, even when

E. coli

formed only 10%

197

of the bacterial exposure thus indicating that sponges may have a higher ability to retain

198

coli

. When sponges were exposed to 10%

E. coli

and 90%

E. faecalis

or assemblages with

199

50% representation of both bacteria, they retained significantly higher abundances of

E. coli

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the same validated filtration test design for gemmule grown juvenile sponges our

E. fluviatilis

268

colonies actually achieved a greater reduction of waterborne

E. coli

concentrations in 96 h

269

filtration tests (Cartwright, 2018). With an obvious dependence on colony size, higher

270

clearance rates and bacteria accumulation have been reported for large colonies of marine

271

sponges (Milanese

et al.

2003; Fu

et al.

2006). Yet our study was conducted with juvenile

272

sponges as unlike sponge colonies the gemmules these are grown from can be surface

273

sterilised to exclude any associated bacteria which were not added to the microcosm for

274

exposure.

275

276

Our study showed that

E. fluviatilis

preferably retained

E. coli

E. faecalis

regardless of the

277

level of experimental exposure to bacteria. Previous research has led to varying conclusions.

278

Some marine sponges appear to have no selectivity in their retention ability (Pile

et al.

, 1996;

279

Wehrl

et al.

, 2007). However, more recent evidence for selective feeding in sponges has

280

documented that some species may indeed be able to filter specific strains or groups of

281

bacteria at a faster rate (Maldonado

et al.

, 2010; McMurray

et al.

, 2016) and there is evidence

282

that differences in digestibility could encourage selective feeding (Maldonado

et al

., 2010).

283

Our study appears to be the first to provide information on the rates of the filtration or

284

retention by sponges for enterococcus and bacteria assemblages which include this faecal

285

indicator organism, while most previous research has been conducted on

E. coli

. As Gram

286

positive and Gram negative bacteria these test organisms differ in their cell wall structure and

287

this may also lead to different digestibility.

288

289

The observed low retention of bacteria could also result from an infection of the sponge

290

tissue which may compromise a

colony’s

filtration ability and digestive system. Sponges can

291

succumb to bacterial infection where their cells become overrun with bacteria causing death

292

to the organism (Böhm

et al.

, 2001). Fu

et al.

(2013) demonstrated the infection of the marine

293

sponge

Hymeniacidon perleve

with

Vibrio

spp. through genetic markers for cell death.

294

However,

E. coli

did not infect the sponge species they investigated. Many studies have

295

successfully used

E. coli

for the investigation of sponge filtration rates (Reiswig, 1975;

296

Willenz

et al.

, 1986; Milanese

et al.

, 2003) and our own filtration tests (Cartwright, 2018) do

297

not suggest that exposure to these bacteria had a negative effect on freshwater sponges. While

298

there appears to be no information on the pathogenicity of

E. faecalis

for sponges, it has been

299

demonstrated by fluorescent

in situ

hybridization

that

E. faecalis

cells attach and aggregate

300

on the external and internal structure of gemmule grown

E. fluviatilis

(Cartwright

et al.

2020)

301

which may indicate an infection capacity. Biofilms of enterococci have been found to release

302

surface aggregation proteins by quorum sensing regulators that can trigger a virulence mode

303

thus causing infection (Arias and Murray, 2012; Zheng

et al.

2017). If

E. faecalis

can infect

304

sponges, there could be an evolutionary advantage in ejecting or egesting these bacteria

305

selectively and this may indeed explain their low retention by sponges throughout our study.

306

Such a response would compromise the suitability of sponges for quantitative time-integrated

307

monitoring.

308

309

When exposed to only one of the two bacteria species in these trials before subsequent

310

exposure to the other species,

E. fluviatilis

retained more

E. coli

regardless of the sequential

311

order of exposure. This confirms that sponges can be qualitative indicators of bacterial

312

presence for an extended time period; however, the abundance of retained bacteria in a

313

sponge also does not appear indicative of the sequence of exposure to different bacteria. Yet

314

this does not prevent applications of sponges for sampling a historic pollution signature. For

315

example, if a faecal pollution episode occurred two days before sampling, a sustained

316

presence of faecal indicator bacteria would be unlikely in a water sample while a sponge

317

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sample could allow for its detection. Future studies should therefore investigate the length of

318

time sponges can retain filtered bacteria post exposure and the minimal concentration and

319

load of bacteria required in exposure to be detectable in a sponge sample. This would test the

320

suitability of sponges for time-integrated qualitative surveillance monitoring of water quality.

321

322

5. Conclusion

323

This study presents evidence that sponges may be suitable for the detection of microbes

324

associated with current and historic faecal pollution episodes. Investigated juvenile

Ephydatia

325

fluviatilis

colonies retained a lower proportion of waterborne bacteria especially when these

326

had been in very high abundance thus demonstrating that sponge tissue analysis will only

327

yield qualitative but not quantitative data for

the presence of indicator bacteria for faecal

328

pollution in aquatic systems for biomonitoring purposes. This study also presents first

329

evidence for the preferential retention of

E. coli

over

E. faecalis

by freshwater sponge

330

fluviatilis.

Sponges can thus provide a time integrated qualitative record of the presence of

331

indicator bacteria from episodic faecal pollution in aquatic systems. They may therefore be

332

suitable for surveillance monitoring of water quality where only low frequency sampling is

333

feasible. Monitoring of natural bathing water sites may be a potential application as

334

conventional monitoring relies on water sampling intervals of one week or longer. Future

335

studies should therefore validate detection limits, i.e. the minimal bacterial exposure required

336

for retention by sponges and the duration of detectable retention as evidence of past pollution

337

events. These would help to determine the suitability of sponges for qualitative time

338

integrated monitoring of faecal indicator bacteria in aquatic systems. If the required minimal

339

bacteria concentrations for retention by sponges were aligned to health relevant water quality

340

thresholds that would enable useful applications.

341

342

Conflicts of Interests

343

The authors declare that there are no conflicts of interests.

344

345

All work was conducted by the authors. Kindly note that much of this work has been reported

346

previously in my PhD thesis cited below (Cartwright, 2018).

347

348

Ethical approval

349

No ethical approval was required for this research.

350

351

Funding information

352

The laboratory work was funded by the Northern Ireland Department for Education and

353

Learning as part of a PhD studentship.

354

355

Author contribution

356

AC investigation, methodology, writing; JSGD conceptualization, supervision, writing; CM

357

supervision; JA conceptualization, supervision, writing.

358

359

Data summary

360

The raw data for the bacterial counts, sponge sizes, bacteria per mm

of sponge and % of

361

added bacteria retained are given for each treatment in each trial. Trial 1 was the bacteria

362

retained when sponges were exposed to

E. coli

and

E. faecalis

in different ratios. Trail 2 was

363

the bacteria retained when sponges were exposed to

E. coli

E. faecalis

first and the

364

opposite

second.

All

data

used

available

from

365

https://figshare.com/s/da59abc86c8d0e7887d7

366

367

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